Changes in cytochrome P450 side chain cleavage expression in the rat hippocampus after kainate injury

Our previous study showed an increase in total cholesterol level of the hippocampus after kainate-induced injury, but whether this is further metabolized to neurosteroids is not known. The first step in neurosteroid biosynthesis is the conversion of cholesterol to pregnenolone by the enzyme cytochrome P450 side chain cleavage (P450scc). This study was carried out to elucidate the expression of this enzyme in the kainate-lesioned rat hippocampus. A net decrease in P450scc protein was detected in hippocampal homogenates by Western blots at 2 weeks post-kainate injection (time of peak cholesterol concentration after kainate injury). Immunohistochemistry showed decreased labeling of the enzyme in neurons, but increased expression in a small number of astrocytes. The level of pregnenolone was also analyzed using a newly developed gas chromatography–mass spectrometry (GC–MS) method, optimized for the rat hippocampus. A non-significant tendency to a decrease in pregnenolone level was detected 2 weeks post-lesion. This is in contrast to a large increase in oxysterols in the lesioned hippocampus at this time (He et al. 2006). Together, they indicate that increased cholesterol in the kainate lesioned hippocampus is mostly metabolized to oxysterols, and not neurosteroids. Wan-Jie Chia and Andrew M. Jenner contributed equally to this work.     

Effects of cholesterol oxidation products on exocytosis

Increase in levels of oxysterols or cholesterol oxidation products have been detected in brain areas undergoing neuroinflammation after excitotoxic injury, and the present study was carried out to elucidate possible effects of these products on exocytosis in rat pheochromocytoma-12 (PC12) cells. An increase in vesicle fusion with the cell membrane indicating exocytosis was observed by total internal reflection microscopy (TIRFM), and confirmed by capacitance measurements, after addition of 7 ketocholesterol, 24 hydroxycholesterol or cholesterol 5, 6 beta epoxide. 7 ketocholesterol induced exocytosis was attenuated by pretreatment with a disruptor of cholesterol-rich domains or “lipid rafts”, methyl-β-cyclodextrin (MβCD) as demonstrated by capacitance and amperometry measurements of neurotransmitter release. Moreover, treatment of cells with thapsigargin to deplete intracellular calcium, or treatment of cells with lanthanum chloride to block calcium channels resulted in attenuation of 7 ketocholesterol induced exocytosis. Fura-2 imaging showed that 7 ketocholesterol induced rapid and sustained increases in intracellular calcium concentration, and that this effect was attenuated in cells that were pre-treated with MβCD, thapsigargin or lanthanum chloride. Together, the results suggest that neurotransmitter release triggered by 7 ketocholesterol is dependent on the integrity of cholesterol rich lipid domains on cellular membranes and a rise in intracellular calcium, either through release from internal stores or influx via calcium channels. Increased cholesterol oxidation product concentrations in brain areas undergoing neuroinflammation may enhance exocytosis and neurotransmitter release, thereby aggravating excitotoxicity.     

Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14

Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure, as a result of protein kinase C (PKC)-zeta activation. However, the mechanism responsible for this remains unknown. The purpose of this study is to determine if LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide produced by phosphatidylcholine-specific phospholipase C (PC-PLC) or CD14. To study this, THP-1 cells were stimulated with LPS. Selected cells were pretreated with the PC-PLC inhibitor D609, exogenous C2 ceramide, CD14 neutralizing antibody, or TLR4 neutralizing antibody. LPS led to production of ceramide, phosphorylation of PKC-zeta, and assembly of the TLR4 within lipid rafts. This was followed by activation of the mitogen-activated protein kinase family and the liberation of cytokines. Pretreatment with D609 or CD14 blockade was associated with attenuated LPS-induced ceramide production, TLR4 assembly on lipid rafts, and cytokine production. Pretreatment with TLR4 blockade did not affect LPS-induced ceramide production but was associated with significant attenuation in cytokine production. Treatment with C2 ceramide prior to LPS reversed the inhibitory effects induced by D609 but not of CD14 or TLR4 blockade. C2 ceramide alone induced the activation of PKC-zeta and the assembly of TLR4 but was not associated with cytokine liberation. This study demonstrates that TLR4 assembly and activation following LPS exposure require the production of ceramide by PC-PLC, which appears to be CD14-dependent.     

