0022-5347/01/1661-0329/0

THE JOURNAL OF UROLOGY® Vol. 166, 329 –334, July 2001

Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.® Printed in U.S.A.

CHARACTERIZATION OF THE SPONTANEOUS ELECTRICAL AND

CONTRACTILE ACTIVITY OF SMOOTH MUSCLE CELLS IN THE RAT
UPPER URINARY TRACT
R. J. LANG, H. TAKANO, M. E. DAVIDSON, H. SUZUKI AND M. F. KLEMM
From the Departments of Physiology, Monash University, Clayton Victoria, Australia, and Nagoya City University, Nagoya, Japan

ABSTRACT

Purpose: We morphologically and electrophysiologically identified the cells that generate the
electrical activity underlying the peristaltic contractions of the rat upper urinary tract.
Materials and Methods: Electron microscopy and tension recording techniques were used to
characterize the smooth muscle cells underlying spontaneous contractions in the wall of the rat
ureter, and proximal and distal renal pelvis. Intracellular microelectrodes, containing 4% neu-
robiotin were used to record data from the cells of the renal pelvis, which were later viewed on
a confocal microscope.
Results: Spontaneous myogenic contractions (average 22.3 ⫾ 2.2 minutes⫺1) originated in the
proximal renal pelvis and propagated into the distal renal pelvis and ureter in 6 preparations.
Smooth muscle cells in the renal pelvis and ureter were typical in appearance with greater than
85% of their sectional area containing clumped contractile filaments. In contrast, contractile
fibrils occupied only 65% of the sectional area of the smooth muscle cells within the most
proximal region of the renal pelvis (pelvicaliceal junction). In strips of the renal pelvis spindle
shaped cells 83 to 200 ␮m. long fired spontaneous action potentials (6 minutes⫺1) consisting of an
initial spike, a quiescent plateau phase and abrupt hyperpolarization to a peak diastolic potential
of ⫺60 mV. Other spindle shaped cells 94 to 112 ␮m. long displayed small membrane transients
(15 minutes⫺1) 9 to 19 mV. in amplitude, firing from a diastolic potential of ⫺40 mV.
Conclusions: It is likely that the spontaneous contractile activity of the rat upper urinary tract
arises from the discharge of action potentials in typical smooth muscle cells of the proximal renal
pelvis that are directly driven by the spontaneous membrane oscillations of atypical smooth
muscle cells.
KEY WORDS: peristalsis; action potentials; rats, Wistar; urinary tract; muscle, smooth

