EVALUATION OF HYDROGEN ION CONCENTRATIONS IN PROSTATES FROM RATS AND DOGS USING FLUORESCENT
Barry S. Levine1, Alexander V. Lyubimov1, Seraya N. Carr1, Alan P. Brown1, Jonathan J. Art2, James A. Crowell3
1Toxicology Research Laboratory, University of Illinois at Chicago, Chicago, IL; 2Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, IL;
3Division of Cancer Prevention, National Cancer Institute, Bethesda, MD.
Figure 2. Mean Value of the Ratio of Intensity of SNARF-1
Loaded In Vitro Onto Rat Prostate Tissue
The knowledge of mechanisms of pH regulation and ion exchange in prostate
cancer cells may help in the development of new drugs and/or enhance the Approximately 150 male CD (Virus Antibody Free) rats, ~400-600 g, and 13 2.5 n=6
anticancer potency of current drugs due to selective delivery, increased influx, male Beagle dogs (6-10 kg) 2
and prevention of extrusion from tumor cells. As a first step in this direction, the n=13 g/ r
objective of this study was to develop in vitro and in vivo methods of pH
LOADING OF THE FLUORESCENT INDICATORS (IN VITRO)
1.5 Linear (g/ r)
measurements in prostate tissue and characterize intracellular pH gradients in
Linear (r/ g)
The microdissected (rat) or sectioned (dog) prostate tissue was loaded for 30 1
epithelial cells of normal rat and dog prostate. Freshly dissected prostate tissues minutes with the dual emission fluorescent indicators (Carboxy–SNARF-1,
(in vitro) or the entire prostate gland (in vivo) were loaded with fluorescent dyes SNARF calcein, and SNAFL calcein) dissolved in loading buffer (RPMI-1640
for 30 or 50 min, respectively. After loading, tissues were viewed using a Zeiss Medium without sodium bicarbonate and amino acids). After 30 minutes, the 0
LSM 510 Laser Scanning Confocal Microscope. Confocal images were initially taken tissues were rinsed twice with RPMI-1640 Medium containing 250 mM
6.4 6.6 6.8 7 7.2 7.4 7.6 7.8
in tissues perfused with RPMI-1640 medium. Calibration in situ was performed in pH
sulfinpyrazone and viewed on the LSM 510 confocal microscope with the 25X
the same tissue with high potassium buffers of known pH containing nigericin. objective. For measurements in the lysosomes, the prostate tissues was loaded
Carboxy-SNARF-1 was the most useful fluorescent indicator for determining the with the single excitation and single emission indicators (Lyso Sensor Green
distribution of hydrogen ions in epithelial cells in rat and dog prostates. SNARF-1 DND-153 and DND 189) for 30 and 60 minute incubation periods and were
was visible only in the cytoplasm of the epithelial cells. The fluorescent indicator viewed on the confocal microscope with the 63 X objective. Figure 3. Calibration Curve of the Mean Ratio of Intensity of
Lyso Sensor Green DND-189, which is only fluorescent in internal cellular SNARF-1 Plotted Over pH (Dog Prostate Tissue)
compartments (lysosomes) at pH < 5.2, was visible inside epithelial cells of the IN VIVO LOADING
prostate tissue. Therefore, it was concluded that the pH of lysosomes in prostate In Vivo loading in rats was performed using a 25 G needle to load SNARF-1 3
tissue was < 5.2. Various procedures were established, which included rat dissolved in DMSO which was diluted in 3.5 mL of (0.4%) Trypan blue salt 2.5
prostate microdissection, in vivo and in vitro fluorescent probe infusion, optimal solution (0.81% sodium chloride, 0.06% potassium phosphate, dibasic) and 5 y = -1.5124x + 12.368
concentrations of fluorescent probes, appropriate temperature regimen, and mL of loading buffer to a final concentration of SNARF-1 at 100 mM. The 2 R2 = 0.9914
incubation time of cell cultures with fluorescent probes. In addition, experimental
SNARF-1 mixture was loaded underneath the capsule of the rat prostate. g/r
conditions for the confocal microscope to study fluorescent probes were
Trypan blue was used for visual confirmation of the dye uptake.
optimized. Based upon several experiments with loading carboxy-SNARF-1 either In Vivo loading of the Beagle dog prostate was performed using a final 1
y = 0.4443x - 2.508
in vitro or in vivo for rat and dog prostates, the intracellular pH of the epithelial concentration of 100 mM of SNARF-1 dissolved in DMSO and diluted in 1.8 mL R2 = 0.9733
cells in both species was ~ 7.0. Besides the measurement of the pH in rat and dog
Trypan Blue Salt solution along with 7 mL of loading buffer. One 10 mL syringe
tissues, a method of pH measurement in prostate tissue (rather than in cell was used to load a total volume of 8.8 mL of the dye into the Beagle dog 0
culture) was developed with fluorescent dye infusion both in vitro and in vivo. prostate. A clamp was placed between the base of the bladder and the prostate
6.2 6.4 6.6 6.8 7 7.2 7.4 7.6
This method may now be used for the measurement of hydrogen ion to prevent the dye from being lost through the urethra. SNARF-1 was loaded pH
concentrations and other ion measurements (cellular ion metabolism) either in for 50 minute into the prostate.
freshly excised tumor tissue or maintained in tissue culture prostate carcinoma.