Ceramide and Toll-like receptor 4 are mobilized into membrane rafts in response to Helicobacter pylori infection in gastric epithelial cells

Helicobacter pylori infection is thought to be involved in the development of several gastric diseases. Two H. pylori virulence factors (vacuolating cytotoxin A and cytotoxin-associated gene A) reportedly interact with lipid rafts in gastric epithelial cells. The role of Toll-like receptor (TLR)-mediated signaling in response to H. pylori infection has been investigated extensively in host cells. However, the receptor molecules in lipid rafts that are involved in H. pylori-induced innate sensing have not been well characterized. This study investigated whether lipid rafts play a role in H. pylori-induced ceramide secretion and TLR4 expression and thereby contribute to inflammation in gastric epithelial cells. We observed that both TLR4 and MD-2 mRNA and protein levels were significantly higher in H. pylori-infected AGS cells than in mock-infected cells. Moreover, significantly more TLR4 protein was detected in detergent-resistant membranes extracted from H. pylori-infected AGS cells than in those extracted from mock-infected cells. However, this effect was attenuated by the treatment of cells with cholesterol-usurping agents, suggesting that H. pylori-induced TLR4 signaling is dependent on cholesterol-rich microdomains. Similarly, the level of cellular ceramide was elevated and ceramide was translocated into lipid rafts after H. pylori infection, leading to interleukin-8 (IL-8) production. Using the sphingomyelinase inhibitor imipramine, we observed that H. pylori-induced TLR4 expression was ceramide dependent. These results indicate the mobilization of ceramide and TLR4 into lipid rafts by H. pylori infection in response to inflammation in gastric epithelial cells.     

Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons

Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.     

Synaptic vesicle endocytosis at a CNS nerve terminal: faster kinetics at physiological temperatures and increased endocytotic capacity during maturation

Synaptic vesicle membrane must be quickly retrieved and recycled after copious exocytosis to limit the depletion of vesicle pools. The rate of endocytosis at the calyx of Held nerve terminal has been measured directly using membrane capacitance measurements from immature postnatal day P7-P10 rat pups at room temperature (RT: 23-24 degrees C). This rate has an average time constant of tens of seconds and becomes slower when the amount of exocytosis (measured as capacitance jump) increases. Such slow rates seem paradoxical for a synapse that can operate continuously at high-input frequencies. Here we perform time-resolved membrane capacitance measurements from the mouse calyx of Held in brain stem slices at physiological temperature (PT: 35-37 degrees C), and also from more mature calyces after the onset of hearing (P14-P18). Our results show that the rate of endocytosis is strongly temperature dependent, whereas the endocytotic capacity of a nerve terminal is dependent on developmental stage. At PT we find that endocytosis accelerates due to the addition of a kinetically fast component (time constant: tau = 1-2 s) immediately after exocytosis. Surprisingly, we find that at RT the rate of endocytosis triggered by short (1- to 5-ms) or long (> or =10-ms) depolarizing pulses in P14-P18 mice are similar (tau approximately 15 s). Furthermore, this rate is greatly accelerated at PT (tau approximately 2 s). Thus endocytosis becomes faster and less saturable during synaptic maturation, making the calyceal terminal more capable of sustaining prolonged high-frequency transmitter release.     

