Chemosensing at the carotid body. Involvement of a HERG-like potassium current in glomus cells.

Currently, it is not clear what type of K+ channel(s) is active at the resting membrane potential (RMP) in glomus cells of the carotid body (CB). HERG channels produce currents that are known to contribute to the RMP in other neuronal cells. The goal of the present study was to determine whether CB glomus cells express HERG-like (HL) K+ current, and if so, to determine whether HL currents regulate the RMP. With high [K+]o, depolarizing voltage steps from -85 mV revealed a slowly deactivating inward tail current indicative of HL K+ current in whole-cell, voltage clamped glomus cells. The HL currents were blocked by dofetilide (DOF) in a concentration-dependent manner (IC50 = 13 nM) and high concentrations (1 and 10 mM) of Ba2+. The steady-state activation properties of the HL current (Vh = -45 mV) suggest that it is active at the RMP in glomus cells. Whole-cell, current clamped glomus cells exhibited a RMP of -48 mV. 150 nM DOF caused a significant (14 mV) depolarizing shift in the RMP. In isolated glomus cells, [Ca2+]i increased in response to DOF (1 microM). In an in-vitro CB preparation, DOF increased basal sensory discharge in a concentration-dependent manner and significantly attenuated the sensory response to hypoxia. These results suggest that the HERG-like current is responsible for controlling the RMP in glomus cells of the rabbit CB, and that it is involved in the chemosensory response to hypoxia of the CB.

HERG-Like potassium current regulates the resting membrane potential in glomus cells of the rabbit carotid body.

Direct evidence for a specific K(+) channel underlying the resting membrane potential in glomus cells of the carotid body has been absent. The product of the human ether-a-go-go-related gene (HERG) produces inward rectifier currents that are known to contribute to the resting membrane potential in other neuronal cells. The goal of the present study was to determine whether carotid body glomus cells express HERG-like K(+) current, and if so, to determine whether a HERG-like current regulates the resting membrane potential. Freshly dissociated rabbit glomus cells under whole cell voltage clamp exhibited slowly decaying outward currents that activated 20-30 mV positive to the resting membrane potential. Raising extracellular K(+) revealed a slowly deactivating inward tail current indicative of HERG-like K(+) current. HERG-like currents were not found in cells resembling type II cells. The HERG-like current was blocked by dofetilide (DOF) in a concentration-dependent manner (IC(50) = 13 +/- 4 nM, mean +/- SE) and high concentrations of Ba(2+) (1 and 10 mM). The biophysical and pharmacological characteristics of this inward tail current suggest that it is conducted by a HERG-like channel. The steady-state activation properties of the HERG-like current (V(h) = -44 +/- 2 mV) suggest that it is active at the resting membrane potential in glomus cells. In whole cell, current-clamped glomus cells (average resting membrane potential, - 48 +/- 4 mV), DOF, but not tetraethylammonium, caused a significant (13 mV) depolarizing shift in the resting membrane potential. Using fluorescence imaging, DOF increased [Ca(2+)](i) in isolated glomus cells. In an in-vitro carotid body preparation, DOF increased basal sensory discharge in the carotid sinus nerve in a concentration-dependent manner. These results demonstrate that glomus cells express a HERG-like current that is active at, and responsible for controlling the resting membrane potential.

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.

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.

Reduction of intracellular pH is not the mechanism for the synergistic interaction between photodynamic therapy and nigericin.

Previous studies showed that photodynamic therapy (PDT) sensitized by aluminum phthalocyanine can be dramatically potentiated by the K+/H+ ionophore nigericin. Nigericin equilibrates intracellular pH (pHi) and extracellular pH (pHe) and is most effective in potentiating PDT damage when cells are in an acidic environment (pH 6.5-6.7). We therefore hypothesized that the ability of nigericin to lower pHi is causally related to its ability to potentiate PDT. To test this, the pHi of A549 cells was reduced using pHe-adjusted growth medium, with or without addition of amiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibitors of the membrane-based exchangers responsible for regulating pHi. Using fluorescence ratio imaging, we found that pHi can be equilibrated to within +/- 0.05 pH unit, in the pH range of 6.0-6.8, for up to 1 h after pHe adjustment. Cells equilibrated to various pHi were subjected to PDT at various light fluences, then plated for clonogenic survival immediately after PDT treatment. There is no significant effect of lowering pHi, to values as low as 6.23, on the toxicity of PDT, regardless of whether pHi is lowered by adjustment of the medium alone or by addition of exchange inhibitors. However, cells equilibrated to pHi 6.0 are more sensitive to PDT, with survival reduced by 1 log at 20 kJ/m2 and 1.5 log at 30 kJ/m2, relative to cells treated at a pHi of 6.8 (controls). In contrast, 20 microM nigericin in medium at pHe 6.7 reduces pHi to 6.55, but reduces the surviving fraction at 20 kJ/m2 by nearly 3 logs relative to controls. These data conclusively demonstrate that the ability of nigericin to potentiate PDT is not directly related to its ability to lower pHi. Furthermore, they show that the expression of PDT damage is independent of pHi, except at the very low value of 6.0. Photodynamic therapy does not induce apoptosis in A549 cells, at surviving fractions of 0.1 to 0.01, under any of the treatment conditions used in this study.

Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies.

A method is described to introduce by electroporation membrane-impermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based dyes (e.g., fluorescein-labeled dextran), large macromolecules (e.g., antibodies), and metabolic precursors (e.g., 32P-ATP) with high efficiency. Conditions are relatively independent of cell type. Electroporation with three pulses of 300 volts at 540 microF capacitance at 4 degrees C is a good starting point for many cell types. Electrode distance from the adherent cells was critical at 1.0 +/- 0.15 mm. Suitable poration medium includes calcium-magnesium free phosphate buffered saline (PBS), PBS-buffered 0.25-3.0 M sucrose, Hepes-buffered sucrose, or unbuffered sucrose. Potential use in fluorescence imaging and metabolic studies is shown with DNA synthesis, cell replication, cell substratum attachment, 32P-ATP phosphorylation, and insulin-mediated increases in glucose uptake and its suppression by antiphosphotyrosine and antiglucose transporter protein antibodies. The ability to load foreign molecules into large numbers of adherent cells provides a means of studying these cells individually via microscopic approaches, such as fluorescence spectroscopic imaging, as well as with conventional biochemical and physiological techniques.

Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells.

Previous investigators have reported that intracellular pH responds to hypoxia with a heterogenous pattern in individual glomus cells of the carotid body. The aim of the present study was to examine whether hypoxia had similar effects on cytosolic calcium ([Ca2+]i) in glomus cells, and if so, whether a heterogenous response pattern is also seen in other cell types. Experiments were performed on glomus cells from adult rat carotid bodies, rat pheochromocytoma (PC12) and vascular smooth muscle (A7r5) cells. Changes in [Ca2+]i in individual cells were determined by fluorescence imaging using Fura-2. Glomus cells were identified by catecholamine fluorescence. [Ca2+]i in glomus cells increased in response to hypoxia (pO2 = 35 +/- 8 mmHg; 5 min), whereas hypoxia induced decreases in [Ca2+]i were not seen. Increases in [Ca2+]i were observed in 20% of the isolated cells and strings of cells, but clustered glomus cells never responded. The magnitude of the calcium change in responding cells was proportional to the hypoxic stimulus. Under a given hypoxic challenge, there were marked variations in the response pattern between glomus cells. The response pattern characteristic of any given cell was reproducible. At comparable levels of hypoxia, PC12 cells also responded with an increase in [Ca2+]i with a heterogenous response pattern similar to that seen in glomus cells. In contrast, increases in [Ca2+]i in A7r5 cells could be seen only with sustained hypoxia (approximately 20 min), and little heterogeneity in the response patterns was evident. These results demonstrate that: (a) hypoxia increases cytosolic calcium in glomus cells; (b) response patterns were heterogeneous in individual cells; and (c) the pattern of the hypoxia-induced changes in [Ca2+]i is cell specific. These results suggest that hypoxia-induced increases in [Ca2+]i are faster in secretory than in non-secretory cells.

Heterogeneity of the changes in cytoplasmic pH upon serum stimulation of quiescent fibroblasts.