Spontaneous contractions occur in the mammalian upper cyclooxygenase-2, on the motility of whole mount prepara-
urinary tract to propel urine from the renal pelvis through to tions of the upper urinary tract in the guinea pig and rat, and
the ureter and then into the bladder, where it is stored until described a number of striking differences in these 2 species.9
micturition. Strips cut along the circumference of the renal Spontaneous contractions in the rat upper urinary tract in
pelvis display spontaneous contractions with an intrinsic control saline at 30C occurred at a frequency 3-fold larger
frequency that decreases as strips are obtained from regions than in the guinea pig at 35C and increased in amplitude with
more distal to the renal calix. The isolated ureter is quiescent time. Also, exposure to capsaicin had little effect on muscle wall
in most mammals except in humans and pigs.1, 2 In the motility of the rat upper urinary tract, while contraction fre-
mammalian upper urinary tract spontaneous myogenic con- quency and amplitude in the guinea pig preparation were re-
tractile activity has been associated with the presence of duced.9, 10 Furthermore, selective cyclooxygenase-1 inhibition
histologically and morphologically distinct atypical smooth
increased the amplitude of contractions in the rat upper urinary
muscle cells. In unicaliceal mammals, including the dog, cat,
tract but had little effect on the frequency or amplitude of the
rat, rabbit and guinea pig, an outer layer of darker staining
atypical smooth muscle cells extends from the region of pelvic contractions recorded in the guinea pig. In contrast, selective
attachment to the renal parenchyma and to the pelviureteral cyclooxygenase-2 blockade had little effect on the motility of the
junction, which is absent in the ureter.3, 4 In mammals with rat upper urinary tract but reduced motility of the guinea pig in
multicaliceal kidneys (humans and pigs) a layer of these a concentration dependent manner.9 Cyclooxygenase-2 inhibi-
atypical cells is also found on the inner aspect of the muscle tion has also recently been shown to reduce the increases in the
wall, interconnecting the minor calices to each other and to activity of the rat afferent renal nerve and prostaglandin E2
the renal parenchyma.5, 6 production induced by increased renal pelvic pressure, suggest-
Although the rat upper urinary tract has been extensively ing that prostaglandins contribute to the stimulation of mech-
studied in terms of its morphology, sensory neuropeptide anosensitive neurons in the pelvic wall.8 In this report we made
distribution7 and afferent renal nerve function,8 there is a detailed examination of the contractile, electrical and morpho-
surprisingly little known about the mechanisms underlying logical properties of the smooth muscle cells within the wall of
pyeloureteral motility. Recently we reported a comparison of the rat upper urinary tract. As in the guinea pig, contractions
the effects of specific inhibitors of the 2 isoforms of cycloox- originated in the proximal renal pelvis, correlating with an
ygenase, constitutive cyclooxygenase-1 and inducible increased presence of atypical smooth muscles in the pelvical-
iceal junction. Contractions arose from the firing of driven ac-
Accepted for publication January 5, 2001.
Supported by the Australian National Health and Medical Re- tion potentials, which were of a much simpler time course
search Council. compared with driven cells in the guinea pig. 11, 12
329
330 SPONTANEOUS ELECTRICAL AND CONTRACTILE ACTIVITY IN RAT RENAL PELVIS

