Isolation and receptor profiling of ileal enterochromaffin cells.

AIM AND METHODS: Enterochromaffin (EC) cells, interspersed throughout the gastrointestinal mucosa, provide most of the serotonin of the body and control intestinal motility, secretion and absorption. We purified EC cells from the rat ileum by a combination of elutriation and density gradient centrifugation in order to characterize the function of this important cell type. RESULTS: Immunostaining showed that there were 84% serotonin-positive cells in the highly enriched EC fraction as compared with 12% in unfractionated cells, yielding a approximately sevenfold enrichment. Serotonin measurements in the cell suspensions indicated a seven to 14-fold enrichment. Presence of alpha- and beta-adrenoreceptor isoforms, muscarinic M3 and gama-aminobutyric acid (GABA)-A receptors was confirmed by RT-PCR and cytochemistry. Increased expression of VMAT-1 and GABA-A mRNA was also shown by quantitative TaqMan PCR using EC cell RNA. Serotonin release in isolated EC cells was stimulated by noradrenaline, and to a smaller extent, by carbachol, while GABA addition was without effect. CONCLUSION: Our data provide a basis for a new approach to characterize receptors on this unique cell type.

Stationary-state oxidized platinum microsensor for selective and on-line monitoring of nitric oxide in biological preparations.

Despite the multifaceted biomedical significance of NO, little progress has been achieved so far in the quantitative understanding of the signal transduction mechanisms where NO is involved. To help progress in this area, we propose a simple electrochemical NO sensor here, consisting of a glass sealed platinum microdisk electrode coated with cellulose acetate to reduce both surface fouling by proteins and response to potential interferences. A differential amperometry protocol is optimized to improve selectivity and provide a stationary oxidation state of the platinum surface, which prevents loss in sensitivity during long-term use. We found the oxidation of NO by O2 second order in [NO] with a rate constant of (8.0 +/- 0.4) x 10(6) M(-2) s(-1), in good agreement with literature data obtained by other than electrochemical methods. The release rates of NO detected in cultures of activated macrophages were on the order of 20 pmol/ (10(6)cells s) and correlated well with the nitrite content determined by the spectrophotometric Griess assay.

Optical detection in microscopic domains. 2. Inner filter effects for monitoring nonfluorescent molecules with fluorescence.

In this research, we test whether optical detection techniques show different characteristics in microscopic solution volumes (nano-, pico-, and femtoliter range) compared to the usual macroscopic samples. In part 1 (Lu, H.; et al. Anal Chem. 2000, 72, 1569-1576.) absorption spectra of high quality were obtained, quantitatively obeying both Beer-Lambert's law and the law of superposition, despite the micrometer optical path lengths and the curvatures of the droplets studied. Addition and subtraction of absorbing molecules with diffusional microburets (DMBs), as well as more complex operations (simultaneous addition of one and subtraction of another molecule, and a consuming scheme), have been monitored with good spectral and temporal resolution. Despite the unexpectedly good performance of absorption microspectrometry, fluorescence-based detection schemes are considered more sensitive for microscopic studies (e.g., cell physiology). In this paper, we test whether fluorescence-based schemes can be used to indirectly measure nonfluorescent chemicals in microscopic domains. Absorption by such molecules will cause a corresponding decrease in overall fluorescence intensity of the added standard fluorescent dye. This phenomenon, the inner filter effect (IFE), was tested using Lucifer Yellow CH (LY) as the fluorescent standard dye. Its effective irradiation was absorbed by Orange G (primary IFE) or its emission by Bromophenol Blue (secondary IFE). By utilizing these phenomena, (1) we measured the concentration of absorbing molecules in microscopic samples by adding a standard amount of LY by a DMB, and (2) we monitored DMB delivery of nonfluorescent reagents into droplets preloaded with LY. The results prove that IFEs are sensitive indirect means of detection of absorbing molecules in microscopic domains. The techniques presented are expected to find applications in cellular studies where absorption spectrometry is usually not considered.

Expression of vesicular monoamine transporters, synaptosomal-associated protein 25 and syntaxin1: a signature of human small cell lung carcinoma.

