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Passive membrane properties and inward calcium current of human uterine smooth muscle cells
Passive membrane properties and inward calcium current of human uterine smooth muscle cells Roger C. Young, MD, PhD, Lea Herndon Smith, and Nels C. Anderson,Jr., PhD Durham, North Carolina, and Charleston, South Carolina Human myometrium was obtained at the time of cesarean delivery by means of excisional biopsy from the upper margin of the uterine incision through the lower uterine segment. Isolated smooth muscle cells were obtained by collagenase treatment. With the whole-cell patch clamp technique, voltage-clamp and current-clamp experiments were performed. Regenerative action potentials were seen in 3 mmollL calcium bathing solution. Voltage-clamp experiments demonstrated the presence of transient inward currents and large outward currents. Inward currents were isolated with the use of tetraethylammonium ion as a blocking agent of outward current. In 3 mmollL calcium inward current began at - 60 mV and maximum inward current occurred at - 40 mV. Maximum inward current density during the rising phase of the action potential was 1.0 ILA/cm 2 in 3 mmollL calcium and was unaffected by reduction of sodium concentration in the bathing solution. (AM J OBSTET GYNECOL 1991 ;164:1132-9.)
Key words: Human uterine smooth muscle, electrophysiology, voltage-activated calcium channel Membrane excitability properties play an important role in the control of uterine contractility. I The rise in intracellular calcium that is necessary for smooth muscle contraction is a direct result of an action potential, which activates calcium ion flow across the surface membrane and possibly triggers release of intracellular calcium stores .•-. Single-channel calcium conductances have recently been observed 5 in human uterine tissue with the use of the patch clamp technique.6 The observation of two distinct conductance levels implied the existence of two types of calcium channels. We report here a characterization, including activation and inactivation properties, of the whole-cell calcium currents that were observed in human uterine smooth muscle cells freshly isolated from term pregnant women. Material and methods Preparation of cells. Cell isolation techniques were similar to those reported previously.7 Small strips of human myometrium (0.5 cm x 0.5 cm x 2.0 cm) were excised from the upper margin of the uterine incision through the lower uterine segment at the time of ceFrom the Division of Physiology, Department of Cell Biology, Duke University Medical Center, and the Department of Obstetrics and Gynecology, Medical University of South Carolina. This work was supported by grant HD-00827 from the National Institute of Child Health and Human Development. Received for publication March 5, 1990; revised September 14, 1990; accepted October 29, 1990. Reprint requests: Roger C. Young, MD, PhD, Department of Obstetrics and Gynecology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. 611 126410
1132
sarean delivery (Medical University of South Carolina Institutional Review Board Approval No. 3605). The tissue was transported and stored for up to 2 hours in 120 mmol/L sodium chloride, 5 mmol/L potassium chloride, 10 mmol/L HEPES (N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid) buffer at pH 7.2. The serosa and endometrium were dissected free and discarded. The myometrium was minced into 1 mm cubes and washed in calcium-free, magnesium-free Hank's balanced salt solution (Sigma Chemical Co., St. Louis). The washed myometrial pieces were placed into an enzyme solution that was composed of 0.4% crude collagenase (type CLS 3, Worthington, Freehold, N.].) in calcium-free magnesium-free Hank's balanced salt solution and agitated at 35° C for 20 minutes . The tissue was then placed into fresh enzyme solution under the same conditions for 30 minutes. The enzyme solution was then removed and the partially digested tissue was gently washed in 120 mmol/L sodium chloride, 5 mmol/L potassium chloride, 10 mmol/L HEPES buffer at pH 7.2. Free cells were liberated by repeated trituration of the tissue through a Pasteur pipette. These freshly dispersed cells are large, typically 150 to 300 JLm in length and 10 to 15 JLm in width. An aliquot of solution that contained free cells was then placed into the recording chamber. Most cells appeared to be elongated, with length-to-width ratios of approximately 20: 1, and exhibited tapered ends. Cells with rounded ends or low length-to-width ratios were not considered appropriate for study. Recording. The myometrial cells were allowed to settle and adhere to the glass bottom of the recording chamber. The recording chamber was then flooded
Electrophysiology of human uterine myometrium
Volume 164 I\umber 4
1133
2.0 1.5 -
« .s
E 1.0
~ ::I t.l
0.5 0
o I -100
20 I -80
40
I
60
80
I -60
-40
I -20
I
I
100
I 0
120 (msec) I 20 (mV)
Fig. 1. Whole-cell voltage-clamp experiment. Current responses to voltage ramp. Bathing solution I (:\ mmollL calci um . 0 mmol / L TEA +j. Holding potential is - 100 mY.