MATERIALS AND METHODS Electrophysiological experiments. Recordings from the

Tension experiments. Wistar rats weighing 200 to 500 gm. proximal renal pelvis were made from 5 ⫻ 10 mm. circum-
were anesthetized with chloroform and then decapitated. The ferential strips of renal pelvis dissected free of the rat kidney
kidneys were removed through a midline incision and imme- and pinned to the bottom of a 1 ml. organ bath. The organ
diately placed in physiological saline, as described. The upper bath was then mounted on an inverted microscope (Olympus
urinary tract was dissected free from the surrounding paren- Optical Corp., Melville, New York) and perfused with bicar-
chyma and a longitudinal cut was made extending from the bonate buffered physiological saline, as described, at 3 to 4
proximal renal pelvis through to the mid region of the ureter, ml. per minute at 34 to 35C. Electrophysiological recordings
enabling the whole preparation to be laid flat. Cuts directed were made using standard intracellular microelectrode tech-
around the circumference and extending to the midline were niques and glass microelectrodes with resistances of 50 to 80
then made. The first cut divided the proximal portion of the m⍀ when filled with 0.5 to 2 M. KCl. Changes in membrane
renal pelvis from the distal renal pelvis, while 2 smaller cuts potential were recorded with a standard unity-gain pream-
were made in the ureter approximately 1 cm. from the pel- plifier and stored digitally using a Labmaster TL-1 (Axon,
viureteral junction to create a short circumferential strip. Union City, California) analog-to-digital converter at a sam-
Threads were then tied around the 3 separated regions and ple rate of 400 to 1,000 Hz., a personal computer and Axotape
the preparation was pinned into a 1.3 ml. organ bath per- 1.1 software (Axon). Various parameters of the spontaneous
fused with physiological saline and maintained at a temper- potentials were measured, including the frequency of the
ature of 30C or 35C. The threads were attached to isometric action potential discharge, amplitude of the initial spike
when present, absolute potential during the plateau, dura-
force transducers, which were connected to a MacLab/4s
tion of the action potential measured from the time the initial
analog-to-digital converter driven by a Macintosh LC (Apple,
spike was half maximal and peak negative diastolic mem-
Sunnyvale, California), thus, allowing the simultaneous re-
brane potential reached during the after-hyperpolarization
cording of tension developed in the 3 regions of the upper
period. In each experiment the parameters of 3 or 4 action
urinary tract. Approximately 1 mN. of tension was placed on
potentials were averaged. A number of similar experiments
each region and the tissue was then left to equilibrate for 30
were then averaged, as indicated. One-way analysis of vari-
to 60 minutes, after which all 3 regions generated contrac-
ance or the paired Student t test was done to test for signif-
tions that were regular in amplitude and frequency.9, 10
icance with p ⬍0.05 considered statistically significant.11, 12
Electron microscopy. Three female rats weighing 150 to
Confocal microscopy and solutions. Neurobiotin (4%) (Vec-
180 gm. were anesthetized with 60 mg./kg. pentobarbitone tor Laboratories, Burlingame California), a commonly used
given intraperitoneally. The abdomen was opened and a can- cell tracer, was dissolved in the microelectrode saline. After
nula was inserted into the dorsal aorta. The animals were successful impalement the tracer was injected into the cell
initially perfused with saline (0.9% NaCl containing 1% so- for more than 1 minute. The tissue was removed from the
dium nitrite) for 1 minute at 80 mm. Hg, followed by fixative bath, re-pinned onto a small piece of dental wax and fixed
for 2 minutes at 80 mm. Hg and 6 to 8 minutes at 50 mm. Hg. overnight at 4C in 4% paraformaldehyde in 0.1 M. phosphate
The fixative consisted of 2% glutaraldehyde and 2% parafor- buffer, pH 7.2. The tissues were rinsed 3 ⫻ 15 minutes in
