Gary R. Bright

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.

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 (Po 2 of medium ∼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 Po 2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was ∼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 μM), 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 Po 2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.

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.

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.

Carbon Monoxide and Carotid Body Chemoreception

It is being increasingly recognized that endogenously generated carbon monoxide (CO) functions as a chemical messenger in the nervous system. CO is released during the breakdown of heme to biliverdin by the enzyme heme oxygenase (HO). Two isoforms of HO have been identified: HO-1 is an inducible form and is found predominantly in spleen and liver; HO-2 is constitutive and is widely distributed in brain and nervous tissues (Verma et al, 1993). We have recently reported that HO-2 is present in the carotid bodies where it is found primarily in the glomus cells (Prabhakar et al, 1995). More importantly, we showed that zinc protoporphyrin-9 (ZnPP-9), an inhibitor of HO, increased chemoreceptor activity, suggesting that endogenous CO is inhibitory to carotid body (CB) activity. In the present study we assessed cellular mechanisms by which endogenous CO modulates CB activity. It is generally accepted that Ca2+-dependent neurotransmitter release from glomus cells plays an important role in transduction of a hypoxic stimulus by the CB. Therefore, we tested the idea that endogenous CO exerts its effects on carotid body sensory activity in part by regulating cytosolic calcium ([Ca2+]I) in glomus cells. To test this possibility we monitored [Ca2+]I and ion channel activities in glomus cells in response to ZnPP-9, an inhibitor of CO synthesis.

Multiparameter fluorescence spectroscopic imaging of cell function

The ability to quantitate physiological parameters in single living cells using fluorescence spectroscopic imaging has expanded our understanding of many cell regulatory processes. Previous studies have focussed on the measurement of single parameters, such as the concentration of calcium, and more recently two parameters, such as calcium and pH using fluorescence ratio imaging. The complexity of the interrelationships among cell biochemical reactions suggests a need to extend the measurement scheme to several parameters. Expansion of the number of parameters involves several complexities associated with fluorescent probe selection and instrumentation design as well as the processing and management of the data. A system has been assembled which provides maximum flexibility in multiparameter fluorescence imaging measurements. The system provides multiple combinations of excitation, dichroic mirror, and emission wavelengths. It has automatic acquisition of any number of parameters. The number of parameters is primarily limited by the selection of fluorescent probes with nonoverlapping spectra. We demonstrate the utility of the system by the coordinated monitoring of stimulated changes in the concentrations of calcium, magnesium, and pH using fluorescence ratio imaging coupled with a conventional transmitted light image of single smooth muscle cells. The results demonstrate coordinated changes in some instances but uncoordinated changes in others.