Edward L. Stuenkel

A Pleckstrin Homology Domain Specific for Phosphatidylinositol 4,5-Bisphosphate (PtdIns-4,5-P2) and Fused to Green Fluorescent Protein Identifies Plasma Membrane PtdIns-4,5-P2 as Being Important in Exocytosis

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca2+. One of these priming steps involves the maintenance of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P2is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P2 important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P2 that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4,5-P2 and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P2, showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca2+ entry and suggest that plasma membrane PtdIns-4,5-P2 is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P2 in some manner alters calcium channel function in chromaffin cells.

Role of Q-type Ca2+ Channels in Vasopressin Secretion From Neurohypophysial Terminals of the Rat

1 The nerve endings of rat neurohypophyses were acutely dissociated and a combination of pharmacological, biophysical and biochemical techniques was used to determine which classes of Ca2+ channels on these central nervous system (CNS) terminals contribute functionally to arginine vasopressin (AVP) and oxytocin (OT) secretion. 2 Purified neurohypophysial plasma membranes not only had a single high‐affinity binding site for the N‐channel‐specific ω‐conopeptide MVIIA, but also a distinct high‐affinity site for another ω‐conopeptide (MVIIC), which affects both N‐ and P/Q‐channels. 3 Neurohypophysial terminals exhibited, besides L‐ and N‐type currents, another component of the Ca2+ current that was only blocked by low concentrations of MVIIC or by high concentrations of ω‐AgaIVA, a P/Q‐channel‐selective spider toxin. 4 This Ca2+ current component had pharmacological and biophysical properties similar to those described for the fast‐inactivating form of the P/Q‐channel class, suggesting that in the neurohypophysial terminals this current is mediated by a ‘Q’‐type channel. 5 Pharmacological additivity studies showed that this Q‐component contributed to rises in intraterminal Ca2+ concentration ([Ca2+]i) in only half of the terminals tested. 6 Furthermore, the non‐L‐ and non‐N‐component of Ca2+‐dependent AVP release, but not OT release, was effectively abolished by the same blockers of Q‐type current. 7 Thus Q‐channels are present on a subset of the neurohypophysial terminals where, in combination with N‐ and L‐channels, they control AVP but not OT peptide neurosecretion.

Ghrelin stimulates neurogenesis in the dorsal motor nucleus of the vagus

Ghrelin, a gastric peptide hormone, has been reported to regulate growth hormone secretion and energy homeostasis. Here we show that ghrelin promotes neural proliferation in vivo and in vitro in the rat dorsal motor nucleus of the vagus (DMNV). Ghrelin receptor mRNA and immunoreactivity were detected in tissues from DMNV. Systemic administration of ghrelin (130 nmol kg−1) significantly increased 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation in the DMNV in adult rats with cervical vagotomy (BrdU positive cells; from 27 ± 4 to 69 ± 14 n= 5, P < 0.05). In vitro, exposure of cultured DMNV neurones to ghrelin significantly increased the percentage of BrdU incorporation into cells in both dose‐dependent (10−9–10−6m), and time‐dependent (6 h to 48 h) manners. Ghrelin significantly increased voltage‐activated calcium currents in isolated single DMNV neurones from a mean maximal change of 141 ± 26 pA to 227 ± 37 pA. Upon removal of ghrelin, calcium currents slowly returned to baseline. Blocking L‐type calcium channels by diltiazem (10 μm) significantly attenuated ghrelin‐mediated increments in BrdU incorporation (n= 5, P < 0.05). Ghrelin acts directly on DMNV neurones to stimulate neurogenesis.

Regulation of intracellular calcium and calcium buffering properties of rat isolated neurohypophysial nerve endings.

1. Electrophysiological measurements of Ca2+ influx using patch clamp methodology were combined with fluorescent monitoring of the free intracellular calcium concentration ([Ca2+]i) to determine mechanisms of Ca2+ regulation in isolated nerve endings from the rat neurohypophysis. 2. Application of step depolarizations under voltage clamp resulted in voltage‐dependent calcium influx (ICa) and increase in the [Ca2+]i. The increase in [Ca2+]i was proportional to the time‐integrated ICa for low calcium loads but approached an asymptote of [Ca2+]i at large Ca2+ loads. These data indicate the presence of two distinct rapid Ca2+ buffering mechanisms. 3. Dialysis of fura‐2, which competes for Ca2+ binding with the endogenous Ca2+ buffers, reduced the amplitude and increased the duration of the step depolarization‐evoked Ca2+ transients. More than 99% of Ca2+ influx at low Ca2+ loads is immediately buffered by this endogenous buffer component, which probably consists of intracellular Ca2+ binding proteins. 4. The capacity of the endogenous buffer for binding Ca2+ remained stable during 300 s of dialysis of the nerve endings. These properties indicated that this Ca2+ buffer component was either immobile or of high molecular weight and slowly diffusible. 5. In the presence of large Ca2+ loads a second distinct Ca2+ buffer mechanism was resolved which limited increases in [Ca2+]i to approximately 600 nM. This Ca2+ buffer exhibited high capacity but low affinity for Ca2+ and its presence resulted in a loss of proportionality between the integrated ICa and the increase in [Ca2+]i. This buffering mechanism was sensitive to the mitochondrial Ca2+ uptake inhibitor Ruthenium Red. 6. Basal [Ca2+]i, depolarization‐induced changes in [Ca2+]i and recovery of [Ca2+]i to resting levels following an induced increase in [Ca2+]i were unaffected by thapsigargin and cyclopiazonic acid, specific inhibitors of intracellular Ca(2+)‐ATPases. Caffeine and ryanodine were also without effect on Ca2+ regulation. 7. Evoked increases in [Ca2+]i, as well as rates of recovery from a Ca2+ load, were unaffected by the extracellular [Na+], suggesting a minimal role for Na(+)‐Ca2+ exchange in Ca2+ regulation in these nerve endings. 8. Application of repetitive step depolarizations for a constant period of stimulation resulted in a proportional frequency (up to 40 Hz)‐dependent increase in [Ca2+]i. On the other hand, for a constant number of stimuli a reduction in the [Ca2+]i. On the other hand, for a constant number of stimuli a reduction in the [Ca2+]i increase per impulse was observed at higher frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

