From the Department of Pharmacology, State University of New York Health Science Center, Brooklyn, New York 11203
Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both INa and ICa that are vastly different
from those in tissue-cultured material. INa is abolished by Na+-free medium and by 1 µM tetrodotoxin. It first appears at ~40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent
of
0.2 ms at 20 mV, and inactivates as a single exponential with a
of 0.4 ms. Na+ conductance is half activated at
21.5 mV, and half inactivated at
59 mV. INa reactivates with a
of 20 ms. ICa is abolished by Ca2+-free medium,
Co2+ (5 mM), or nisoldipine (2 µM), and enhanced in 30 mM Ca2+, Ba2+, or BAY-K 8644. It first appears at ~
30
mV and reaches maximum at +10 mV. It activates with a voltage-dependent
of 1.5 ms at 20 mV, and inactivates
in two exponential phases, with
's of 33 and 133 ms. Ca2+ conductance is half activated at
7.4 mV, and half inactivated at
34 mV. ICa reactivates with
's of 27 and 374 ms. INa and ICa are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak INa/ICa changes
from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of INa, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum,
both currents decrease markedly, with INa vanishing from most myocytes. Estrogen-enhanced genomic influences
may account for the emergence of INa, and increased densities of INa and ICa as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy.
Smooth muscles comprise a diverse group of involuntary excitable tissues, which are dispersed widely throughout the body, subserving important physiological functions. Because individual smooth myocytes are small,
their ionic channel functions have not been studied in
earnest until the introduction of the tight-seal patch-clamp method (Hamill et al., 1981), and successful regimens of dissociating individual myocytes from tissue
assemblies (Bagby et al., 1971; Fay and Delise, 1973;
Momose and Gomi, 1980). However, the usefulness of
enzyme-dissociated smooth myocytes as physiological models has not been tested. The uterine smooth muscle is singularly suitable for addressing this issue because
it has been studied as multicellular preparations (Anderson, 1969; Mironneau, 1974
; Kao and McCullough,
1975
), as dissociated cells (references below), and in
tissue culture (e.g., Mollard et al., 1986
; Amedee et al., 1987
; Toro et al., 1990
; Rendt et al., 1992
).
Because the structure and function of uterine smooth
muscle change markedly under the influence of ovarian hormones and during pregnancy (see Kao, 1989),
it is also suitable for studying hormonal regulation of
ionic channels. Although several papers on freshly dissociated uterine myocytes have appeared (Ohya and
Sperelakis, 1989
; Inoue et al., 1990
; Inoue and Sperelakis 1991
; Miyoshi et al., 1991
; Piedras-Renteria et al.,
1991
), each dealt with a limited aspect of myometrial
function. In this and another paper (Wang, S.Y., M. Yoshino, and C.Y. Kao, manuscript submitted for publication), we will provide a wider coverage of ionic channel functions of uterine myocytes in the nonpregnant
state and throughout pregnancy. There are significant
changes in these functions during pregnancy that not
only profoundly influence the ultimate physiological functions, but also illustrate some hormonal influence
on ionic channels. In this paper, we address the inward
currents and changes in them during pregnancy; in the
other paper, we will deal with the outward currents.
Preliminary accounts have been given (Suput et al.,
1989
; Kao et al., 1989
; Yoshino et al., 1989
, 1990
; Wang and Kao, 1993
; Wang et al., 1996
).
Female rats (Sprague-Dawley; Harlan Sprague Dawley Inc., Indianapolis, IN) were mated individually, and pregnancy was dated from the morning when cervical plugs were found. Dating was
confirmed at the experiment from fetal size (Witschi, 1956).
Uteri from days 2-22 (term) of pregnancy and postpartum were
used. For nonpregnant rats, the estrus status was ascertained by
cytological examinations of vaginal washing.
