Ronald W. Holz

α-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor

alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxins effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.

Intracellular Ca2+ activates phospholipase C

Abstract It is well established that a receptor-mediated mechanism, perhaps involving a guanine nucleotide binding protein, directly activates polyphosphoinositide-specific phospholipase C. Recent evidence indicates that in excitable tissues a rise in cytosolic Ca 2+ can also activate the phospholipase C. The activation of phospholipase C by Ca 2+ can be a direct effect rather than a result of the Ca 2+ -dependent release of neurotransmitters which activate phospholipase C through a receptor-mediated mechanism. Ca 2+ -activated phospholipase C may represent a positive feedback system for Ca 2+ : small increases in cytosolic Ca 2+ induced by Ca 2+ influx across the plasma membrane may result in higher cytosolic Ca 2+ concentrations due to IP 3 -induced release of Ca 2+ from intracellular stores. The activation of phospholipase C by Ca 2+ may also provide a mechanism for diacylglycerol generation and protein kinase C activation following Ca 2+ influx. Thus, the regulation of phospholipase C activity by Ca 2+ may be physiologically important in regulating cytosolic Ca 2+ and protein kinase C in excitable tissues.

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K+ stimulated 45Ca2+ uptake into chromaffin cells two‐ to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca2+ uptake and catecholamine secretion induced by the mixed nicotinic‐muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca2+ uptake occurred within 15 s of stimulation by carbachol or elevated K+ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K+ was similar to that of Ca2+ uptake. There was a close correlation between Ca2+ uptake and catecholamine secretion at various concentrations of Ca2+. The concentration dependencies for inhibition of both processes by Mg2+ or Cd2+ were similar. Ca2+ uptake saturated with increasing Ca2+ concentrations, with an apparent Km for both carbachol‐induced and elevated K+‐induced Ca2+ uptake of approximately 2 mM. The Ca2+ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca2+ entry and a presumed increase in cytosolic Ca2+ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca2+‐ dependent processes associated with catecholamine secretion. Ca2+ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage‐sensitive Ca2+ channels.

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.

Muscarinic receptors in chromaffin cell cultures mediate enhanced phospholipid labeling but not catecholamine secretion.

Abstract: The addition of either carbachol or muscarinic agonists to cultured bovine adrenal chromaffin cells results in a selective stimulation of phosphatidate (PhA) and phosphatidylinositol (PhI) labeling from 32Pi and [3H]glycerol that can be inhibited by the inclusion of atropine, but not d‐tubocurarine. In contrast, increased catecholamine secretion is observed on the addition of carbachol or nicotinic agonists and is inhibited by d‐tubocurarine but not by atropine. Added calcium is essential for catecholamine secretion but not for stimulated phospholipid labeling. Chelation of endogenous Ca2+ with EGTA does, however, inhibit the stimulated phospholipid labeling. These results suggest that stimulated phospholipid labeling in the bovine chromaffin cell and catecholamine secretion are separate and distinct processes.

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

Abstract: The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophos‐phate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mMCa2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1‐dimethyl‐4‐phenyl‐piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+‐dependent [3H]inositol phosphate production, presumably by a non‐receptor‐mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3–10 μM) directly stimulated the release of inositol phosphates from digitonin‐permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catechol‐amine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and wyo‐inositol 1,4,5‐trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.

Retrograde regulation of synaptic vesicle endocytosis and recycling

Sustained release of neurotransmitter depends upon the recycling of synaptic vesicles. Until now, it has been assumed that vesicle recycling is regulated by signals from the presynaptic bouton alone, but results from rat hippocampal neurons reported here indicate that this need not be the case. Fluorescence imaging and pharmacological analysis show that a nitric oxide (NO) signal generated postsynaptically can regulate endocytosis and at least one later step in synaptic vesicle recycling. The proposed retrograde pathway involves an NMDA receptor (NMDAR)-dependent postsynaptic production of NO, diffusion of NO to a presynaptic site, and a cGMP-dependent increase in presynaptic phosphatidylinositol 4,5-biphosphate (PIP2). These results indicate that the regulation of synaptic vesicle recycling may integrate a much broader range of neural activity signals than previously recognized, including postsynaptic depolarization and the activation of NMDARs at both immediate and nearby postsynaptic active zones.

The C2 Domains of Rabphilin3A Specifically Bind Phosphatidylinositol 4,5-Bisphosphate Containing Vesicles in a Ca2+-dependent Manner IN VITRO CHARACTERISTICS AND POSSIBLE SIGNIFICANCE

In the present study we investigated the lipid binding characteristics of the C2 domains of Rabphilin3a. We found that the tandem C2 domain of Rabphilin3a specifically bound lipid vesicles containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in a Ca2+-dependent manner. There was little binding to vesicles containing PtdIns(3,4)P2 in the presence or absence of Ca2+. Binding to phosphatidylinositol 3,4,5-triphosphate-containing vesicles was similar to binding to PtdIns(4,5)P2-containing vesicles. The presence of physiological amounts of phosphatidylserine (PS) greatly potentiated the ability of PtdIns(4,5)P2 to cause vesicle binding. As with the C2 domains together, the binding of individual C2 domain of Rabphilin3a was much greater to PtdIns(4,5)P2-containing vesicles than PtdIns(3,4)P2-containing vesicles. Both C2 domains also bound 29 mol % PS-containing vesicles in a Ca2+-dependent manner. Because of the importance of the C2B domain in the enhancement of secretion from chromaffin cells by Rabphilin3a, its biochemistry was further investigated. The mutation of aspartates 657 and 659 to asparagines in C2B decreased Ca2+-dependent and increased Ca2+-independent vesicle binding, indicating the Ca2+ dependence of the domain is provided by aspartic acid residues in the putative Ca2+-binding pocket. A peptide from the COOH-terminal region of the C2B domain specifically inhibited ATP-dependent secretion from permeabilized chromaffin cells and the binding of Rabphilin3a to phosphatidylcholine/PS/PtdIns(4,5)P2-containing lipid vesicles, suggesting a role of this sequence in secretion through its ability to interact with acidic lipid vesicles.

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors.

Poisoning with α-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of α-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind α-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to α-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of α-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of α-latrotoxin, cells of other tissues are also susceptible to the toxic effects of α-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin‐treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin‐treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin‐treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N‐ethylmaleimide (NEM) or p‐bromophenacyl bromide (BPB) resulted in dose‐dependent inhibition of 10 μM Ca2+‐stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 μM. The IC50 of BPB for both events was 25 μM. High concentrations (5–20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin‐treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.