Vesicular monoamine transporters (VMATs) are a prerequisite for the uptake of biogenic amines into intracellular storage organelles, whereas soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs; such as SNAP-25 and syntaxin1) are essential for exocytosis of biogenic amines by neurons and endocrine cells. In this study, we examined whether these proteins exist in high-grade malignant small cell lung carcinomas (SCLCs), large cell carcinomas, adenocarcinomas, and squamous cell carcinomas of the lung. We analyzed two established human SCLC cell lines, one adenocarcinoma cell line, paraffin-embedded tumors (SCLC, n = 25; large cell carcinoma, n = 10; adenocarcinoma, n = 10; squamous cell carcinoma, n = 10), and snap-frozen SCLC samples (n = 2). Using immunocytochemistry, Western blotting, Northern blotting, RT-PCR, and sequencing, we identified VMAT1, VMAT2, SNAP-25, and syntaxin1 in cultured SCLC cells. Immunohistochemistry carried out on paraffin sections revealed that all SCLC tumors express VMAT1, VMAT2, SNAP-25, and syntaxin1. The presence of SNAP-25 and syntaxin1 in SCLC was confirmed by RT-PCR performed with material extracted from paraffin sections. Western blot analysis and RT-PCR carried out with snap-frozen SCLC tumors revealed the presence of SNAREs and VMATs. Immunohistochemistry showed that non-SCLC tumors were negative for SNAREs and VMATs, with the exception of immunostaining for SNAP-25 and syntaxin1 in 3 of 10 adenocarcinomas. Our findings indicate that SCLC cells are endowed with transporters necessary for intracellular storage of biogenic amines and with proteins required for exocytosis of secretory products. These proteins may be used as markers of differentiation of human lung tumors. Moreover, the presence of VMATs provides the basis for a diagnostic application of biogenic amine-derived tracers in positron emission tomography of SCLC tumors.

Synaptosome-associated protein of 25 kilodaltons in oocytes and steroid-producing cells of rat and human ovary: molecular analysis and regulation by gonadotropins.

The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially involved in exocytosis in neurons. The aim of this study was to investigate whether it is present in the ovary. We found SNAP-25 to be expressed in nonneuronal cells of the rat and human ovary, namely in all oocytes and in steroidogenic cells, including granulosa cells (GC) of large antral follicles and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary. Oocytes obtained by laser capture microdissection were shown to express SNAP-25b, whereas SNAP-25a was found in rat GC and human luteinized GC. Immunohistochemical observations of strong SNAP-25 staining in GC of large growing antral follicles compared with absent or weak staining in small follicles suggested a role in folliculogenesis. To study a presumed regulation of SNAP-25, we used a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing human GC, which express LH receptors. FSH elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in GFSHR-17 cells. The cAMP analogue dibutyryl-cAMP (db-cAMP) mimicked this action of FSH. The effects of both db-cAMP and FSH were inhibited by the protein kinase A (PKA) inhibitor H89. In contrast, SNAP-25 protein and mRNA-levels were not altered by LH/hCG in luteinized human GC. Our results for the first time identify SNAP-25b in oocytes and SNAP-25a in steroidogenic cells of the mammalian ovary. SNAP-25a and b may be involved in different exocytotic processes in these cell types.

Identification of an ovarian voltage-activated Na+-channel type: hints to involvement in luteolysis.

An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24-48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24-48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.

Optical detection in microscopic domains. 1. Monitoring chemical manipulations with absorption microspectrometry.

In this research, we test whether optical detection techniques, such as absorption microspectrometry, fluorescence detection, and inner filter effects, show different characteristics in microscopic domains (nano-, pico- and femtoliter range) with respect to usual solution volumes. In this part 1, characterization of absorption microspectrometry is facilitated by the use of a novel microscopic tool, the diffusional microburet (DMB), suitable for fine chemical manipulations of microscopic liquid samples. Since diffusional delivery of substances will not induce appreciable changes in volume and shape of the sample, a DMB makes it possible to obtain spectral recordings and a reference spectrum from the same microscopic droplet. Thus, good quantitative spectra of microscopic domains can be assessed. With this approach, despite the curvature of the sample boundaries, the large surface-to-volume ratios, and microscopic optical path lengths, both Beer-Lambert's law and the law of superposition were found to be directly applicable in microscopic domains without any corrections for absorption at sample boundaries. Use of a combination of microspectrometry and chemical manipulations by DMBs made it possible for the first time to record in real time, in truly microscopic domains, spectral evolution upon addition or subtraction of chemicals, as well as monitor the progress of chemical reactions. This approach is expected to contribute to the optical exploration of microchemistry and microanalysis.

Fine chemical manipulations of microscopic liquid samples. 2. Consuming and nonconsuming schemes.