Quantal ATP release in rat β-cells by exocytosis of insulin-containing LDCVs

Quantal release of adenosine triphosphate (ATP) was monitored in rat pancreatic β-cells expressing P2X₂ receptors. Stimulation of exocytosis evoked rapidly activating and deactivating ATP-dependent transient inward currents (TICs). The unitary charge (q) of the events recorded at 0.2 μM [Ca²⁺]_i averaged 4.3 pC. The distribution of the ³√q of these events could be described by a single Gaussian. The rise times averaged ∼5 ms over a wide range of TIC amplitudes. In β-cells preloaded with 5-hydroxytryptamine (5-HT; accumulating in insulin granules), ATP was coreleased with 5-HT during >90% of the release events. Following step elevation of [Ca²⁺]_i to ∼5 μM by photo release of caged Ca²⁺, an increase in membrane capacitance was observed after 33 ms, whereas ATP release first became detectable after 43 ms. The step increase in [Ca²⁺]_i produced an initial large TIC followed by a series of smaller events that echoed the changes in membrane capacitance (ΔC _m). Mathematical modeling suggests that the large initial TIC reflects the superimposition of many unitary events. Exocytosis, measured as ΔC _m or TICs, was complete within 2 s after elevation of [Ca²⁺]_i with no sign of endocytosis masking the capacitance increase. The relationship between total charge (Q) and ΔC _m was linear with a slope of ∼1.2 pC/fF. The latter value predicts a capacitance increase of 3.6 fF for the observed mean value of q, close to that expected for exocytosis of individual insulin granules. Our results indicate that measurements of ATP release and ΔC _m principally (≥85–95%) report exocytosis of insulin granules.     

FcγRIIa requires lipid rafts, but not co-localization into rafts, for effector function

Objective

To determine if receptor localization into lipid rafts, or the lipid rafts themselves, are important for FcγRIIa effector functions.

Material

Wild-type FcγRIIa or mutant FcγRIIa(C208A) that does not translocate to lipid rafts were transfected into Chinese hamster ovary (CHO) cells which have been shown to be reliable cells for studying FcγR function.

Treatment

Cells were treated with buffer or methyl-β-cyclodextrin (MβCD) to deplete cholesterol and dissolve the structure of lipid rafts.

Methods

To evaluate lipid raft association, transfected CHO cells were lysed and centrifuged over a sucrose gradient. Fractions were run on SDS-PAGE and blotted for FcγRIIa or sphingolipid GM1 to illustrate the lipid raft fractions. Lateral mobility of GFP-tagged wild-type or mutant FcγRIIa was assessed using fluorescence recovery after photobleaching (FRAP) microscopy. Internalization of IgG-opsonized erythrocytes was assessed by fluorescence microscopy and uptake of heat-aggregated IgG (haIgG) was measured using flow cytometry.

Results

We observed that FcγRIIa(C208A) did not localize into lipid rafts. However, the mutant FcγRIIa retained lateral mobility and effector function similar to wild-type FcγRIIa. However, mutant FcγRIIa function was abolished upon treatment with MβCD.

Conclusions

Lipid rafts provide an essential component required for effector activities independent of receptor localization.     

Presynaptic kainate receptor facilitation of glutamate release involves protein kinase A in the rat hippocampus

We have explored the mechanisms involved in the facilitation of glutamate release mediated by the activation of kainate receptors in the rat hippocampus using isolated nerve terminal (synaptosome) and slice preparations. In hippocampal nerve terminals, kainate (KA) produced an increase of glutamate release at concentrations of agonist ranging from 10 to 1000 μ m . In hippocampal slices, KA at low nanomolar concentrations (20–50 n m ) also produced an increase of evoked excitatory postsynaptic currents (eEPSCs) at mossy fibre–CA3 synapses. In both, synaptosomes and slices, the effect of KA was antagonized by CNQX, and persisted after pretreatment with a cocktail of antagonists for other receptors whose activation could potentially have produced facilitation of release. These data indicate that the facilitation of glutamate release observed is mediated by the activation of presynaptic glutamate receptors of the kainate type. Mechanistically, the observed effects of KA appear to be the same in synaptosomal and slice preparations. Thus, the effect of KA on glutamate release and mossy fibre–CA3 synaptic transmission was occluded by the stimulation of adenylyl cyclase by forskolin and suppressed by the inhibition of protein kinase A by H-89 or Rp-Br-cAMP. We conclude that kainate receptors present at presynaptic terminals in the rat hippocampus mediate the facilitation of glutamate release through a mechanism involving the activation of an adenylyl cyclase–second messenger cAMP–protein kinase A signalling cascade.     