maldehyde in 0.1 M. sodium cacodylate buffer, pH 7.2. After phosphate buffered saline (PBS) containing 0.5% Triton
perfusion the kidneys and ureters were gently removed and X-100 (PBS/Triton), unpinned and incubated in streptavidin-
placed in containers of fresh fixative. While immersed in CY3 (Jackson Immunoresearch, West Grove, Pennsylvania)
fixative, the renal pelvis and ureters were separated from the and diluted 1:1,000 in PBS/Triton for 24 to 48 hours. After 3
kidney parenchyma and small pieces were cut from the pel- final 10-minute rinses in PBS tissues were mounted on glass
vicaliceal junction, an area immediately distal to the attach- slides using Dako mounting medium (Dako Corp., Carpinte-
ment of the renal pelvis to the kidney parenchyma as well as ria, California) and examined using a TCS NT confocal mi-
from the renal pelvis and ureter about 1 to 2 cm. from the croscope (Leica, Leica Mikroskope und Systeme GmbH, Wet-
pelviureteral junction. The small tissue pieces were incu- zlar, Germany) equipped with 16{times], 40⫻ and 63⫻ oil
bated in fresh fixative for a further 2 hours, rinsed 3 times for immersion lenses. Specimens were excited using the 568 nm.
15 minutes each time in 0.1 M. sodium cacodylate buffer, pH line of the Kr:Ar laser and emissions were detected through
7.2, fixed in buffered 1% osmium tetroxide for 2 hours before a 665 nm. pass filter. Preparations were scanned and XYZ
dehydration in ethanol and epoxy propane, and embedding in stacks were collected, so that the whole volume of neurobi-
EPON. Sections were cut from randomly selected blocks from otin filled cells was imaged. Stacks of images were saved to a
each region using a diamond knife on a Reichert Ultracut computer hard disk and 3-dimensional reconstructions were
ultramicrotome (Leica Microsystems, Vienna, Austria), made using Voxblast software (Vaytek, Fairfield, Iowa).
mounted on copper grids and then examined using a Jeol Physiological saline composition was 120 mM. NaCl, 5 mM.
TEM100 transmission electron microscope (Joel, Tokyo, Ja- KCl, 2.5 mM. CaCl2, 1 mM. MgCl2, 1 mM. NaH2PO4, 25 mM.
pan).12 Grid squares containing smooth muscle cells were NaHCO3 and 11 mM. glucose, pH 7.3 to 7.4 after bubbling
photographed at 5,000⫻ magnification. Photographs were with a 95% O2-5% CO2 gas mixture.
enlarged to approximately 15,000⫻ magnification and the
outlines of a minimum of 30 smooth muscle cells per animal
were traced onto acetate sheets and digitized into a com- RESULTS
puter. The areas of the smooth muscle cell profile occupied by General morphology. Low magnification electron micros-
mitochondria, clumps of contractile filaments and the cell copy and immunostaining for ␣-smooth muscle actin revealed
nucleus (if present) were outlined. Areas of close apposition the general arrangement of the cells that composed the wall
between cells were marked and grouped as 50 to 80 nm. of the rat upper urinary tract (figs. 1 and 2). A single layer of
appositions where the apposing cell membranes were sepa- closely packed epithelial cells lined the upper urinary tract of
rated by intervening basal lamina, 20 nm. junctions where the rat (fig. 1, A). Lamina propria, which contained blood
the cell membranes were closely opposed and the basal lam- vessels, axon bundles and fibroblast-like cells, separated the
ina was absent, and adherence-type junctions, where closely epithelial cells from the main smooth muscle wall. In the
apposed cell membranes were identified by the presence of ureter smooth muscle cells were arranged into an inner cir-
membrane specializations. Sectional area and cell length cular and an outer longitudinal layer. In both muscle layers
were calculated and expressed as a percent of the total sec- the smooth muscle cells were tightly packed, as evidenced by
tional area of each cell or as a percent of the sectional perim- the dense immunoreactivity for ␣-smooth muscle actin ob-
eter, respectively. served under confocal microscopy (fig. 2, C). In the renal
 SPONTANEOUS ELECTRICAL AND CONTRACTILE ACTIVITY IN RAT RENAL PELVIS 331