Microscopic liquid particles can be manipulated chemically using a suitable diffusional microburet (DMB), whose tiny tip plugged with a diffusion membrane acts as a well-defined diffusional transfer channel. In part 1 of this work (Gratzl et al. Anal. Chem. 1999, 71, 2751-2756), we discussed the simplest DMB-based operation: addition, i.e., loading a droplet with a chemical that accumulates there without any chemical reaction occurring. Since in this process no consumption of the delivered molecules in the target droplet takes place, addition is a nonconsuming scheme. In this work, another type of nonconsuming scheme is explored, which is the subtraction of a substance from droplets via a DMB. This process has no analogy among macroscopic chemical operations. Both addition and subtraction occur according to an exponential asymptotic process when diffusion is at quasisteady state inside the DMB tip. These nonconsuming operations were characterized using the transport of microscopic quantities of Lucifer Yellow CH, a fluorescent dye, under a fluorescent microscope. The third basic type of chemical manipulation is when the substance delivered by a DMB is consumed in the target droplet instantaneously by a fast chemical reaction. This consuming scheme was studied by delivering EDTA into droplets containing Pb2+ ions and a color indicator. These microscopic titrations were monitored using gray scale transmittance images of the droplets as recorded versus time. A unified theory of the three basic DMB operations is also presented.

The mechanism of histamine secretion from gastric enterochromaffin-like cells.

Enterochromaffin-like (ECL) cells play a pivotal role in the peripheral regulation of gastric acid secretion as they respond to the functionally important gastrointestinal hormones gastrin and somatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the key stimulus of histamine release from ECL cells in vivo and in vitro. Voltage-gated K(+) and Ca(2+) channels have been detected on isolated ECL cells. Exocytosis of histamine following gastrin stimulation and Ca(2+) entry across the plasma membrane is catalyzed by synaptobrevin and synaptosomal-associated protein of 25 kDa, both characterized as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein. Histamine release occurs from different cellular pools: preexisting vacuolar histamine immediately released by Ca(2+) entry or newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized by cytoplasmic HDC and accumulated in secretory vesicles by proton-histamine countertransport via the vesicular monoamine transporter subtype 2 (VMAT-2). The promoter region of HDC contains Ca(2+)-, cAMP-, and protein kinase C-responsive elements. The gene promoter for VMAT-2, however, lacks TATA boxes but contains regulatory elements for the hormones glucagon and somatostatin. Histamine secretion from ECL cells is thereby under a complex regulation of hormonal signals and can be targeted at several steps during the process of exocytosis.

Fine chemical manipulations of microscopic liquid samples. 1. Droplet loading with chemicals.

Physically and chemically stable microscopic aqueous droplets of nano-, pico-, and femtoliter volumes were made and kept under heptane. Such droplets can contain chemicals of interest in pico-, femto-, and attomole amounts or less. Their fine chemical manipulation was achieved by a diffusional microburet (DMB), which consists of a pulled glass capillary whose microscopic tip is filled with a tiny diffusion membrane of agar or polyacrylamide gel. Once this tip is moved into a target droplet by a fine micromanipulator under a microscope, diffusional reagent delivery from the DMB body begins, driven by the concentration gradient within the tip. This system was tested in this work by delivering an inert fluorescent dye, Lucifer Yellow CH, into buffer droplets while fluorescence intensity in each droplet was recorded. An exponential decay of delivery rate was observed corresponding to an exponential saturation process for the accumulation of the delivered chemical inside the target droplet.

Adjusting the distance of electrochemical microsensors from secreting cell monolayers on the micrometer scale using impedance.

There are emerging applications of electrochemical microsensors where the distance of the sensor from an insulating plane needs to be adjusted and/or accurately known. The plane may be merely an obstruction or the source of a species whose release rate needs to be measured. An example is in cell secretion studies where a monolayer of cells is stimulated to secrete ions and/or other biochemical species which then diffuse away from the cells while being measured by a microsensor. Sensor response will thus depend on both the rate of release and the distance of the sensor from the cells. To obtain accurate release rates, the precision of the scheme to control electrode distance from the monolayer needs to be on the micrometer scale for species with ionic diffusivities. Optical (stereomicroscope and microruler) and mechanical (precalibrated micrometer screw) methods to precisely position the electrode are difficult to use under realistic circumstances (due to opaqueness of the chamber, and/or the medium, or irreproducible chamber depth). In this work we propose to correlate electrochemical cell impedance with sensor distance. This scheme has been used to adjust the distance of a chloride (tip diameter approximately 250 microns) and a potassium (tip diameter approximately 1000 microns) ion-selective microelectrode in the 0-250- and 0-2500-micron range, respectively, from a planar obstruction as well as from a monolayer of cells with a best precision of +/- 5 microns (n = 6) for the chloride and about +/- 20 microns for the potassium sensor. Larger electrodes have a broader range of distances over which they are sensitive, albeit with a poorer spatial resolution. This was verified by using Ag disk electrodes of 250 and 500 microns in diameters in AgNO3 solution.