Isolation of Uterine Myocytes
The procedures for cell isolation are similar to those used for taenia coli myocytes (Yamamoto, et al., 1989). Briefly, strips (~2 × 15 mm) of endometrium-free longitudinal myometrium were incubated in 0.1% collagenase (Wako Bioproducts, Richmond,
VA) in a Ca2+-free modified Krebs solution for 30 min. They were
then washed free of collagenase before being triturated. Use of
higher concentrations of collagenase or other additional proteolytic enzymes were associated with more cells with large leakage conductance and poor ionic currents, whereas longer incubations were associated with higher incidences of myocytes coated
with a layer of optically transparent material that interfered with
cell settling and seal formation with electrodes. In practice, concentrations of 200-300 cells/µl (by hemocytometer counting) have
been obtained (see also Fig. 1 A). The myocytes, kept in a high
K+, Ca2+-free medium ("KB" medium, Isenberg and Klöckner,
1982) at 5-8°C, retained good electrophysiological properties for
3 d. Most data were obtained within 6 h after isolation. All myocytes used for this work were relaxed and adhered to the bottom
of the plastic or glass chamber with no additional substrate. More
than 90% remained relaxed when exposed to Ca2+; after visual
screening, 70-80% of the cells selected for study would have good
ionic currents.
Table I. Some Morphometric Parameters of Rat Uterine Smooth Myocytes |
Electrophysiological Methods
The methods are similar to those used for the work on taenia myocytes (Yamamoto et al., 1989), in which the quality of the voltage
clamp and the isopotentiality of the whole cell were demonstrated experimentally. Adequacy of control of the uterine myocytes can be surmised from the gradual current-voltage (I-V)1 relations in the negative resistance region. For this work, the range
of capacitance cancellation in the amplifier (EPC-7; List Electronic, Darmstadt, Germany) was extended to cover 200 pF to accommodate the larger pregnant uterine myocytes. Because of the presence of fast INa, series-resistance compensation and capacitance cancellation were essential, as these procedures introduced
some positive feedback, accelerating the charging of the membrane capacity (Sigworth, 1986
). For the usual recording, the settling time was <800 µs. For small myocytes under optimal compensation, the transient artifact could be reduced to <150 µs. In
selecting results for inclusion, we looked for a state of zero current between the end of the transient and the beginning of INa
(e.g., Fig. 3 B). Leakage currents and residual transient artifacts
after hardware correction were corrected with a p/4 protocol.
Table III. Stage of Pregnancy and INa and ICa of Uterine Myocyte |
Table II. Stage of Pregnancy and Total Myocyte Capacitance |
All experiments were conducted at room temperature (~22°C).
Solutions
The bath contained (mM): 135 NaCl, 5.4 KCl, 3 CaCl2, 1 MgCl2, 5 glucose, 10 HEPES, pH adjusted to 7.25 with NaOH. Ca2+ was
sometimes increased (as specified), with an equimolar reduction in Na+. The pipette solution contained (mM): 140 KCl, 1 EGTA,
2 Na2ATP, 10 HEPES, pH adjusted to 7.25 with KOH. Osmolarity
of all solutions was routinely maintained at 275-290 mosM. For
studying the inward current, the K+ in the pipette solution was
replaced with Cs+, and the bath contained 30 mM tetraethylammonium chloride (TEA) and 5.4 mM CsCl with equimolar reduction of Na+. Run-down of ICa, which was a problem only in small
myocytes of nonpregnant or early pregnant uteri, could be obviated by incorporating 5 mM each of potassium salts of pyruvate,
oxaloacetate, and succinate in the pipette solution (Klöckner
and Isenberg, 1985).
PREPARATION
Cell Morphology
Because the uterine myocyte hypertrophies during pregnancy, cellular morphometric data at different stages of pregnancy are needed to correlate with physiological observations. Although freshly dissociated uterine myocytes were usually long, slender, and fusiform, pleomorphism was common, especially in late pregnancy when cells with irregularly swollen central regions or terminal arms (Fig. 1) constituted ~10% of the population. In the present study, to avoid possible inadequate space clamp in cells with such complex geometry, we used only long and relaxed myocytes, assuming that the pleomorphic cells shared similar basic electrophysiological properties.
Morphometric data at three stages were collected: nonpregnant (also representing early pregnancy from days 1-8), midpregnancy (14 d, also representing days 9-16), and late pregnancy (days 17-21). Table I shows that during pregnancy, the maximum diameter of the individual myocyte increased 2.8-fold from 8 to 22 µm, and the length increased 1.7-fold from 130 to 225 µm. Such changes led to a fourfold increase of surface area to 7,600 µm2, and an eightfold increase of cell volume to 21 pl in the term myocyte.