Layer-specific sulfatide localization in rat hippocampus middle molecular layer is revealed by nanoparticle-assisted laser desorption/ionization imaging mass spectrometry

Lipids are major structural component of the brain and play key roles in signaling functions in the central nervous system (CNS), such as the hippocampus. In particular, sulfatide is an abundant glycosphingolipid component of both the central and the peripheral nervous system and is an essential lipid component of myelin membranes. Lack of sulfatide is observed in myelin deformation and neurological deficits. Previous studies with antisulfatide antibody have investigated distribution of sulfatide expression in neurons; however, this method cannot distinguish the differences of sulfatide lipid species raised by difference of carbon-chain length in the ceramide portion in addition to the differences of sulfatide and seminolipid. In this study, we solved the problem by our recently developed nanoparticle-assisted laser desorption/ionization (nano-PALDI)-based imaging mass spectrometry (IMS). We revealed that the level of sulfatide in the middle molecular layer was significantly higher than that in granule cell layers and the inner molecular layer in the dentate gyrus of rat hippocampus. H. Ageta and S. Asai contributed equally to this work.     

Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms

The integrin CD11b/CD18 plays a central role in neutrophil phagocytosis. Although CD11b/CD18 binds a wide range of ligands, including C3bi and beta-glucan, and transmits outside-in signaling, the mechanism of this signaling responsible for phagocytosis remains obscure. Here, we report that lactosylceramide (LacCer)-enriched lipid rafts are required for CD11b/CD18-mediated phagocytosis of nonopsonized zymosans (NOZs) by human neutrophils. Anti-CD11b and anti-LacCer antibodies inhibited the binding of NOZs to neutrophils and the phagocytosis of NOZs. During phagocytosis of NOZ, CD11b and LacCer were accumulated and colocalized in the actin-enriched phagocytic cup regions. Immunoprecipitation experiments suggested that CD11b/CD18 was mobilized into the LacCer-enriched lipid rafts during phagocytosis of NOZs. DMSO-treated, neutrophil-like HL-60 cells (D-HL-60 cells) lacking Lyn-coupled, LacCer-mediated signaling showed little phagocytosis of NOZs. However, loading of D-HL-60 cells with C24 fatty acid chain-containing LacCer (C24-LacCer) reconstructed functional Lyn-associated, LacCer-enriched lipid rafts, and restored D-HL-60 cell NOZ phagocytic activity, which was inhibited by anti-LacCer and anti-CD11b antibodies. Lyn knockdown by small interfering RNA blocked the effect of C24:1-LacCer loading on D-HL-60 cell phagocytosis of NOZs. CD11b/CD18 activation experiments indicated phosphorylation of LacCer-associated Lyn by activation of CD11b. Taken together, these observations suggest that CD11b activation causes translocation of CD11b/CD18 into Lyn-coupled, LacCer-enriched lipid rafts, allowing neutrophils to phagocytose NOZs via CD11b/CD18.     

