Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers.

Before secretory vesicles undergo exocytosis, they must recruit the proteins syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in the plasma membrane. GFP-labeled versions of both proteins cluster at sites where secretory granules have docked. Single-particle tracking shows that minority populations of both molecules are strongly hindered in their mobility, consistent with their confinement in nanodomains. We measured the fluorescence of granule-associated clusters, the fluorescence of single molecules, and the numbers of unlabeled syntaxin-1 and SNAP-25 molecules per cell. There was a more than 10-fold excess of SNAP-25 over syntaxin-1. Fifty to seventy copies each of syntaxin-1 and SNAP-25 molecules were associated with a single docked granule, many more than have been reported to be required for fusion.

Multivesicular exocytosis in rat pancreatic beta cells.

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 µmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 µm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.

Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells.

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.

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.

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.

Combined polychemotherapy in the treatment of primitive lung cancer of the "non-oat cell" type.

Forty-one patients (30 males, 11 females; mean age 49.7 years) with diagnosis of the "non-oat cell" type of lung cancer were studied. All patients were included in a randomized treatment design (regimens A and B) and completed six full cycles of polychemotherapy. Only 28 of the 41 patients proved evaluable. The performance status was 0-2 in 20 subjects (71.4%) and 3-4 in 8 (28.6%). The total number of patients achieving partial and complete responses was 20 (71.4%), of whom 14 (70%) received CDDP and the other 6 DDR. A progression of the disease was seen in 8 patients (28.6%), of whom 5 (62.5%) received DDR and the other 3 (37.5%) CDDP. An increase in body weight (3-8 kg) was observed in 15 patients (53.6%). Twenty-four of the 28 patients (85.7%) had a subjective feeling of well-being throughout the 6 cycles of chemotherapy. Nausea and vomiting and alopecia were the most frequent side-effects. The most important complications were paralytic ileus in 2 cases and a darkish manifestation in forearm and hand tissue due to vinblastine extravasation in another 2 cases. Haematological toxicity was found to be acceptable: leucopenia (less than 3.000) in 30 patients (73.2%); and thrombocytopenia (less than 100.000) in 12 patients (29.2%), which proved reversible. Some modalities relative to treatment schemes and the incorporation of additional agents are discussed.

Compensatory endocytosis in chromaffin cells.

Exocytosis occurs via fusion of secretory granules with the cell membrane, whereupon the granule content is at least partially released and the granule membrane is temporarily added to the plasma membrane. Exocytosis is balanced by compensatory endocytosis to achieve net equilibrium of the cell surface area and to recycle and redistribute components of the exocytosis machinery. The underlying molecular mechanisms remain a matter of debate. In this review, we summarize and discuss recent progress in the understanding of compensatory endocytosis, with the focus on chromaffin cells as a useful model for studying mechanisms of regulated secretion.

Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors.

The single-channel activity of rabbit skeletal muscle ryanodine receptor (skeletal RyR) and dog cardiac RyR was studied as a function of cytosolic [Ca2+]. The studies reveal that for both skeletal and cardiac RyRs, heterogeneous populations of channels exist, rather than a uniform behavior. Skeletal muscle RyRs displayed two extremes of behavior: 1) low-activity RyRs (LA skeletal RyRs, approximately 35% of the channels) had very low open probability (Po < 0.1) at all [Ca2+] and remained closed in the presence of Mg2+ (2 mM) and ATP (1 mM); 2) high-activity RyRs (HA skeletal RyRs) had much higher activity and displayed further heterogeneity in their Po values at low [Ca2+] (< 50 nM), and in their patterns of activation by [Ca2+]. Hill coefficients for activation (nHa) varied from 0.8 to 5.2. Cardiac RyRs, in comparison, behaved more homogeneously. Most cardiac RyRs were closed at 100 nM [Ca2+] and activated in a cooperative manner (nHa ranged from 1.6 to 5.0), reaching a high Po (> 0.6) in the presence and absence of Mg2+ and ATP. Heart RyRs were much less sensitive (10x) to inhibition by [Ca2+] than skeletal RyRs. The differential heterogeneity of heart versus skeletal muscle RyRs may reflect the modulation required for calcium-induced calcium release versus depolarization-induced Ca2+ release.

Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms.

In the present study, we compare functional consequences of dissociation and reconstitution of binding proteins FKBP12 and FKBP12.6 with ryanodine receptors from cardiac (RyR2) and skeletal muscle (RyR1). The skeletal muscle RyR1 channel became activated on removal of endogenously bound FKBP12, consistent with previous reports. Both FKBP12 and FKBP12.6 rebind to FKBP-depleted RyR1 and restore its quiescent channel behavior by altering ligand sensitivity, as studied by single-channel recordings in planar lipid bilayers, and macroscopic behavior of the channels (ryanodine binding and net energized Ca2- uptake). By contrast, removal of FKBP12.6 from the cardiac RyR2 did not modulate the function of the channel using the same types of assays as for RyR1. FKBP12 or FKBP12.6 had no effect on channel activity of FKBP12.6-depleted cardiac RyR2, although FKBP12.6 rebinds. Our studies reveal important differences between the two ryanodine receptor isoforms with respect to their functional interaction with FKBP12 and FKBP12.6.