This study was supported by NCI Contract No. N01-CN-95055. CALIBRATION
The probes were calibrated using the high K+/nigericin in situ approach
(Thomas et al., 1979). Nigericin is an K+/H+ ionophore which cause equilibrium SUMMARY
between intracellular and extracellular pH. Each calibration buffer was adjusted
to the appropriate pH (6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, and 7.6) using either 1 M
• Carboxy-SNARF-1 was the most useful fluorescent indicator for pH
KOH or 1 M NaOH.
measurements and was visible only in the cytoplasm of the epithelial cells
collected from rat and dog prostates. However, the Lyso Sensor Green DND-
LASER CONFOCAL SCANNING MICROSCOPY
189 which is only fluorescent in internal cellular compartments (lysosomes)
The dual emission dyes were excited with Argon/Krypton (Ar/Kr) laser light at
at pKa < 5.2 was visible in cells of the prostate tissue. Therefore, it was
488 nm and 568 nm. The fluorescence was measured simultaneously at 585 –
concluded that the pH of lysosomes in prostate tissue was < 5.2.
615 nm (assigned to the green channel in all figures) and > 650 nm (assigned
to the red channel in all figures). For the DND-153, DND-189 and BCESF
• In vivo loading of the prostate tissue in rats and dogs was successfully
INTRODUCTION fluorescent dyes an absorption/emission spectrum of 488/505-550 nm was
achieved for the first time.
Many tumors contain hypoxic areas due to decreased vascular supply which may • Based upon several experiments with loading carboxy-SNARF-1 either in
PERFUSION AND IMAGING OF PROSTATE TISSUE
result in increased glycolysis and lactic acid production, resulting in decreased vitro or in vivo for rat and dog prostates, the intracellular pH of the
A Lambda Double Perfusion tube set was used with the Lambda Perfusion Pump
local pH (Wike-Hooley et al., 1985). Recent studies have demonstrated that the epithelial cells was measured at ~ 7.0. Other cells located in the prostate
in order to simultaneously irrigate fresh media into the confocal dish and to
extracellular pH of solid tumors is more acidic than the intracellular pH of tumor tissue such as the stroma cells were not stained by any fluorescent
aspirate media out of the dish (approximately 35-45 mL/hr). The Bioptechs
cells (McCoy et al., 1995; Raghunand et al., 1999). The pH gradients in solid indicators including the acetoxymethyl (AM) ester form of SNARF-1 dye
Objective Temperature Control System was used to provide a temperature
tumors may provide a means for more selectively targeting neoplastic cells with which was easily and selectively up taken only by epithelial cells. This fact
controlled environment (Figure 1). RPMI-1640 medium was perfused onto the
cytotoxic or anticancer agents. is opening a theoretical possibility of the synthesis of the AM forms of new
microdissected (rat) or sectioned (dog) tissue while it was in a confocal dish on
constructed therapeutic compounds and its selective delivery to the
the stage of the LSM 510 confocal microscope. Images were obtained every 2
prostate epithelial cells based on the intercellular esterase cleavage.
The objective of this research program was to investigate the hypothesis that minutes until the intensity reached a plateau. The tissue was then perfused
prostate cells contain a relatively acidic intracellular environment and to with the calibration buffers at pHs 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, and 7.6.
• A method of pH measurement in the prostate tissue (rather than in
characterize the organelles that demonstrate low intracellular pH. This work was Images were obtained every 3, 5, 7, and 9 minutes for each buffer.
individual cells) was developed with fluorescent infusion both in vitro and in
of considerable importance because the characterization of intracellular pH of
vivo. This method may now be used for the measurements of hydrogen ion
various cells within the prostate of an animal model reflective of the human DATA ANALYSIS
concentrations and other ion measurements (cellular ion metabolism) in
prostate may have implications for drug development based upon pH-dependent The intensity of the dye was obtained by subtracting each density level from
prostate cancer tissues.
drug delivery and activity. The present study was the first attempt to evaluate the maximum possible level of density (density of absolute black = 255). The
the hydrogen ion concentration of the epithelial cells from the prostate of rats mean intensity and standard deviation of five measurements were calculated.
• A method of viability assessment of the cells in tissue sections was
and dogs. This study created a model for studying the spatial distribution of pH Standard curves of the ratios of intensity (red/green and green/red) of the
developed by loading with the second dye (BCECF AM) after the completion
within normal and tumor epithelial cells in prostate tissues. fluorescent indicator versus pH were plotted. Intracellular pH levels of the
of pH measurements with the primarily fluorescent indicator (SNARF-1).
prostate cell were determined from linear regression of the calibration data.
Both dyes are fluorescent only in live cells.