Membrane Properties
In the present experiments, the seal resistance ranged
from 5 to 40 G. When studied with Cs+-filled pipettes,
the input resistance of the whole myocyte ranged from
0.5 to 3 G
. Resting and action potentials were not routinely measured because the Cs+-pipette solution caused
a significant depolarization. However, myocytes for this
work were selected for their low leakage conductance. If the average input resistance were taken as 1 G
, then
the specific membrane resistance for the averaged size
late-pregnant myocyte would be 76 k
-cm2.
Consistent with hypertrophy, the cell capacitance increased as pregnancy progressed (Table II). In early pregnancy, the average cell capacitance remained ~30 pF, slightly higher than that of the nonpregnant myocyte (25 pF). In midpregnancy, capacitance increased markedly, possibly stimulated by fetal growth and stretch of the uterus. In late pregnancy, capacitance stabilized at ~110 pF, because there were no statistically significant differences among the values listed for days 18-21. Within 18-h postpartum, there were no significant changes in the cell capacitance.
As the amount of caveolae in myocytes at different stages of pregnancy is not known, estimation of specific membrane conductance is based on the morphometric surface. Taking the average of 108 pF as the cell capacitance for the late-pregnant myocyte with an average surface area of 7,600 µm2 (Table I), the specific membrane capacitance works out to be 1.42 µF/cm2. For the nonpregnant myocyte, based on a surface area of 1,928 µm2, the specific capacitance works out to be 1.30 µF/cm2.
Coexistence of INa and ICa
The inward current consists of two distinct components: a fast activating and inactivating component, followed by a more slowly activating and more sustained component (Fig. 2). Although the peak magnitudes of the two components and the degree of overlap vary considerably from cell to cell, the slow component is seen in all myocytes, and the fast component in half of the myocytes from nonpregnant estrus uterus or early pregnancy, and >90% of myocytes from late pregnancy.
The nature of each component is readily identifiable
by ion substitution and by selective blocking agents. In
a Na+-free medium, the fast component was abolished,
whereas the slow component and its associated tail current were unchanged (Fig. 3 A). The fast component
was fully blocked by tetrodotoxin (TTX) at 1 µM concentration (Fig. 3 B, also Ohya and Sperelakis, 1989). In a Ca2+-free medium, the slow component was abolished, leaving the fast component (Fig. 3 C). It was also
abolished when nisoldipine (2 µm) was added to the
bath (Fig. 4 C). Thus, the fast component can be identified as INa and the slow component as ICa.
INa first appeared at ~40 mV, reached a maximum
at 0-10 mV, and reversed at 80-84 mV (Fig. 3 D), in
good agreement with the expected ENa. The magnitude
of INa varied widely, in part because of differences in
cell size, and in part with the stage of pregnancy (see
below). When normalized to cell capacitance, and adjusted for the morphometric surface areas, peak INa
works out to be 2.77 µA/cm2 for nonpregnant myocytes, and 5.10 µA/cm2 for late-pregnant myocytes.
In 3 mM [Ca2+]o, ICa was first seen at ~30 mV, and
reached a maximum at +10 mV (Fig. 4 A). The magnitude of ICa varied because of cell size and stage of pregnancy. When normalized to cell capacitance and the morphometric surface, peak ICa (in 3 mM Ca2+) works out
to be 5.67 µA/cm2 for the nonpregnant myocyte and
3.43 µA/cm2 for the late-pregnant myocyte. In 30 mM
Ca2+, the activation of ICa shifted positive by ~15 mV. The
maximum current increased, and the voltage at which
it was attained also shifted positive by ~20 mV (Fig. 4
A), as expected from a screening effect on surface negative charges (Frankenhauser and Hodgkin, 1957
).
When 3 mM Ba2+ replaced Ca2+ in the bath solution, the inward current activated as rapidly, reached approximately the same maximum but slightly later, and the I-V relation was shifted ~10 mV to the negative. The inactivation was markedly slower (Fig. 4 B), probably because of interference with Ca2+-mediated Ca2+ inactivation (see below). The Ba2+ current, as well as ICa, could be fully blocked by 5 mM Co2+ (not illustrated).
BAY K 8644, a dihydropyridine compound, increased
the magnitude of the ICa without shifting the I-V relations (Fig. 4 D). In contrast to Ba2+, it did not slow the
inactivation, and the larger current was inactivated at
an appreciably faster rate. Nisoldipine (2 µM) readily
blocked the ICa of pregnant (Fig. 3 B, legend) and nonpregnant myocytes (Fig. 4 C), similar to those reported
for 10 µM nifedipine (Miyoshi et al., 1991) and on Ba2+
current (Ohya and Sperelakis, 1989
).