Quinacrine abolishes increases in cytoplasmic phospholipase A<Subscript>2</Subscript> mRNA levels in the rat hippocampus after kainate-induced neuronal injury

The present investigation was carried out to study the possible effects of quinacrine in modulating cytoplasmic phospholipase A₂ (cPLA₂) mRNA levels in rat hippocampus after kainate treatment. Injections of kainate into the right lateral ventricle resulted in significant increases in cPLA₂ mRNA levels in the hippocampus, at 3 days and 7 days after injection. The elevation in cPLA₂ mRNA levels is consistent with previous observations of increased cPLA₂ immunoreactivity in degenerating neurons and astrocytes at these times. Rats that received once daily intraperitoneal injections of quinacrine (5 mg/kg) after the intracerebroventricular kainate injections showed almost complete attenuation of increased cPLA₂ expression, at both 3 and 7 days after kainate injection. These results show that in addition to its well-known effect of inhibition of PLA₂ activity, quinacrine could also inhibit cPLA₂ expression, and further supports a role for PLA₂ in kainate-induced neuronal injury. Electronic Publication     

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells

 Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca²⁺ current (I _Ca) and exocytosis. The dependency of exocytosis on I _Ca evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of I _Ca, implying the existence of a Ca²⁺-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca²⁺ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca²⁺ concentration. Pertussis toxin blocked the inhibitory effects of ATP on I _Ca and exocytosis. Although RB-2, a P_2Y antagonist, blocked the inhibitory effect of ATP on I _Ca, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited I _Ca via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of I _Ca by activation of a pertussis-toxin-sensitive G-protein linked to P_2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis. Received: 20 February 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Kiss-and-run exocytosis and fusion pores of secretory vesicles in human β-cells

Exocytosis of secretory vesicles results in the release of insulin from pancreatic β-cells, although little is known about this process in humans. We examined the exocytosis of single secretory vesicles and their associated fusion pores in human β-cells by cell-attached capacitance and conductance measurement. Unitary capacitance steps were observed, consistent with the exocytosis of single secretory vesicles. These were often coincident with increases in patch conductance representing the presence of a stable fusion pore. In some events, the fusion pore closed, mediating kiss-and-run, which contributed 20% of the exocytotic events. The cAMP-raising agent forskolin (5 μM) doubled the relative contribution of kiss-and-run. This effect was confirmed visually in MIN6 cells expressing a fluorescent granule probe. Thus, we demonstrate the unitary capacitance steps and fusion pores during single vesicle exocytosis in human β-cells. Furthermore, these secretory vesicles can undergo rapid recycling by kiss-and-run, and this process is up-regulated by cAMP.     

Distribution of ferritin in the rat hippocampus after kainate-induced neuronal injury

A gradual increase in iron occurs in the lesioned hippocampus after neuronal injury induced by the excitotoxin kainate, and the present study was carried out to investigate whether this increase in iron might be associated with changes in expression of the iron binding protein, ferritin. An increase in ferritin immunoreactivity was observed in glial cells of the hippocampus, as early as three days after intracerebroventricular injections of kainate. The number of ferritin positive cells peaked four weeks after the kainate injection, and decreased eight and twelve weeks after injection. They were found to be mostly microglia and oligodendrocytes by double immunofluorescence labeling with glial markers. A number of ferritin-labeled endothelial cells were also observed via electron microscopy. The decline in ferritin immunoreactivity four weeks after the injection of kainate is accompanied by an increase in the number of ferric and ferrous iron positive cells in the lesioned tissue. A substantial non-overlap between ferritin and iron-containing cells was observed. In particular, spherical ferric or ferrous iron-laden cells in the degenerating hippocampus were unlabeled for ferritin for long time periods after the kainate injection. An increase in iron, together with a reduced expression of iron binding proteins such as ferritin at long time intervals after kainate lesions, could result in a relative decrease in ferritin-induced ferroxidase activity and the presence of some of the iron in the ferrous form. It is postulated that this may contribute to chronic neuronal injury, following acute kainate-induced neurodegeneration.     