FIG. 3. High magnification electron micrographs of rat renal pel-

FIG. 1. Electron micrographs shows arrangement of smooth mus- vis. A, examples of nerve bundles surrounded by Schwann cell. B,
cle cells and other elements in rat. A, pelvicaliceal region. Bar rep- typical and atypical smooth muscle cells. C, ICC-like fibroblasts
resents 5 ␮m. Inset reveals 3 adherence-type junctions at higher containing several caveolae, discontinuous basal laminar material
magnification B, renal pelvis. Arrows indicate typical 20 nm. junc- and no contractile filaments. Bars represent 1 ␮m.
tion. smc, smooth muscle cells. Bar represents 2 ␮m. Inset shows
typical 20 nm. junction at higher magnification. C, ureter. Arrows
indicate 50 to 80 nm. appositions filled with basal lamina. Bar
represents 1 ␮m. average sectional area of 222 and 151 individual cells con-
taining contractile fibrils was 85% ⫾ 1% (range 55% to 100%)
in the ureter and 86% ⫾ 1% (range 54% to 100%) in the renal
pelvis. In the renal pelvis and ureter mitochondrial profiles
occupied approximately an average of 4% (4% ⫾ 0.3% and
4.6% ⫾ 0.2%, respectively) of the cell sectional area (fig. 1, B
and C, and fig. 3, B). Bundles of unmyelinated nerve axons
and varicosities often surrounded by a Schwann cell were
observed within the lamina propria and muscle layers of the
ureter (fig. 1, C), renal pelvis (fig. 3, A) and pelvicaliceal
junction.
In the most proximal region of the renal pelvis (pelvical-
iceal junction) smooth muscle cells were arranged in an open
network of individual cells (fig. 1, A) with little ␣-smooth
muscle actin immunostaining (fig. 2, A). Only 3, 50 to 80 nm.
junctions were observed in this region, reflecting this sparse
bundling of cells. Pelvicaliceal muscle cells were widest at the
nucleus and had many long narrow processes that formed
FIG. 2. Confocal images of sections of rat tissue stained to show specialized junctions with other similar cells (fig. 1, A and 3,
relative immunoreactivity for ␣-smooth muscle actin. A, pelvicaliceal B). These junctions were of the 20 nm. and the adherence
region. B, renal pelvis. C, ureter. Bars represent 50 ␮m. type, occupying an average of 7.3% ⫾ 1.2% and 1.18% ⫾
0.22% of the perimeter of 29 and 17 cells, respectively. A total
of 76 smooth muscle cells of the pelvicaliceal region also had
pelvis smooth muscle cells were arranged in smaller, ran- significantly less average sectional area occupied by mito-
domly oriented bundles, which were connected with each chondria compared with smooth muscle cells of the ureter
other to form a plexiform layer and separated by connective and renal pelvis (2.6% ⫾ 0.38%, Student’s unpaired t test p
tissue (fig. 1, B). This looser packaging of muscle cells was ⬍0.05). In 76 cells examined contractile fibrils in the muscle
also evidenced by the reduced immunoreactivity for ␣-smooth cells of the pelvicaliceal junction occurred in only small
muscle actin (fig. 2, B). clumps, separated by large areas of cytoplasm rich in endo-
Smooth muscle cell bundling within the renal pelvis and plasmic reticulum. The average sectional area of 76 cells
ureter was reflected by the large percentage of cell perime- occupied by contractile fibrils was 66.4% ⫾ 3.4%, which was
ters in close apposition with a neighboring cell in 133 and 184 significantly smaller than the sectional areas of muscle cells
cells examined (mean 29.4% ⫾ 1.6%, range 0% to 84% and in the renal pelvis or ureter (unpaired t test p ⬍0.05).
24.2% ⫾ 1.2%, range 0% to 75%, respectively), and separated In the guinea pig upper urinary tract smooth muscle cells
by a 50 to 80 nm. gap filled with basal lamina (fig. 1, A and have been divided into a typical and an atypical type based
B). Muscle cells of the upper urinary tract also formed more on the skewed distribution frequency profiles of the percent
tightly coupled junctions of 20 nm. with absent basal lamina of sectional area of cells filled with contractile fibrils.12 In our
(fig. 1, B, inset). In the renal pelvis and ureter these 20 nm. experiments the frequency distributions of the percent of
junctions occupied an average of 2.8% ⫾ 0.4% and 4.08% ⫾ sectional area of cells filled with contractile fibrils in the rat
0.71% of the perimeters of 20 and 37 cells, respectively. renal pelvis and ureter were such that 99% of the cells had
Specialized adherence-type junctions occupying an average greater than 60% of the sectioned area occupied by contrac-
of 2.2% ⫾ 0.3% of the perimeter of 11 cells were also occa- tile filaments and were termed typical smooth muscle cells.
sionally observed in the renal pelvis and ureter (fig. 1, A, Atypical smooth muscle cells with less than 40% of the sec-
inset). tional area occupied by contractile filaments12 represented
When the smooth muscle cells of the renal pelvis and only 22% of the smooth muscle cells examined in the pelvi-
ureter were analyzed at higher magnification, they were caliceal junction (fig. 3, B), while the remaining 78% had
mostly filled with clumped contractile fibrils (fig. 3, B). The typical morphology.
332 SPONTANEOUS ELECTRICAL AND CONTRACTILE ACTIVITY IN RAT RENAL PELVIS

