H. Clive Palfrey

CFTR regulates phagosome acidification in macrophages and alters bactericidal activity

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl− channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr−/− mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

Quantal Size Is Dependent on Stimulation Frequency and Calcium Entry in Calf Chromaffin Cells

To what extent the quantal hypothesis of transmitter release applies to dense-core vesicle (DCV) secretion is unknown. We determined the characteristics of individual secretory events in calf chromaffin cells using catecholamine amperometry combined with different patterns of stimulation. Raising the frequency of action potential trains from 0.25-10 Hz in 2 mM [Ca(2+)]o or [Ca(2+)]o from 0.25-7 mM at 7 Hz elevated the amount released per event (quantal size). With increased stimulation, quantal size rose continuously, not abruptly, suggesting that release efficiency from a single population of DCVs rather than recruitment of different-sized vesicles contributed to the effect. These results suggest that catecholamine secretion does not conform to the quantal model. Inhibition of rapid endocytosis damped secretion in successive episodes, implying an essential role for this process in the recycling of vesicles needed for continuous secretion.

High-affinity nerve growth factor receptor (Trk) immunoreactivity is localized in cholinergic neurons of the basal forebrain and striatum in the adult rat brain

Trk-immunoreactivity was observed in basal forebrain and striatal cholinergic neurons, whereas low-affinity NGF receptor immunoreactivity was observed in basal forebrain but not striatal cholinergic neurons. Since NGF exerts trophic actions on both basal forebrain and striatal cholinergic populations, the presence of Trk in these neurons lends strong support for an essential role of Trk in NGF-responsive neurons, but suggests that the low affinity receptor is not necessary for NGF actions in the striatum.

HORMONE‐SENSITIVE ION TRANSPORT SYSTEMS IN ERYTHROCYTES AS MODELS FOR EPITHELIAL ION PATHWAYS

216. Sci. USA. 78: 1057-1061. 1980. Nature 284: 281-283. Palfrey & Greengard : Hormone-Sensitive Transport 307

Sustained stimulation shifts the mechanism of endocytosis from dynamin-1-dependent rapid endocytosis to clathrin- and dynamin-2-mediated slow endocytosis in chromaffin cells

Transient stimulation of secretion in calf chromaffin cells is invariably followed by rapid endocytosis (RE), a clathrin- and K+-independent process with a half time of several seconds. Here we show that when exocytosis is triggered in a more sustained manner, a much slower form of endocytosis (SE) replaces RE. SE is complete within 10 min and is abolished when anticlathrin antibodies are introduced into the cell or when intracellular K+ is removed. RE, but not SE, is blocked by intracellular administration of antidynamin-1 antibodies; the inverse specificity was found for antidynamin-2 antibodies. Replacement of extracellular Ca2+ by Ba2+ or Sr2+ completely blocked RE but had little effect on SE. Thus chromaffin cells exhibit two kinetically and mechanistically distinct forms of endocytosis that are coupled to different extents of exocytosis and are mediated by different isoforms of dynamin. We surmise that RE is associated with the transient fusion (“kiss-and-run”) mechanism of transmitter release and is the prevalent means of vesicle recapture and recycling under normal physiological conditions, whereas the clathrin-based SE mechanism comes into play only at higher levels of stimulation and may be associated with complete fusion of vesicles with the plasma membrane.

Calmodulin is the divalent cation receptor for rapid endocytosis, but not exocytosis, in adrenal chromaffin cells.

Exocytosis and the ensuing rapid endocytosis in adrenal chromaffin cells are both Ca(2+)-dependent phenomena but differ in their divalent cation specificity, implying distinct Ca2+ receptors for the two processes. To ascertain whether calmodulin is the Ca2+ receptor for either process, we blocked its function by introducing calmodulin-binding peptides or anti-calmodulin antibodies into these cells. Exo/endocytosis was followed by measurement of cell membrane capacitance. Rapid endocytosis, but not exocytosis, was abolished by these treatments, indicating that calmodulin is the Ca2+ receptor for rapid endocytosis but is not involved in exocytosis. The principal calmodulin target is not protein phosphatase-2B, as antagonism of this enzyme did not inhibit but accelerated rapid endocytosis. Calmodulin may thus regulate both the rate and extent of rapid endocytosis by distinct pathways.

Specific role for the PH domain of dynamin‐1 in the regulation of rapid endocytosis in adrenal chromaffin cells

Dynamin plays a key role in the scission event common to various types of endocytosis. We demonstrate that the pleckstrin homology (PH) domain of dynamin‐1 is critical in the process of rapid endocytosis (RE) in chromaffin cells. Introduction of this isolated PH domain into cells at concentrations as low as 1 μM completely suppressed RE. PH domains from other proteins, including that from the closely related dynamin‐2, were ineffective as inhibitors, even at high concentrations. Mutational studies indicated that a pair of isoform‐specific amino acids, located in a variable loop between the first two β‐strands, accounted for the differential effect of the two dynamin PH domains. Switching these amino acids in the dynamin‐2 PH domain to the equivalent residues in dynamin‐1 (SL→GI) generated a molecule that blocked RE. Thus, the PH domain of dynamin‐1 is essential for RE and exhibits a precise molecular selectivity. As chromaffin cells express both dynamin‐1 and ‐2, we speculate that different isoforms of dynamin may regulate distinct endocytotic processes and that the PH domain contributes to this specificity.