INa and ICa in Relation to Stages of Pregnancy
ICa was recordable in all uterine myocytes. INa was additionally seen in 19 of 38 myocytes taken from nonpregnant estrus, metestrus, or diestrus, but not from proestrus, animals. INa was seen in 11 of 22 myocytes from 2-d pregnant uterus, and almost all myocytes from day 5 of pregnancy to term.
Table III summarizes the INa and ICa at different stages of pregnancy. The data are based on currents that were stable for at least 15-20 min. For comparing cellular properties, only myocytes with both INa and ICa were included. Several features are apparent in the data, (a) from baseline values at the beginning of pregnancy, the densities of both INa and ICa increased during the first trimester; (b) they then declined during the second trimester; (c) whereas INa reached its lowest density on the 17th d and began to recover by the 18th d, ICa continued to decline and reached its lowest value on the 18th d; (d) from days 2 to 17, peak INa density was less than peak ICa density, but in late-pregnant myocytes, this relation was reversed; and (e) the net changes over the course of pregnancy are that the density of peak INa increased by 1.8-fold, while that of ICa decreased by 1.7-fold.
In 12 myocytes from uteri that were 14-18 h postpartum, ICa was observed in every myocyte, but INa was observed in only one. This myocyte was large, with a cell capacitance of 200 pF. Both the average peak ICa density and the peak INa density of the single myocyte were lower than the least values recorded during pregnancy. The decline in the current densities occurred even as the cell capacitance remained the same (Table II). Also, peak INa/ICa in this single myocyte was 0.57, similar to that in nonpregnant myocytes.
ACTIVATION AND INACTIVATION Kinetics of Activation and Inactivation
Activation.To avoid possible errors in time-to-peak
measurements caused by current inactivation, kinetics
of activation was examined by curve fitting the early
part of the ionic currents. With optimum compensation, INa can be distinctly separated from the capacitive current transient (e.g., Figs. 2 and 3). At ~22°C, INa
reached a peak in ~1 ms. The best fit was obtained with
a fourth-power function (Fig. 5 A), with voltage-dependent 's varying between 0.39 ms at
20 mV and 0.18 ms at 20 mV (Fig. 5 B).
In contrast, the activation of ICa was best fitted with a
square function (Fig. 5 C), with 's varying between 2.2 ms at
10 mV to 1.4 ms at 30 mV (Fig. 5 D).
INa inactivated as a single exponential
(Fig. 6 A), with a voltage-dependent that varied from
0.77 ms at
10 mV to 0.41 ms at 30 mV (Fig. 6 B).
For ICa, inactivation was more complex. In a small
number of myocytes, a small fraction (<5%) of ICa remained even at the end of a 400-ms step (Fig. 6 C, inset). The inactivation time course can be fitted with two
exponential phases, with 's of ~32 ms (
f) and 133 ms
(
s) (Fig. 6 C).
f was strongly voltage dependent, showing a U-shaped relation with the fastest rate at 10 mV
(usually <40 ms) and significantly slower rates at either more negative or more positive voltages (Fig. 6 D).
s
may also be voltage dependent (175 ms at
10 mV, 81 ms at +20 mV, 289 ms at +40 mV), but there was too
much variability in the small sampling to support any
statistically significant differences.
Deactivation of both INa and ICa follow
single exponential time constants. In five myocytes, in
the presence of a high concentration of nisoldipine (20 µM), INa, when repolarized from 0 to 80 mV, decreased
with a
of 0.87 ± 0.10 ms. In another group of five myocytes in which INa had been abolished by depolarizing
steps of >10 ms, ICa (repolarizing from +10 to
80 mV)
deactivated with a
of 2.71 ± 0.23 ms.
Steady State Activation and Inactivation
Fig. 7 shows the steady state activation and inactivation
of INa and ICa. These observations were made on four to
five different cells from 17-, 18-, and 20-d pregnant
uteri that show both INa and ICa. To facilitate the experiments, one current was blocked so that the other could
be studied.
For both INa and ICa, the activation relations followed
Boltzmann distributions. For INa (Fig. 7 A), significant
conductance was first seen at ~40 mV. Half activation
occurred at
21 mV, full activation at ~+20 mV. The
slope factor was 5 mV. For ICa (Fig. 7 B), the first detectable conductance appeared at ~
30 mV; half activation at
8 mV, full activation at ~+30 mV. The slope
factor was 6.6 mV.