A new role for the dynamin GTPase in the regulation of fusion pore expansion

The role of dynamin GTPase activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated. The experiments show that, in addition to playing a role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion.

Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that chronic increase of cytosolic calcium concentration in hepatocytes upon obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome-lysosome fusion in liver, preventing accumulation of protein inclusions and lipid droplets and suppressing inflammation and insulin resistance. As calcium channel blockers have been safely used in clinics for the treatment of hypertension for more than thirty years, our results suggest they may be a safe therapeutic option for restoring autophagic flux and treating metabolic pathologies in obese patients.

Fluorescence Resonance Energy Transfer Reports Properties of Syntaxin1A Interaction with Munc18-1 in Vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal α-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced ∼70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca2+-regulated secretory events.

Facilitation of Ca2+-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1‐V12, Rac1‐L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin‐permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1‐N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21‐activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1‐V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1‐V12‐mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin‐A inhibited secretion and eliminated the secretory effects of Rac1‐V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca2+ influx, partly through regulation of cytoskeletal organization.

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational “opening” of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.

Regulation of secretory granule recruitment and exocytosis at rat neurohypophysial nerve endings.

1. Time‐resolved cell membrane capacitance (Cm) measurements were used in combination with fura‐2 microfluorometry under whole‐cell patch clamp recording to investigate the kinetics and Ca2+ sensitivity of exocytotic granule fusion evoked by depolarizing stimuli at single, isolated nerve endings of the rat neurohypophysis. 2. Single step depolarizations or trains of depolarizing pulses evoked voltage‐dependent, inward Ca2+ currents (ICa) and induced both Ca(2+)‐dependent and Ca(2+)‐independent changes in Cm. Three distinct Cm responses were observed and were differentiated by their kinetics and Ca2+ sensitivity: a non‐exocytotic transient (delta Cm,t) and an exocytotic Cm ‘jump’ (delta Cm,J) and a slower, often latent, exocytotic Cm rise (delta Cm,s) that outlasted the depolarizing pulse stimulus. 3. The delta Cm,t was characterized by a rapid, transient component observed in 70% of nerve endings and a voltage‐activation relationship that preceded that of the ICa. The amplitude and kinetics of the delta Cm,t were unaffected by ICa block by Cd2+, Ca2+ load reduction, or alterations in intracellular Ca2+ buffering. 4. In contrast to the delta Cm,t, both the delta Cm,J and delta Cm,s were Ca2+ dependent as evidenced by their sensitivity to Cd2+ block of ICa, intraterminal application of 10 mM BAPTA and reduced [Ca2+]o or replacement of Ca2+ as the charge carrier with Ba2+. 5. The delta Cm,J was proportional to depolarization‐evoked Ca2+ influx with initial exocytotic rate of approximately 350 granule fusions s‐1. The amplitude of the delta Cm,J rose exponentially (tau = 40 ms) and approached an asymptote (15.5 fF) with longer duration depolarizations indicating the fusion from and depletion of an immediately releasable pool (IRP) estimated at nineteen docked and primed secretory granules. 6. The delta Cm,s was induced by the application of repetitive long duration pulses and defined as the exocytosis of secretory granules from a readily releasable granule pool (RRP). The delta Cm,s response occurred only after exceeding a [Ca2+]i threshold value and rose thereafter in proportion to Ca2+ influx with a mean initial secretory rate of 36 granule fusions s‐1. The mean latency for delta Cm,s activation was 850 ms following the initiation of the step depolarizations. The delta Cm,s response magnitude, reflecting the size of the RRP, was dependent on the resting [Ca2+]i and the nerve ending size, and was depletable using repetitive depolarizations of long duration. 7. Recruitment into and release from the RRP and IRP were differentially sensitive to changes in intraterminal Ca2+ buffering conditions. For example, introduction of 5 mM EGTA was shown to have no effect on the evoked IRP but significantly reduced the RRP. In comparison, diminishment of the endogenous Ca2+ buffering capacity of nerve endings by treatment with the mitochondrial Ca2+ uniporter blocker Ruthenium Red (10 microM) potentiated the RRP size but had no significant effect on the IRP size. 8. The present study indicates that the Ca(2+)‐dependent recruitment of and release from functionally distinct pools of peptide‐containing secretory granules in combination with the [Ca2+]i regulatory properties of neurohypophysial nerve endings may explain both the depletion of peptide release under prolonged stimulus and the potentiation of peptide release observed to occur during recurrent phasic action potential activity in this system.