A third population of 7 cells was also occasionally identi- control value of contractions at 30C in all 3 regions of the
fied within the lamina propria, pelvicaliceal junction and upper urinary tract (data not shown).
renal pelvis but never in the ureter. These fibroblast-like Electrical recordings in the rat renal pelvis. We made some
cells displayed numerous caveolae and a discontinuous basal preliminary recordings of the electrical activity underlying
lamina material, but no visible bundles of contractile fila- the spontaneous contractions in the rat renal pelvis using
ments. These cells also had an oval shaped nuclear region intracellular microelectrodes filled with a high KCl saline
and many narrow extended processes. However, these cells containing 4% of the intracellular cell marker neurobiotin.
were not immunoreactive for ␣-smooth muscle actin or c-Kit, Electrical recordings were generally made from cells for 1 to
the proto-oncogene for tyrosine kinase and specific marker 10 minutes. Tissues were the removed from the bath, fixed,
for interstitial cells of Cajal (ICC) in the intestine.12 processed for immunostaining for neurobiotin and then
Spontaneous contractile activity. At 35C contractions of the viewed on a confocal microscope. XYZ stacks of images (5 to
whole mount preparations of the rat upper urinary tract 15 slices at 1 to 4 ␮m. thick) of fluorescent cells were captured
originated in the proximal renal pelvis and propagated dis- and reconstructed on a computer.
tally into the distal renal pelvis and then into the ureter. Spontaneous membrane potential discharges were of 2
Moreover, these contractions often occurred from an oscillat- general types. In 2 cells small transient oscillations of mem-
ing baseline (fig. 4, B). Contraction amplitudes decreased brane potential 9 to 19 mV. in amplitude and 816 to 1020 ms.
distally along the rat upper urinary tract. For example, the in duration were recorded at a frequency of 15 minutes⫺1 (fig.
average contraction amplitude in the proximal and distal 5, A) These potential transients were separated by a period of
renal pelvis of 6 rats was 0.45 ⫾ 0.09 mN. and 0.3 ⫾ 0.07, and membrane hyperpolarization with a peak negative (diastolic)
in the ureter of 3 it was 0.18 ⫾ 0.11 mN. Average contraction potential of ⫺40 mV. Examination of these tissues under the
frequency also decreased distally in the proximal and distal confocal microscope revealed that these transient potentials
renal pelvis of 6 rats, and in the ureter of 3 (22.3 ⫾ 2.2 had been recorded in spindle-shaped immunoreactive cells 94
minutes⫺1 and 14.1 ⫾ 2.6, respectively). In contrast, contrac- to 112 ␮m. long (fig. 5, B).
tions in these 3 regions of the upper urinary of 9 guinea pigs In 6 cells a distinct action potential discharge at an aver-
tract recorded under the same conditions at 35C were gen- age of 6.3 ⫾ 1.2 minutes⫺1 consisted of an average initial
erally 2 to 5-fold larger in amplitude and occurred at a mean spike of 49.2 ⫾ 4.2 mV. in amplitude, followed by a long
of 7.1 ⫾ 0.4 minutes⫺1 in all regions.9, 10 quiescent plateau with an average half-amplitude duration
When the temperature was reduced to 30C, the contrac- of 981 ⫾ 83 ms. at an average absolute potential of ⫺18.1 ⫾
tions of the rat upper urinary tract became more regular in 1.3 mV. The plateau phase in the 6 cells was terminated by
amplitude and frequency, allowing more reliable meas- abrupt repolarization, which was followed by after-
urements of parameters during experiments into the effects hyperpolarization to an average peak diastolic potential of
of applied excitatory or inhibitory agents.9 In the proximal ⫺61.4 ⫾ 4.2 mV. In 3 cells in which action potentials dis-
and distal renal pelvis this 5C decrease in the bathing tem- charged at higher frequencies (10 to 14 minutes⫺1) after-
perature to 30C significantly increased contraction ampli- hyperpolarization showed distinct, slowly developing repo-
tudes by 20% and 33%, respectively (fig. 4, C). However, the larization before the discharge of the next action potential
frequency of these contractions at 30C was decreased signif- (fig. 5, C and E). In contrast, 3 cells in which action potential
icantly to 59% of the control frequency at 35C (p ⬍0.05, fig. 4, discharge occurred at lower frequencies (4 minutes⫺1) the
C). When these changes in contraction parameters in the after-hyperpolarization was relatively transient and followed
proximal and distal renal pelvis were expressed as a motility by a relatively quiescent membrane potential before the next
index using the formula, amplitude ⫻ frequency, it was ob- discharge (fig. 6, A and C).
served that the calculated motility index at 30C was reduced Confocal micrographs of neurobiotin filled cells in which
to 68% and 76%, respectively, of the control motility index (p
recordings of action potential discharge were relatively short
⬍0.0.5, fig. 4, C). Comparable experiments in 9 guinea pigs
(less than 5 minutes) usually consisted of 1 to 3 dimly fluo-
revealed a similar 20% to 80% increase above the control
amplitude and decrease in frequency to 45% to 85% of the