Neurotrophin‐3 and Brain‐Derived Neurotrophic Factor Activate Multiple Signal Transduction Events but Are Not Survival Factors for Hippocampal Pyramidal Neurons

Abstract: Expression of the neurotrophin‐3 (NT‐3) receptor (TrkC) and the effects of NT‐3 on signal transduction were investigated in highly enriched populations of embryonic rat hippocampal pyramidal neurons grown in bilaminar cultures. PCR analysis revealed that the predominant trkC isoform is K1, which lacks an insert in the kinase domain. Polyclonal TrkC‐specific antibodies stained >90% of the neurons and revealed a single ∼145‐kDa protein in immunoblots of extracts from adult hippocampus and pyramidal neuron cultures. Addition of NT‐3 (50 mg/ml) to these cultures induced the tyrosine phosphorylation of TrkC but not TrkB, as determined by anti‐phosphotyrosine staining of immunoprecipitates; thus, all the effects of NT‐3 are mediated through TrkC. NT‐3 also increased the tyrosine phosphorylation of 42‐, 44‐, 49‐, 55‐, 95‐, and 145‐kDa proteins; the pattern induced by brain‐derived neurotrophic factor (BDNF) was similar but not identical to that induced by NT‐3, suggesting that subtle differences may exist in signaling by TrkB and TrkC receptors. Immunoprecipitation of p21ras from 32P‐prelabeled cells showed that NT‐3 increased the level of the GTP‐bound form of the protein threefold over the control within 5 min. Mitogen‐activated protein (MAP) kinase activity was maximally elevated by NT‐3 within 2 min and then returned slowly toward baseline over the next 60 min. Tyrosine phosphorylation of phospholipase C‐γ increased rapidly after NT‐3, suggesting that this enzyme becomes activated. Consistent with this, the neurotrophin rapidly increased protein kinase C activity as well as intracellular Ca2+ levels. The effects of both NT‐3 and BDNF on Ca2+ levels were attenuated in Ca2+‐free medium, suggesting that both neurotrophins increase Ca2+ flux across the plasma membrane as well as release from internal stores. NT‐3 also increased c‐Fos expression in >80% of the cells; the effect peaked at 30 minand declined to baseline by 120 min. Despite the activation of ras‐MAP kinase and phosphoinositide signaling pathways, neither NT‐3 nor BDNF alone or in combination could sustain hippocampal pyramidal neurons deprived of glial support. We conclude that in this system NT‐3 and BDNF do not appear to be acting as classical “neurotrophic” factors and that activation of the MAP kinase pathway is insufficient for the promotion of neuronal survival.

BDNF-activated Sianal Transduction in Rat Cortical Glial Cells

Cortical glial cells in culture were found to be responsive to the neurotrophin brain‐derived neurotrophic factor (BDNF), as evidenced by activation of multiple signal transduction processes. BDNF produced an increase in mitogen‐activated protein (MAP) kinase tyrosine phosphorylation, MAP kinase activity, intracellular calcium concentration and c‐fos expression in the glial cells. Only a subset of the glial cells responded to BDNF, as reflected in single‐cell analysis of calcium transients and c‐fos expression. BDNF had no detectable effect on glial mitotic activity, as measured by DNA synthesis. In parallel studies, nerve growth factor and neurotrophin‐3 had no effect on signalling in these cultures. BDNF has previously been demonstrated to act via trkB receptors with a cytoplasmic tyrosine kinase domain (gp145trkB). Pretreatment of glial cultures with K252a, which at low concentrations specifically inhibits the trk tyrosine kinases, abolished BDNF effects on MAP kinase stimulation, suggesting that BDNF was acting through gp 145trkB. However, subsequent studies showed that gp 145trkB was expressed at extremely low levels in the cultures: gp145trkB mRNA transcripts could only be detected using the reverse transcription‐polymerase chain reaction, and gp 145trkB protein was not detected by either immunoblotting or immunocytochemistry. On the other hand, the glia expressed significantly higher levels of gp95trkB mRNA and protein, which represent truncated forms of trkB receptors lacking the tyrosine kinase domain. The results of these studies demonstrate that a subset of cultured CNS glia respond to BDNF with the activation of conventional signal transduction processes. The mechanism of BDNF‐initiated signal transduction in glial cells most likely involves a relatively small number of gp 145trkB receptors, but involvement of the more abundant truncated gp95trkB receptors cannot be excluded.

Secretion: Dense-core vesicles can kiss-and-run too

New measurements show that the entire transmitter contents of a dense-core vesicle can be released within a second through a narrow fusion pore that opens transiently. With other results, this raises the possibility that some dense core vesicles may, like small synaptic vesicles, undergo immediate recycling.