Patch-clamp characterisation of somatostatin-secreting -cells in intact mouse pancreatic islets.

The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact mouse pancreatic islets. Three types of electrical activity were observed corresponding to alpha-, beta- and delta-cells. The delta-cells were electrically active in the presence of glucose but lacked the oscillatory pattern seen in the beta-cells. By contrast, the alpha-cells were electrically silent at high glucose concentrations but action potentials could be elicited by removal of the sugar. Both alpha- and beta-cells contained transient voltage-activated K+ currents. In the delta-cells, the K+ currents activated above -20 mV and were completely blocked by TEA (20 mM). The alpha-cells differed from the delta-cells in possessing a TEA-resistant K+ current activating already at -40 mV. Immunocytochemistry revealed the presence of Kv3.4 channels in delta-cells and TEA-resistant Kv4.3 channels in alpha-cells. Thus the presence of a transient TEA-resistant current can be used to functionally separate the delta- and alpha-cells. A TTX-sensitive Na+ current developed in delta-cells during depolarisations beyond -30 mV and reached a peak amplitude of 350 pA. Steady-state inactivation of this current was half-maximal at -28 mV. The delta-cells were also equipped with a sustained Ca2+ current that activated above -30 mV and reached a peak of 60 pA when measured at 2.6 mM extracellular Ca2+. A tolbutamide-sensitive KATP channel conductance was observed in delta-cells exposed to glucose-free medium. Addition of tolbutamide (0.1 mM) depolarised the delta-cell and evoked electrical activity. We propose that the KATP channels in delta-cells serve the same function as in the beta-cell and couple an elevation of the blood glucose concentration to stimulation of hormone release.

Sarcomas of the jaw.

A retrospective analysis is made of 61 patients with clinical and histological diagnosis of pure sarcomas of the jaw treated between 1950 and 1984. Surgery was the treatment of choice in 41 cases. Biopsy or palliative treatment were undertaken in the remaining patients. Twenty-four cases relapsed before 12 months, and 12 relapsed before 3 years. Twenty-four died before 12 months, nine before 2 years, and three after 2 years. At present there is no evidence of disease in eight patients: three out of 25 fibrosarcomas (one more than 1 year, one more than 13 years, and one more than 14 years), three out of 15 chondrosarcomas (one more than 1 year, one more than 13 years, and one more than 14 years), and two out of 15 osteosarcomas (one more than 18 years). The prognosis is gloomy. However, an early diagnosis and radical surgery contribute to an improvement in the course of the disease.

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.

Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels.

The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial glucagon-secreting alpha-cells in intact mouse pancreatic islets. alpha-cells were distinguished from the beta- and delta-cells by the presence of a large TTX-blockable Na+ current, a TEA-resistant transient K+ current sensitive to 4-AP (A-current) and the presence of two kinetically separable Ca2+ current components corresponding to low- (T-type) and high-threshold (L-type) Ca2+ channels. The T-type Ca2+, Na+ and A-currents were subject to steady-state voltage-dependent inactivation, which was half-maximal at -45, -47 and -68 mV, respectively. Pancreatic alpha-cells were equipped with tolbutamide-sensitive, ATP-regulated K+ (KATP) channels. Addition of tolbutamide (0.1 mM) evoked a brief period of electrical activity followed by a depolarisation to a plateau of -30 mV with no regenerative electrical activity. Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mM glucose, concentrations up to 2 microM stimulated glucagon secretion to the same extent as removal of glucose. We conclude that electrical activity and secretion in the alpha-cells is dependent on the generation of Na+-dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage-dependent inactivation of voltage-gated membrane currents. Glucose may inhibit glucagon release by depolarising the alpha-cell with resultant inactivation of the ion channels participating in action potential generation.

Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets.

1. The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin-secreting B-cells and 65 % non-B-cells (A- and D-cells). 2. Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mM glucose and had the electrophysiological characteristics of isolated B-cells maintained in tissue culture. 3. The Ca2+ current recorded from B-cells in situ was 80 % larger than that of isolated B-cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4. Voltage-dependent K+ currents were observed during depolarisations to membrane potentials above -20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between -90 and -30 mV. 5. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP-regulated K+ (KATP) channels, amounted to approximately 4 nS. Glucose produced a concentration-dependent reduction of KATP channel conductance with half-maximal inhibition observed with 5 mM glucose. 6. Combining voltage- and current-clamp recording allowed the estimation of the gap junction conductance between different B-cells. These experiments indicated that the input conductance of the B-cell at stimulatory glucose concentrations ( approximately 1 nS) is almost entirely accounted for by coupling to neighbouring B-cells.

The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery.

Although N- and P-type Ca2+ channels predominant in fast-secreting systems, Lc-type Ca2+ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca2+ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic beta-cells, evoked secretion is highly sensitive to Ca2+. These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca2+ channels to allow high Ca2+ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65/syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc753-893, separating repeats II-III of its alpha1C subunit, interacts with p65 and "pulls" down native p65 from rat brain membranes. Lc753-893 injected into single insulin-secreting beta-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca2+, nor does it affect Ca2+ current. These results suggest that Lc753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.