FIG. 4. Typical example of spontaneous contractile activity re-

corded simultaneously. A, in proximal and distal renal pelvis, and FIG. 5. Morphological properties of 3 neurobiotin filled cells gen-
ureter at 30C. B, in proximal and distal renal pelvis, and ureter at erating spontaneous electrical activity recorded in same preparation
35C. C, average amplitude and frequency of contractions in proximal of rat renal pelvis. A, C and E, spontaneous electrical activity re-
and distal renal pelvis were measured when tissues were at 30C or corded for 1 to 2 minutes using intracellular microelectrodes contain-
35C and motility index was calculated using formula, amplitude ⫻ ing 4% neurobiotin. B, D and F, after fixation and immunostaining
frequency. Asterisk indicates statistical significance of changing for neurobiotin, cell shapes were viewed on confocal microscope.
temperature. Calibration bar represents 50 ␮m.
 SPONTANEOUS ELECTRICAL AND CONTRACTILE ACTIVITY IN RAT RENAL PELVIS 333
by the relative number of atypical smooth muscle cells in the
pelvicaliceal junction, the frequency of these contractions is
not directly related to the absolute number of atypical cells
present.
Investigations into the electrical origin of pyeloureteral
motility first involved the use of extracellular sucrose gap
techniques in strips of the guinea pig renal pelvis and indi-
cated that the spontaneous contractions were associated with
simple oscillations in the membrane potential. In contrast,
electrically evoked contractions in the distal renal pelvis and
ureter were associated with action potentials consisting of a
rapid rising phase and a long plateau.15–17 More recently,
intracellular microelectrode recordings from different re-
gions of the guinea pig renal pelvis and ureter have revealed
3 types of cells characterized in terms of their electrical
behavior and morphology, as revealed by electron microscopy
or by neurobiotin injections and confocal microscopy. Simple
pacemaker oscillations of the membrane potential (8 min-
utes⫺1) were recorded in spindle-shaped cells in the pelvical-
FIG. 6. Typical examples of neurobiotin filled driven cells in rat iceal junction (85% of cells)18 and the proximal renal pelvis
renal pelvis. A, driven action potentials were recorded for 1 to 2
minutes. B, after fixation and immunostaining for neurobiotin cell (15% of cells)11 but never in the distal renal pelvis or ure-
shapes, confocal microscopy reveals relative spread of cell marker to ter.12 This finding correlated closely with the relative num-
neighboring cells. C, driven action potentials were recorded for more ber of atypical smooth muscle cells. Most muscle cells of the
than 5 minutes. D, after fixation and immunostaining for neurobi- ureter (100%), and the proximal (83%) and distal (97.5%)
otin cell shapes, confocal microscopy reveals relative spread of cell
marker to neighboring cells. Bar represents 50 ␮m. renal pelvis were typical in appearance. Driven action poten-
tials consisting of an initial spike followed by various num-
bers of potential oscillations superimposed on a plateau
phase were recorded in these regions with a similar relative
rescent immunoreactive cells in close proximity to each other distribution. After filling with neurobiotin cells discharging
(fig. 5, C and E and fig. 6, A). When recordings lasted more driven action potentials were also seen to be spindle-shaped
than 5 minutes, immunoreactive neurobiotin was far more and typically 250 ␮m. long. A third class of intermediate
intense near the point of microelectrode insertion. Moreover,
action potentials with a single spike followed by a quiescent
this immunoreactivity was observed to have spread consid-
plateau and abrupt repolarization was recorded in the pelvi-
erable distances into neighboring cells or cell bundles (fig. 6,
caliceal junction, and in the proximal and distal renal pelvis
D). These 6 cells also appeared spindle-shaped, 5 to 10 ␮m.
in 11% to 17% of cells. These action potentials were recorded
wide at the nucleus and an average of 147 ⫾ 21 ␮m. long. In in irregular shaped cells with an oval-shaped nucleus and
addition, complex action potentials consisting of an initial many branching interconnecting processes, which were not
spike and a plateau phase with many potential oscillations immunoreactive for ␣-smooth muscle actin or c-Kit, the se-
superimposed, which have been routinely recorded in the