For the steady state inactivation curves, the relations
also followed Boltzmann distributions well. For INa (Fig.
7 A), half inactivation occurred at 59 mV, and the
slope was 8.7 mV. For ICa (Fig. 7 B), half inactivation occurred at
34 mV, and the slope was 5.4 mV. Therefore, at the usual resting potential of ~
50 mV, 24% of
INa and 95% of ICa are available.
For both INa and ICa, there was a small overlap of the
activation and inactivation relations (window current),
with that for INa peaking to ~5% of the maximum current at 35 mV, and that for ICa peaking to 10% of the
maximum at
25 mV.
Recovery from Inactivation
Because the spontaneous electrical activity of the myometrium consists typically of bursts of action potentials
that lead to contractions (see Kao, 1989), the influence
of an action potential on those following it can be important. Fig. 8 shows the recovery of INa from inactivation. The data are closely clustered even though the
myocytes came from different stages of pregnancy. They are also well fitted by a single exponential curve with a
time constant of 20 ms.
Fig. 9 shows the recovery of ICa from inactivation. The
recovery is best fitted by two exponential components;
the smaller component (12%) has a of 27 ms, and the
larger component (86%) has a
of 374 ms.
Viewed differently, for INa, 50% recovery is attained in <15 ms and 80% recovery (possibly needed to generate propagated action potentials) is attained in ~30 ms. For ICa, 50% recovery is attained in ~200 ms, and 80% recovery in ~600 ms. Since the rates of recovery from inactivation are important determinants of the rates of repetitive action potentials, the widely different reactivation of the two types of channels must affect their respective contributions to myometrial excitability.
Dissociated Smooth Myocytes as Physiological Models
The tight-seal patch-clamp method and improved cell-isolation procedures have fostered a surge of recent
studies on dissociated single smooth muscle cells, which
are unencumbered by complex intercellular connections, ion accumulation in extracellular clefts, and insurmountable cleft resistance encountered in multicellular preparations. Although the procedures of enzyme-aided dissociation are conceded as potentially injurious
(Bolton et al., 1985; Sanders, 1989
), the suitability of such
myocytes as models has rarely been tested. The pregnant rat myometrium provides an opportunity for a
critical scrutiny of this issue, because it possesses some
unique tissue-specific properties that can serve as markers, and because it has been studied in a wide spectrum
of organization levels and in tissue culture. The small
multicellular preparations, against which dissociated
myocytes can be compared, were subjected only to being dissected free from the uterus but not to any enzymes or artificial intracellular milieu. Hence, their ionic
channel functions are likely to be as nearly physiological as any isolated tissue or cells can be.
Using new information obtained on dissociated uterine myocytes (specific capacitance, 1.42 µF/cm2; surface:volume ratio, 0.36 µm1; average cell capacitance,
108 pF; see RESULTS), the active region of a previous
study (with a total capacitance of 0.13 µF; Kao and McCullough, 1975
) can be estimated to contain ~1,000
(867-1,204) cells. In such preparations, the presence
of a Na+ and a Ca2+ component in the inward current
had already been shown (also Anderson et al., 1971
;
Kao, 1978
), as had an increasing relative contribution
of the Na+ component in late pregnancy as term approached (Nakai and Kao, 1983
). Phenotypic expression of a Na+ channel is rare among visceral smooth
muscles. Confirmation in dissociated uterine myocytes
not only of that rare phenotype but also of its participation in highly tissue-specific functional changes demonstrate that such cells, if properly prepared, do not deviate substantially from physiological norms.
Because of such similarities, the freshly dissociated
uterine myocytes can also serve as a basis for assessing
some tissue-cultured preparations as physiological models (e.g., Mollard et al., 1986; Amedee et al., 1987
; Toro
et al., 1990
; Rendt et al., 1992
). Such a comparison reveals so many differences as to suggest that the observed channel functions pertain to different cells.