Addition of mitogens to quiescent cells results in rapid ionic changes in the cytoplasm, including pH. We studied the changes in cytoplasmic pH in single Swiss 3T3 cells upon serum stimulation using fluorescence ratio imaging microscopy. Quiescence was attained using two approaches, serum deprivation of subconfluent cells and confluence. All measurements were made in the presence of bicarbonate and the absence of other organic buffers. We also used BCECF coupled to dextran to avoid several artifacts associated with using BCECF-AM, including leakage and phototoxicity. Analysis of the changes in cytoplasmic pH demonstrated a dramatic heterogeneity in the responses of single cells. There were six basic classes of responses, 1) a fast alkalinization, reaching a maximum pH in approximately 2-5 min; 2) a slow alkalinization, reaching a maximum pH in 10-20 min; 3) a very slow alkalinization, not reaching a plateau pH within the measurement time; 4) no apparent change in pH during the measurement time; 5) an early transient acidification, followed by either a fast or slow alkalinization; and 6) an acidification, followed by alkalinization and then by a decrease to some intermediate pH. Subconfluent cells exhibited greater heterogeneity in response than confluent cells, with no single dominant class of response. The dominant (55%) response for confluent cells was a gradual alkalinization of approximately 0.01 pH units/min. A larger proportion (52%) of subconfluent cells exhibited an early transient acidification compared to confluent cells (7%). A significant proportion of both types of cells (23% subconfluent, 36% confluent) exhibited no change in cytoplasmic pH upon stimulation. In general, the kinetics of changes in cytoplasmic pH were significantly different from the published results with population averaging methods.

Evaluation of algorithms for ratio imaging in fluorescence microscopy.

Ratio imaging in fluorescence microscopy is used in measuring parameters such as pH, pCa, cytoplasmic porosity, and the relative concentration of fluorescent analogs within single cells. The fastest method for ratio imaging is to use lookup tables on special-purpose image processors. Since lookup tables store integers in integer addresses, using a lookup table will generate rounding errors. The magnitude of the error will depend on the transformation performed and on the number of levels used in the lookup table. We examined ratio imaging by lookup table and computed the errors generated by both inversion and log subtraction methods. Both uniformly fluorescing fields and fluorescing cell images were employed to provide data for use in confirming our calculations and illustrating both the magnitude and spatial incidence of errors. It is shown that, through proper design of lookup tables, a significant reduction can be made in the errors generated in comparison with common methods available in most image processors.

Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells.

Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.

Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH.

Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.

Preparation and reactions of an iodinated imidoester reagent with actin and alpha-actinin.

The chemical iodination of an imidoester (methyl-p-hydroxybenzimidate, Wood et al. (1975) Anal. Biochem. 68, 339) and subsequent coupling of iodinated imidoester (IIE) to protein is an indirect method of iodinating proteins that is specific for the epsilon amino group of lysine residues and maintains the positive charge on the amino group at physiological pH. Purification of the IIE from chloramine-T and free iodine by benzene extraction eliminates the need for isoelectric precipitation and produces a more time- and cost-efficient IIE preparation and purification protocol. The separation of free from protein-bound label by chromatography, using centrifugal elution, provides a separation method that is rapid and efficient, without the generation of large volumes of radioactive wastes characteristic of conventional chromatographic and dialysis methods. To optimize the parameters of labeling protein with IIE, a systematic assessment of the effects of pH, reactant concentrations, and reaction time was made using purified cardiac actin and gizzard alpha-actinin. The parameters were defined to achieve an average labeling ratio of one IIE per protein polypeptide. The data demonstrate that both proteins appear to be labeled at the same rate and define several determining factors that limit the rate and extent of IIE incorporation into protein.

Immunofluorescence comparisons of anti-actin specificity.

The abilities of antibody populations against brain actin and two immunogenic forms of cardiac actin to react with sarcomeric muscle actin and cytoplasmic non-muscle actin were tested by indirect immunofluorescence, by using isolated skeletal muscle myofibrils and cultured non-neuronal dorsal root ganglion cells as the test systems. All three antibody preparations stained the I-bands of myofibrils, a result that demonstrated the presence of antigenic determinants shared among skeletal, cardiac, and brain actins. However, although antibodies against cytoplasmic brain actin stained the stress fibers of cultured cells, those against glutaraldehyde cross-linked cardiac actin did not, a result that implies that cardiac actin possesses determinants common to sarcomeric actins but not present on cytoplasmic actin. Finally, antibodies against SDS-treated cardiac actin readily stained the stress fibers of cultured cells, in contrast to those against glutaraldehyde cross-linked cardiac actin, a result that suggests that the state of the original immunogen can affect the actin type specificity of the resulting antibody population.