lective marker of ICC.12
renal pelvis (greater than 75% of cells) and ureter (100% of
The smaller frequency of contraction in the proximal re-
cells) of the guinea pig,11, 12 were never recorded in the rat
gions of the guinea pig renal pelvis may perhaps correlate
renal pelvis. with the relatively higher number of ICC-like cells present
compared with the rat renal pelvis. We previously speculated
DISCUSSION
that guinea pig ICC-like cells were electrically activated by 1
When viewed by electron microscopy, the general arrange- or more pacemaker atypical smooth cell bundles. Moreover,
ment of the smooth muscle cells within the wall of the rat not every pacemaker potential invading an ICC-like cell was
upper urinary tract progressed from tightly packed bundles successful in triggering an intermediate action potential due
in the ureter to loosely packed bundles in the renal pelvis and to their longer refractory periods. This refractory process was
mostly individual cells in the pelvicaliceal junction. In the repeated when intermediate action potentials invaded typi-
pelvicaliceal junction the average cross-sectional area (65%) cal smooth muscles, which fire the driven action potentials
of the smooth muscle cells occupied by contractile filaments responsible for muscle contraction.19 This refractoriness
was also significantly smaller than cells in the renal pelvis among cell types may well explain the distally decreasing
(86%) and ureter (85%). These results are consistent with the frequency of driven action potential discharge and contrac-
relatively little immunoreactivity for ␣-smooth muscle actin tion along the renal pelvis and ureter as the cell profile
observed in the pelvicaliceal junction when examined by con- changes in each region.12 In the rat only 7 ICC-like cells were
focal microscopy (fig. 2) and 3, 50 to 80 nm. junctions when observed and they did not appear to form the complex net-
viewed by electron microscopy. work of cells observed in the guinea pig. In the rat we believe
It has often been postulated that the spontaneous contrac- that the pacemaker potentials (15 minutes⫺1) of a number of
tions underlying pyeloureteral motility in mammals arise atypical smooth muscle cells propagate directly to the typical
from the proximal regions of the renal pelvis, where there is smooth muscle cells and combine to initiate the action poten-
a relatively high number of atypical smooth muscles.3, 4, 12 tial discharge. Thus, the absence of the integrating function
Moreover, these propagating contractions are little affected of the ICC-like cells in the rat may well give rise to the
by tetrodotoxin or blockers of sympathetic and parasympa- observed higher frequency of contraction.
thetic motor nerves, indicating that these contractions are In the rat renal pelvis the majority of recorded action
myogenic in origin.9, 10, 13, 14 In the rat and guinea pig pelvi- potentials came from spindle shaped cells 150 ␮m. long.
caliceal junction 22% and 80%, respectively, of the smooth These cells are presumably the smooth muscle cells charac-
muscle cells were deemed to be atypical. However, the fre- terized as typical by electron microscopy. Short injections of
quency of contraction in the proximal renal pelvis of the rat neurobiotin resulted in only dimly immunoreactive cells
was significantly higher than in the guinea pig (22 minutes⫺1 when viewed by confocal microscopy. Longer injection peri-
versus 8, unpaired t test p ⬍0.05). Thus, although the origin ods revealed that neurobiotin had spread to neighboring cells
of spontaneous contractile activity is likely to be determined and bundles. The time course of the action potentials in the
334 SPONTANEOUS ELECTRICAL AND CONTRACTILE ACTIVITY IN RAT RENAL PELVIS

typical smooth muscle cells was far simpler than that of the 2. Constantinou, C. E.: Velocity gradient and contraction frequency
action potentials recorded in similar regions of the guinea of the pyeloureteral system. Experientia, 35: 791, 1979
pig. The peak diastolic and resting membrane potential be- 3. Gosling, J. A. and Dixon, J. S.: Morphologic evidence that the
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CONCLUSIONS imal renal pelvis of the guinea pig. J Urol, 155: 332, 1996
We have demonstrated that, as in the guinea pig, it is 12. Klemm, M. F., Exintaris, B. and Lang, R. J.: Identification of the
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