Some of the key differences are: (a) most fresh myocytes used in this work had a readily recordable INa,
which could be elicited from holding potentials of 90
to
60 mV (also Ohya and Sperelakis, 1989
). It was
fully blocked by 1 µM TTX (Fig. 3 B), which had a Kd of
27 nM (Ohya and Sperelakis, 1989
), characterizing the TTX receptor as a high affinity type (references in Kao
and Levinson, 1986
). In cultured cells, with the exception of the human uterine myocyte (Young and Herndon-Smith, 1991
), none showed any Na+ action potentials (Mollard et al., 1986
) or any INa, all the inward current being attributed to ICa (Amedee et al., 1987
; Rendt et al., 1992
). Some cultured cells had no inward currents at all (Toro et al., 1990
). In cultured cells, INa
could be elicited only after interference with Na+ inactivation; when elicited, the Kd of TTX blockade was 2 µM (Amedee et al., 1986
), characterizing the receptor
as a low affinity type. The different TTX sensitivities
could reflect different amino acid compositions of the
channel molecules. (b) In fresh (Fig. 4 C), but not cultured (Rendt et al., 1992
) nonpregnant myocyte, ICa
was susceptible to blockade by dihydropyridines. (c) In
fresh myocytes, half activation of the Ca2+ conductance
in 3 mM of Ca2+ was at
8 mV (Fig. 7 B). In cultured
cells, half activation in 10 mM Ca2+ was at
14 mV
(Amedee et al., 1987
), or at
7 mV (Rendt et al., 1992
), in spite of a known screening effect of 10 mM
Ca2+ on surface negative charges (Frankenhauser and
Hodgkin, 1957
; Yamamoto et al., 1989
; Sui and Kao,
1997
; and Fig. 4 A), which should have caused a significant positive displacement. (d) In fresh myocytes, the
steady state ICa inactivation was almost all voltage dependent, with half inactivation at
34 mV. In cultured cells, 10-12% of the ICa did not inactivate, and the
remainder had a half inactivation voltage of
17
(Amedee et al., 1987
) or
55 (Rendt et al., 1992
) mV.
Hence, the cultured myometrial cells described so far possess none of the unique cell-specific functional properties of the freshly dissociated uterine myocytes, and therefore are less likely to provide physiologically relevant information on ionic channel functions of uterine myocytes.
Charge Carriers and Channels for Inward Currents
The density of Na+ channels in uterine myocytes (3-5 µA/cm2) is two to three orders of magnitude lower than those in peripheral nerves, skeletal, or cardiac muscles. Kinetic data suggest the presence of a single class of Na+ channels in uterine myocytes.
The Ca2+ channels in rat uterine myocytes are mostly
of the L-type (also Ohya and Sperelakis, 1989; Miyoshi
et al., 1991
), resembling the case in other visceral smooth
myocytes (urinary bladder, Klöckner and Isenberg,
1985
; ureter, Sui and Kao, 1997
; taenia coli, Yamamoto et al., 1989
). However, in human uterine myocytes,
T-type Ca2+ channels have been described (Inoue et al.,
1990
; Young et al., 1993
). Inactivation of ICa involves
both voltage-dependent and Ca2+-mediated mechanisms.
A voltage-dependent mechanism is demonstrated in
the steady state voltage-inactivation relation, whereas a
Ca2+-mediated mechanism is seen in the U-shaped relation between
f of inactivation and ICa (Fig. 6 D), in a
slowing of the rate when Ba2+ replaced Ca2+ (Fig. 4 B),
and in an increased rate when ICa was enhanced by BAY-K 8644 (Fig. 4 D).
The density of ICa in uterine myocytes (3-11 µA/cm2)
is approximately midway in a spectrum among different
visceral smooth myocytes (urinary bladder, 20 µA/cm2,
Klöckner and Isenberg, 1985; taenia coli, 20 µA/cm2,
Yamamoto et al., 1989
; ureter, 3 µA/cm2, Sui and Kao,
1997
). In taenia coli myocytes (Yamamoto et al., 1989
)
and urinary bladder myocytes (Sui et al., 1993
), Ca2+
influx during an action potential is capable of discharging the membrane capacity and raising [Ca2+]i to 8-13
µM. Thus, the influx is potentially adequate for initiating various physiological functions. In the non- and
early-pregnant uterine myocytes, the situation is similar
and the same conclusion may apply. In the late-pregnant myocyte, the situation is more complex. Although
the cell capacitance is larger, the combined influx of
Na+ and Ca2+ may still discharge it to initiate the action
potential. However, the cell volume is much larger while
the density of ICa is lower (Tables II and III). Under
such circumstances, whether influx of Ca2+ alone is adequate to supply the needed Ca2+ or whether internal
sources become more important are problems that require further study.