Monitoring drug efflux from sensitive and multidrug-resistant single cancer cells with microvoltammetry.

Multidrug resistance (MDR) is the eventual cross-resistance of certain cancer cells to a series of chemically unrelated drugs. It is attributed to a number of possible biophysical processes, one of them being increased drug efflux from resistant cells which leads to a decreased intracellular drug accumulation and retention. In this work, a carbon fiber microdisk electrode was used to monitor directly doxorubicin efflux from single preloaded cancer cells. Electrochemical cleaning, adsorptive preconcentration, and an electrocatalytic effect due to ambient oxygen made it possible to detect eventually very low drug concentrations (down to 1 nM) at good temporal resolution (down to 30 s/measurement) very close (< or = 1 micron) to single cancer cells for the first time. The results from a sensitive (AUXB1) and a drug-resistant (CHRC5) version of Chinese hamster ovarian cancer cells show that resistant cells exhibit a much higher initial efflux rate and shorter efflux time constant when both cell lines are preloaded up to the same intracellular drug concentration. These observations are consistent with results obtained from populations of the same cells by conventional techniques, proving that microvoltammetry can be used to monitor doxorubicin efflux at the single-cell level. Compared with existing methodologies, however, whose data represent only average cell behavior at typically low temporal resolution, the technique described here can provide information on the microheterogeneity of cancer cell populations in terms of drug efflux at high temporal resolution. The actual driving force of efflux is obtained since concentrations are measured directly at individual cells. This approach may lead to important new information on the mechanisms and prospective treatments of MDR.

SNAP-25 requirement for dendritic growth of hippocampal neurons.

Structure and dimension of the dendritic arbor are important determinants of information processing by the nerve cell, but mechanisms and molecules involved in dendritic growth are essentially unknown. We investigated early mechanisms of dendritic growth using mouse fetal hippocampal neurons in primary culture, which form processes during the first week in vitro. We detected a key component of regulated exocytosis, SNAP-25 (synaptosomal associated protein of 25 kDa), in axons and axonal terminals as well as in dendrites identified by the occurrence of the dendritic markers transferrin receptor and MAP2. Selective inactivation of SNAP-25 by botulinum neurotoxin A (BoNTA) resulted in inhibition of axonal growth and of vesicle recycling in axonal terminals. In addition, dendritic growth of hippocampal pyramidal and granule neurons was significantly inhibited by BoNTA. In contrast, cleavage of synaptobrevin by tetanus toxin had an effect on neither axonal nor dendritic growth. Our observations indicate that SNAP-25, but not synaptobrevin, is involved in constitutive axonal growth and dendrite formation by hippocampal neurons.

Single cell model for simultaneous drug delivery and efflux.

Multidrug resistance (MDR) of some cancer cells is a major challenge for chemotherapy of systemic cancers to overcome. To experimentally uncover the cellular mechanisms leading to MDR, it is necessary to quantitatively assess both drug influx into, and efflux from, the cells exposed to drug treatment. By using a novel molecular microdelivery system to enforce continuous and adjustable drug influx into single cells by controlled diffusion through a gel plug in a micropipet tip, drug resistance studies can now be performed on the single cell level. Our dynamic model of this scheme incorporates drug delivery, diffusive mixing, and accumulation inside the cytoplasm, and efflux by both passive and active membrane transport. Model simulations using available experimental information on these processes can assist in the design of MDR related experiments on single cancer cells which are expected to lead to a quantitative evaluation of mechanisms. Simulations indicate that drug resistance of a cancer cell can be quantified better by its dynamic response than by steady-state analysis.

