EVALUATION OF HYDROGEN ION CONCENTRATIONS IN PROSTATES FROM RATS AND DOGS USING FLUORESCENT CONFOCAL

1
EVALUATION OF HYDROGEN ION CONCENTRATIONS IN
PROSTATES FROM RATS AND DOGS USING FLUORESCENT
CONFOCAL MICROSCOPY 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.
METHODS ANIMALS Approximately 150 male CD (Virus
Antibody Free) rats, 400-600 g, and 13 male
Beagle dogs (6-10 kg) LOADING OF THE
FLUORESCENT INDICATORS (IN VITRO) The
microdissected (rat) or sectioned (dog) prostate
tissue was loaded for 30 minutes with the dual
emission fluorescent indicators (CarboxySNARF-1,
SNARF calcein, and SNAFL calcein) dissolved in
loading buffer (RPMI-1640 Medium without sodium
bicarbonate and amino acids). After 30 minutes,
the tissues were rinsed twice with RPMI-1640
Medium containing 250 mM sulfinpyrazone and
viewed on the LSM 510 confocal microscope with
the 25X objective. For measurements in the
lysosomes, the prostate tissues was loaded with
the single excitation and single emission
indicators (Lyso Sensor Green DND-153 and DND
189) for 30 and 60 minute incubation periods and
were viewed on the confocal microscope with the
63 X objective. IN VIVO LOADING In Vivo loading
in rats was performed using a 25 G needle to load
SNARF-1 dissolved in DMSO which was diluted in
3.5 mL of (0.4) Trypan blue salt solution (0.81
sodium chloride, 0.06 potassium phosphate,
dibasic) and 5 mL of loading buffer to a final
concentration of SNARF-1 at 100 mM. The SNARF-1
mixture was loaded underneath the capsule of the
rat prostate. Trypan blue was used for visual
confirmation of the dye uptake. In Vivo loading
of the Beagle dog prostate was performed using a
final concentration of 100 mM of SNARF-1
dissolved in DMSO and diluted in 1.8 mL Trypan
Blue Salt solution along with 7 mL of loading
buffer. One 10 mL syringe was used to load a
total volume of 8.8 mL of the dye into the Beagle
dog prostate. A clamp was placed between the
base of the bladder and the prostate to prevent
the dye from being lost through the urethra.
SNARF-1 was loaded for 50 minute into the
prostate. 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 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 KOH or 1 M
NaOH. LASER CONFOCAL SCANNING MICROSCOPY The
dual emission dyes were excited with
Argon/Krypton (Ar/Kr) laser light at 488 nm and
568 nm. The fluorescence was measured
simultaneously at 585 615 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 fluorescent dyes
an absorption/emission spectrum of 488/505-550 nm
was used. PERFUSION AND IMAGING OF PROSTATE
TISSUE A Lambda Double Perfusion tube set was
used with the Lambda Perfusion Pump in order to
simultaneously irrigate fresh media into the
confocal dish and to aspirate media out of the
dish (approximately 35-45 mL/hr). The Bioptechs
Objective Temperature Control System was used to
provide a temperature controlled environment
(Figure 1). RPMI-1640 medium was perfused onto
the microdissected (rat) or sectioned (dog)
tissue while it was in a confocal dish on the
stage of the LSM 510 confocal microscope. Images
were obtained every 2 minutes until the intensity
reached a plateau. The tissue was then perfused
with the calibration buffers at pHs 6.2, 6.4,
6.6, 6.8, 7.0, 7.2, 7.4, and 7.6. Images were
obtained every 3, 5, 7, and 9 minutes for each
buffer. DATA ANALYSIS The intensity of the dye
was obtained by subtracting each density level
from the maximum possible level of density
(density of absolute black 255). The mean
intensity and standard deviation of five
measurements were calculated. Standard curves of
the ratios of intensity (red/green and green/red)
of the fluorescent indicator versus pH were
plotted. Intracellular pH levels of the prostate
cell were determined from linear regression of
the calibration data.
ABSTRACT 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 anticancer potency of current
drugs due to selective delivery, increased
influx, and prevention of extrusion from tumor
cells. As a first step in this direction, the
objective of this study was to develop in vitro
and in vivo methods of pH measurements in
prostate tissue and characterize intracellular pH
gradients in epithelial cells of normal rat and
dog prostate. Freshly dissected prostate tissues
(in vitro) or the entire prostate gland (in vivo)
were loaded with fluorescent dyes for 30 or 50
min, respectively. After loading, tissues were
viewed using a Zeiss LSM 510 Laser Scanning
Confocal Microscope. Confocal images were
initially taken in tissues perfused with
RPMI-1640 medium. Calibration in situ was
performed in the same tissue with high potassium
buffers of known pH containing nigericin.
Carboxy-SNARF-1 was the most useful fluorescent
indicator for determining the distribution of
hydrogen ions in epithelial cells in rat and dog
prostates. SNARF-1 was visible only in the
cytoplasm of the epithelial cells. The
fluorescent indicator Lyso Sensor Green DND-189,
which is only fluorescent in internal cellular
compartments (lysosomes) at pH inside epithelial cells of the prostate tissue.
Therefore, it was concluded that the pH of
lysosomes in prostate tissue was procedures were established, which included rat
prostate microdissection, in vivo and in vitro
fluorescent probe infusion, optimal
concentrations of fluorescent probes, appropriate
temperature regimen, and incubation time of cell
cultures with fluorescent probes. In addition,
experimental conditions for the confocal
microscope to study fluorescent probes were
optimized. Based upon several experiments with
loading carboxy-SNARF-1 either in vitro or in
vivo for rat and dog prostates, the intracellular
pH of the epithelial cells in both species was
7.0. Besides the measurement of the pH in rat and
dog tissues, a method of pH measurement in
prostate tissue (rather than in cell culture) was
developed with fluorescent dye infusion both in
vitro and in vivo. This method may now be used
for the measurement of hydrogen ion
concentrations and other ion measurements
(cellular ion metabolism) either in freshly
excised tumor tissue or maintained in tissue
culture prostate carcinoma. This study was
supported by NCI Contract No. N01-CN-95055.