An immunocytochemical study of calpain II in the hippocampus of rats injected with kainate

The distributions of the kainate/dl-alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (KA/AMPA) receptors GluR1 and calcium-activated neutral protease II (calpain II) in the hippocampus of normal and kainate-lesioned rats were studied by immunocytochemistry. There was a reduction in GluR1 immunoreactivity and a slight increase in calpain II immunoreactivity on the dendrites of pyramidal neurons in CA fields affected by the kainate at 18 h postinjection. Calpain II immunoreactivity was associated with amyloid fibrils at electron microscopy. These fibrils were most often intracellular, in membrane-bound profiles, some of which were contacted by axon terminals and were identified as degenerating dendrites. There was extensive destruction of mitochondrial membranes in degenerating profiles, and accumulations of amyloid fibrils were often localised in mitochondria in a calpain-positive profile. This was unlike other, calpain-negative degenerating profiles, that contained tubulovesicular profiles or multilamellar bodies, where mitochondrial membranes were preserved. Many more calpain-positive profiles were observed at electron microscopy 6 days after kainate injection. The enzyme was present in macrophages and astrocytes in lesioned areas.     

Mitochondrial superoxide production in kainate-induced hippocampal damage

The objective of this study was to determine the role of mitochondrial superoxide radical-mediated oxidative damage in seizure-induced neuronal death. Using aconitase inactivation as an index of superoxide production, we found that systemic administration of kainate in rats increased mitochondrial superoxide production in the hippocampus at times preceding neuronal death. 8-Hydroxy-2-deoxyguanosine, an oxidative lesion of DNA, was also increased in the rat hippocampus following kainate administration. Manganese(III) tetrakis(4-benzoic acid)porphyrin, a catalytic antioxidant, inhibited kainate-induced mitochondrial superoxide production, 8-hydroxy-2-deoxyguanosine formation and neuronal loss in the rat hippocampus. Kainate-induced increases of mitochondrial superoxide production and hippocampal neuronal loss were attenuated in transgenic mice overexpressing mitochondrial superoxide dismutase-2.We propose that these results demonstrate a role for mitochondrial superoxide production in hippocampal pathology produced by kainate seizures.     

Diversity and ecological remarks of ectocommensals and ectoparasites (Annelida, Crustacea, Mollusca) of echinoids (Echinoidea: Mellitidae) in the Sea of Cortez, Mexico

The diversity of symbiotic invertebrates on intertidal and shallow water echinoids of the northwestern Gulf of California was studied. Five ectosymbionts were found: three pinnotherid crabs, Dissodactylus lockingtoni Glassell (1935), Dissodactylus nitidus Smith (1870), and Dissodactylus xanthusi Glassell (1936); one eulimid gastropod, Turveria encopendema Berry (1956); and one polychaete, Struwela sp. We discovered seasonal fluctuation in D. nitidus Smith (1870) and D. xanthusi Glassell (1936), which result in these species being undetectable during the winter season when the temperature in the head of the Gulf of California drops to 14–18°C; the rest of the species are permanent residents in this area. An updated list of all external macrosymbionts of echinoids of the Mexican Pacific is presented.     

Cell-attached measurements of attofarad capacitance steps in rat melanotrophs

 Capacitance changes in cell-attached patches of rat melanotrophs were measured by a high-frequency lock-in amplifier. The background noise of around 30 aF allowed the detection of discrete steps due to fission (endocytosis) and fusion (exocytosis) of vesicles with diameters as small as 60 nm. The amplitude of both types of steps was similar with a modal value of around 300 aF. The frequency of these steps was not changed, if secretagougues such as ionomycin and/or dibutyril cAMP, were applied to the bathing solution. Moreover, this treatment did not result in an increased appearance of expected 2000 to 3000 aF steps due to exocytosis of secretory granules. We conclude that the likely explanation for recorded capacitance steps is that they represent the constitutive vesicle traffic. From the typical frequency and amplitude of these events (around 1 min^–1, 300 aF) in a membrane patch (26 fF) it is estimated that the whole membrane of a rat melanotroph may be ingested under our conditions in 1 to 2 h. Received: 10 February 1997 / Accepted: 3 April 1997