Changing Densities of INa and ICa in Pregnancy and Their Implications
Using small multicellular preparations from 16-21-d
pregnant rat uterus, Nakai and Kao (1983) described a
changing ratio of the Na+ and Ca2+ components of the
inward current, such that the ratio of peak INa/peak ICa
increased as term approached. Those observations are
now confirmed on single uterine myocytes, and extend
to cover the entire pregnancy.
Although Inoue and Sperelakis (1991) have made a
similar confirmation, they found no INa in any day-5 myocyte, and constant densities of INa and ICa in individual
myocytes from day 9 to term (their Fig. 5 A). They also
found that the fraction of myocytes expressing INa increased with the progression of pregnancy (their Fig. 5
B). By interpreting the frequency of their observing INa as a probability of INa occurrence, and by averaging
data from all cells, including all day-5 cells with no INa,
they concluded that the density of INa increased towards
term (their Fig. 5 C).
Our observations are quite different. We found INa in myocytes from all stages of pregnancy, from day 2 to term, and also in those from nonpregnant uteri under estrogen stimulation. Although only half of the day-2 myocytes had recordable INa, the finding of any INa indicates that conditions for the phenotypic expression of Na+ channels were already in place. Whereas a specific ionic current indicates both the presence and the expression of that channel, its absence only indicates the nonexpression of the channel and not its nonexistence. Because of this consideration, and because of our belief that population phenomena should be supported by more inclusive random sampling than can be provided by the usual limited sampling of whole-cell patch-clamp studies, we focused our interest on the properties of individual myocytes and compared only those myocytes in which both INa and ICa were recorded. Our data show that, in individual myocytes, densities of both INa and ICa change during the course of pregnancy.
Two broad categories of regulation by ovarian hormones can account for the observed changes: genomic
and nongenomic influences. Considering the fourfold
increase of the surface area in the hypertrophied uterine myocyte during pregnancy, new channel proteins
must be synthesized at a rate exceeding that needed for
replacement, because the densities of INa and ICa are always more than a quarter of the original level in the
nonpregnant myocyte. In the simplest sense, these increased current densities might be attributed to estrogen-enhanced transcription, leading to more copies of
Na+ and Ca2+ channels (genomic influence). However,
the regulation may be more complex, and may include
significant contributions from nongenomic influences.
For Na+ channels, it involves bringing forth a previously unexpressed phenotype (as in the estrus-diestrus
phase of nonpregnant uteri), and then rapidly extinguishing it (as in postpartum uteri). The decline in the
densities of INa and ICa in midpregnancy may involve progesterone, which is known to rise at that time and
has an antiestrogen effect on the expression of a K+
channel (Yang et al., 1994). Whatever the regulatory
mechanism might be, the observed changes are probably physiological, because the envelope of their effects
has already been observed in multicellular preparations
(Nakai and Kao, 1983
) that contained enough individual myocytes to provide a reasonably inclusive random
sampling.
The increased role of INa in late pregnancy subserves
well the physiological functions of the parturient uterus.
The more intense sodium currents could contribute to
a faster spread of the electrical impulse throughout the
parturient uterus. More importantly, the faster inactivation and reactivation of INa would permit more frequent repetitive spike discharges than would be possible with the slower ICa. Furthermore, because of a concomitant "de-expression" of some large-conductance
Ca2+-activated K+ channels (Kao et al., 1989; and Wang,
S.Y., M. Yoshino, and C.Y. Yao, manuscript submitted
for publication), the membrane conductance is lowered, and the membrane potential less negative than
otherwise. Perhaps, through a combination of these
processes, the excitability of the parturient uterus is enhanced and coordinated across large areas of the whole
organ to facilitate contraction throughout.
Address correspondence to Dr. C.Y. Kao, Department of Pharmacology (Box 29), SUNY Health Science Center, 450 Clarkson Ave., Brooklyn, NY 11203. Fax: 718-270-3309.
Received for publication 6 January 1997 and accepted in revised form 12 September 1997.
1 Abbreviations used in this paper: I-V, current-voltage; TEA, tetraethylammonium chloride; TTX, tetrodotoxin.This work was supported in part by grants from the National Institutes of Health (HD-00378 and DK-39371).
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