• SUMMARY
• Carboxy-SNARF-1 was the most useful fluorescent
  indicator for pH measurements and was visible
  only in the cytoplasm of the epithelial cells
  collected from rat and dog prostates. However,
  the Lyso Sensor Green DND-189 which is only
  fluorescent in internal cellular compartments
  (lysosomes) at pKa the prostate tissue. Therefore, it was concluded
  that the pH of lysosomes in prostate tissue was 5.2.
• In vivo loading of the prostate tissue in rats
  and dogs was successfully achieved for the first
  time.
• Based upon several experiments with loading
  carboxy-SNARF-1 either in vitro or in vivo for
  rat and dog prostates, the intracellular pH of
  the epithelial cells was measured at 7.0.
  Other cells located in the prostate tissue such
  as the stroma cells were not stained by any
  fluorescent indicators including the
  acetoxymethyl (AM) ester form of SNARF-1 dye
  which was easily and selectively up taken only by
  epithelial cells. This fact is opening a
  theoretical possibility of the synthesis of the
  AM forms of new constructed therapeutic compounds
  and its selective delivery to the prostate
  epithelial cells based on the intercellular
  esterase cleavage.
• A method of pH measurement in the prostate tissue
  (rather than in individual cells) was developed
  with fluorescent infusion both in vitro and in
  vivo. This method may now be used for the
  measurements of hydrogen ion concentrations and
  other ion measurements (cellular ion metabolism)
  in prostate cancer tissues.
• A method of viability assessment of the cells in
  tissue sections was developed by loading with the
  second dye (BCECF AM) after the completion of pH
  measurements with the primarily fluorescent
  indicator (SNARF-1). Both dyes are fluorescent
  only in live cells.

INTRODUCTION Many tumors contain hypoxic areas
due to decreased vascular supply which may result
in increased glycolysis and lactic acid
production, resulting in decreased local pH
(Wike-Hooley et al., 1985). Recent studies have
demonstrated that the extracellular pH of solid
tumors is more acidic than the intracellular pH
of tumor cells (McCoy et al., 1995 Raghunand et
al., 1999). The pH gradients in solid tumors may
provide a means for more selectively targeting
neoplastic cells with cytotoxic or anticancer
agents. The objective of this research program
was to investigate the hypothesis that prostate
cells contain a relatively acidic intracellular
environment and to characterize the organelles
that demonstrate low intracellular pH. This work
was of considerable importance because the
characterization of intracellular pH of various
cells within the prostate of an animal model
reflective of the human prostate may have
implications for drug development based upon
pH-dependent drug delivery and activity. The
present study was the first attempt to evaluate
the hydrogen ion concentration of the epithelial
cells from the prostate of rats and dogs. This
study created a model for studying the spatial
distribution of pH within normal and tumor
epithelial cells in prostate tissues.