Characterization of vesicular membrane-bound alpha-SNAP and NSF in adrenal chromaffin cells.

alpha-SNAP and NSF are thought to act as soluble factors, which transiently bind to a complex formed between syntaxin and SNAP-25 located at the plasma membrane and synaptobrevin at the secretory vesicle membrane, at the moment of exocytosis. Here we present data which permit the novel conclusion that alpha-SNAP and NSF are not soluble in adrenal chromaffin cells but are rather membrane-bound in particular to undocked chromaffin vesicles. Evidence for this new paradigm is derived from several experimental approaches. First, alpha-SNAP and NSF were found predominantly at cellular membranes and not in the cytosol of cracked chromaffin cells. Second, alpha-SNAP and NSF were not released from membranes by Mg2+ATP, which causes priming of vesicles. Third, immune electron microscopy and immunoblotting of chromaffin vesicles purified by immunoisolation or density gradient centrifugation revealed the presence of alpha-SNAP and NSF together with typical vesicular proteins such as synaptobrevin and synaptotagmin. In the sucrose gradient 30% alpha-SNAP and 27% NSF were recovered with chromaffin vesicles. Bound alpha-SNAP was quantified (14 molecules/vesicle), and binding was characterized with recombinant his6-tagged alpha-SNAP. Overlay blots revealed that alpha-SNAP is bound to vesicular SNAP-25 and endogenous NSF. Our data show that mature chromaffin vesicles already contain specifically bound alpha-SNAP and NSF before docking at the plasmalemma.

Continuous in situ electrochemical monitoring of doxorubicin efflux from sensitive and drug-resistant cancer cells.

One of the least well understood problems in cancer chemotherapy is the cross-resistance of certain tumor cells to a series of chemically unrelated drugs. Multidrug resistance (MDR) can be attributed to several different biophysical processes, among them increased drug efflux. This has been found to correlate with overexpression of the cell surface 170-kDa P-glycoprotein that actively excludes cytotoxic drugs against their concentration gradient. To better understand MDR, experimental methods are needed to study drug efflux from cancer cells. Continuous measurement of efflux of nonfluorescent drugs on the same cell culture in situ, or assessing efflux from a few cells or even a single cell, is beyond the capabilities of existing technologies. In this work, a carbon fiber (CF) microelectrode is used to monitor efflux of doxorubicin from a monolayer of two cell lines: an auxotrophic mutant of Chinese hamster ovary cells, AUXB1, and its MDR subline, CHRC5. Because doxorubicin is both fluorescent and electroactive, the results could be validated against existing data obtained optically and with other techniques on the same cell lines, with good agreement found. The electrochemical detection, however, is capable of in situ monitoring with high temporal resolution and is suitable for single-cell studies.

Release of dopamine and norepinephrine by hypoxia from PC-12 cells.

We examined the effects of hypoxia on the release of dopamine (DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cells and assessed the involvement of Ca2+ and protein kinases in stimulus-secretion coupling. Catecholamine release was monitored by microvoltammetry using a carbon fiber electrode as well as by HPLC coupled with electrochemical detection (ECD). Microvoltammetric analysis showed that hypoxia-induced catecholamine secretion (PO2 of medium approximately 40 mmHg) occurred within 1 min after the onset of the stimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysis revealed that, at any level of PO2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was approximately 11-fold greater than NE release. The magnitude of hypoxia-induced NE and DA releases depended on the passage, source, and culture conditions of the PC-12 cells. Omission of extracellular Ca2+ or addition of voltage-gated Ca2+ channel blockers attenuated hypoxia-induced release of both DA and NE to a similar extent. Protein kinase inhibitors, staurosporine (200 nM) and bisindolylmaleimide I (2 microM), on the other hand, attenuated hypoxia-induced NE release more than DA release. However, protein kinase inhibitors had no significant effect on K+-induced NE and DA releases. These results demonstrate that hypoxia releases catecholamines from PC-12 cells and that, for a given change in PO2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.

Electrochemistry in microscopic domains. 1. The electrochemical cell and its voltammetric and amperometric response.

Microscopic aqueous sample droplets of nano- and picoliter volumes were formed on the bottom of a polystyrene dish under water-saturated heptane. The electrochemical cell consisted of a beveled carbon fiber microdisk working electrode and a reference electrode with a miniature junction, both inserted into the studied droplet. Both electrodes had a nominal tip diameter of 7.5 microns. Cyclic voltammetry and chronoamperometry in droplets of 3.3 mM ruthenium hexaammine trichloride in 0.1 M KCl solution were performed with this system. The experiments revealed for the first time major deviations in both voltammetric and amperometric microelectrode behavior in picoliter domains as compared to nanoliter volume and bulk solution. The concept and criteria of electrochemical microscopicity of volume is discussed. This work also provides a simple and robust experimental model system to verify electrochemical experiments in restricted domains such as in or near single biological cells or in microscopic tissue cavities. The methodology developed has, however, more general analytical and physicochemical applications as well.