We have shown that a specific cytosolic pH (pHcyt) regulatory mechanism, i.e., vacuolar type H+-ATPases at the plasma membrane (pmV-ATPases), allows angiogenic and metastatic cells to survive in an acidic and hostile environment. However, a functional evaluation of this pump's activity in situ (i.e., in living animal models) has not been attempted. We developed a mouse model of angiogenesis and metastasis based on the dorsal skin fold chamber, and implanted highly metastatic human tumor cells that have been engineered to express green fluorescent protein (GFP). GFP can be used as a pH reporter because its fluorescence is pH sensitive. Our studies in isolated single cells indicated that there are distinct pHcyt gradients in the invadipodia versus the lamellipodia due to the preferential expression of pmV-ATPases at the leading edge. We hypothesize that in vivo, these pH gradients also exist. We employed spectral imaging and real time confocal imaging microscopy, since these approaches are complementary and exhibited unsurpassed temporal and spectral resolution, thus allowing us to study pHcyt in discrete subcellular regions of the cells expressing GFP. We can acquire a full frame (i.e., 512 x 512 pixels) in real time confocal imaging at ca. 25-50 msec, whereas spectral imaging allow us to obtain spectral information from discrete domains of ca. 10 μm in the x-y plane and every 10 μm from leading to lagging edge within a time frame of 5 msec at 0.4 nm spectral resolution. This is possible because we employ frame transfer cooled CCD cameras and spectrographs. Studies are under way to evaluate proton gradients using multiphoton approaches since this will allow us to evaluate pH deeper into the tissue (i.e., 300-600 μm), and should allow us to follow pHcyt and the progression of tumor metastasis.
Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.
Our understanding of intracellular pH homeostatis in eukaryotic systems has been enhanced since the introduction of carboxyfluorescein diacetate as a useful pH probe more than 20 years ago. BCECF, a derivative of this earlier fluoroprobe has dominated the field. In the past 10 years, SNARF-1 has emerged as an alternative pH probe. Recently, a novel derivative of BCECF, BCPCF has been developed. Green Fluorescent Proteins (GFPs) have also been used recently to monitor pH in a non invasive manner in several cell types. Here, we report that human mammary epithelial cells can be transfected with the gene encoding for cyan (CFP), green (GFP), and yellow (YFP), to study cytosolic pH. The novel red fluorescent protein (DsRed) is not sensitive to pH. Multidrug resistance (MDR) has been associated with altered cytosolic pH homeostasis. We show that experimental maneuvers that decrease pHin enhance the efficacy of chemotherapeutic drugs. We also show that short pulses of UV-B light elicited acidosis in cells, as evaluated by ratio ion cell imaging, and confocal/spectral imaging microscopy. During the course of these experiments we noticed that cells exhibit intrinsic fluorochromes that can be used to monitor pH in living cells.
Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.
We have previously shown the relationship between metastatic potential and plasmalemmal V-H+-ATPase (pmV-ATPase) expression in tumor cells. This led us to hypothesize that pmV-ATPase activity is involved in invasion. Angiogenesis involves invasion of adjacent tissues by microvascular endothelial cells, thus we hypothesized that pmV-ATPases contribute to pHin regulation and invasion in microvascular endothelial cells.
Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.
A spectrograph based imaging system attached to a microscope is described for use in monitoring the fluorescence of multiple probes loaded into single cells. Movements of ions, important in regulating cell function, are interrelated under many conditions. Since we are interested in the mechanisms underlying coupling between ion movements and metabolism, we required a microscope based system to analyze these parameters at the level of a single cell. The spectral imaging system was implemented for two purposes. (1) To resolve heterogeneities which exist in the response of individual cells in a population, and within any given cell due to loading of dye into more than one subcellular compartment, and (2) to study the temporal relations between several physiological parameters from a single cell. Since spectral imaging resolves signal from multiple probes simultaneously, the concentrations of several ions or metabolic factors can be followed in a single cell. Moreover, spatial information is acquired along one axis, providing information from probes located in individual compartments. The spectral imaging microscope and some specific applications are described.
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