with an electrolyte bathing solution that was selected from the group of solutions in Table I. Cells were observed with a Zeiss Axiovert 35 inverted phase-contrast microscope (Carl Zeiss Inc., Thornwood . N.Y.). Electrodes were pulled on a horizontal puller (Flaming-Brown, Sutter Instruments , San Rafael, Calif.) with borosilicate capillary tubes. Electrode resistances ranged from 4 to 7 Mn when filled with electrolyte solution (solution 3, Table I) . Patch clamp recordings were performed with the Axopatch I-B amplifier (Axon Instruments, Foster City, Calif.). A 1 cn feedback resistor was used. and data were filtered through a four-pole Bessel filter that was set at 2 kHz cutoff. After formation of a gigaohm seal between the patch pipette and the cell membrane, intracellular access was accomplished with moderate suction on the pipette at a holding potential of - 60 m V under voltage-clamp conditions. Data were digitized with the Axolab J 100 data acquisition system (Axon Instruments) and analyzed with the p-CLAMP software (Axon Instruments) on a Compaq 286 computer (Com pac Computer Corp., Houston). All recordings were made at 20° to 25° C. Electrode capacitance and stray capacitance were electrically compensated; however, capacitive currents as a result of cell membrane capacitance were not compensated . Voltage-clamp data were obtained by application of voltage-step or voltage-ramp commands from the holding potential to test potentials while the membrane current was monitored. By standard convention, positive ions that flow into the cell are defined as inward current and are represented by a downward deflection of current. The magnitude and time course of membrane currents were stable over time (up to 1 hour). An interval of at leas t 2 seconds between test pulses was necessary for the cell to reequilibrate and to avoid inactivation of membrane currents. For data reported here ,
Table I. Composition of solutions in millimoles per liter Bath
Sodium chloride Potassium chloride Calcium chloride TEA + HEPES EGTA pH
Solution
11 Solution 2
120 5 3 0 10
60 5 3 60 10
7.2
7.2
Pipette -Solution 3
5 120 0.1 10 I
7.2
pH was adjusted to 7.2 :t 0.05 with sodium hydroxide for bathing solutions and with potassi um h ydrox ide for the pipette solution. EGTA . Eth yleneglycol-bis-(f:l-aminoeth yletherjN,N,N',N'-telraacetic acid.
5 seconds was generally allowed as a convenient interval between test pulses. Current-voltage curves were obtained by plotting observed currents against test potentials. Membrane currents were measured from the holding level, and unless specifically noted, were not corrected for leak. Da ta for inactivation curves were obtained with the double-pulse technique-a varying conditioning potential that was immediately followed by a constant test potential. These currents were corrected for leak before analysis. Curve-fitting of the inactivation curves was performed with Marquardt nonlinear regression with the aid of STATCRAPHICS software (STSC. Inc., Rockville, Md.). The equation fit was the Hodgkin-Huxley formulation 8 for inactivation: I;)I;n(max) = (l + exp[(V - Vo;)/kW' Fig. 1 demonstrates the use of a voltage ramp to determine membrane capacitance. At the beginning of the tracing the holding potential is - 100 m V. At time 0, a voltage ramp of 1.07 V Isec is begun and applied for 140 msec. Be-
1134 Young, Smith, and Anderson
April 1991 Am J Obstet Gynecol
-40 -60 >-80 E ~
~-100
(5
>
-120
o
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Time (sec)
Fig_ 2. Passive membrane potential response in the current clamp experiment. Bathing solution I (3 mmollL calcium, 0 mmollL TEA +). A small hyperpolarizing current is applied to the cell lO establish a resting membrane potential of -75 m V.
the voltage ramp between - 90 to - 60 m V through the relation Rm = IlE/lli. Unit membrane resistivities are membrane resistances that are corrected for cell surface area. After the voltage-clamp experiments, current-clamp experiments were performed. Membrane potentials at zero applied current varied between - 30 and -70 m V. Usually a small hyperpolarizing current was required to maintain the membrane potential in the - 70 to - 80 mV range.
-80 -85
:> .§.
-90 -95
Q)
0>
~
(5
>
-100 -105 -1 10 -115
Results
o
20
40
60
80
100
Time (msec)
Fig. 3. Best-fit single exponential function (solid line) of voltage relaxation from data in Fig. 2.
tween - 100 and - 60 m V the response of the cell membrane is "passive" in that the electrical properties of the cell membrane can be described by a resistor (Rm) in parallel with a capacitor (Cm). The current response to the beginning of the voltage ramp is the charging of the membrane capacitance through the electrode resistance (Re) and is the current step that is seen at time 0 in Fig. I. The capacitance is calculated from the height of the current step (Ili) through the relation Ili = Cm x Il V lilt, where Il V lilt is the ramp rate (1.07 V I sec). For the purpose of approximation of cell membrane surface area, we assume a unit membrane capacitance of 1.0 microfarads per square centimeter, which is found in other smooth muscle preparations,9 and calculate the surface area from the capacitance measurement. Membrane resistances were calculated from the slope of the current response to
Passive membrane properties. Surface membrane capacitance and resistance measurements of freshly dispersed human uterine cells were obtained from voltage-ramp experiments (Fig. I). The surface area of six cells varied between 8.0 X 10" and 2.9 x 10-' cm 2 • Measured membrane resistance values varied between I and 2 eil for those cells. Calculated resistivity varied between 0.8 and 2.25 x 105 il-cm 2 • Fig. 2 shows results from a current-damp experiment and demonstrates membrane potential response to a hyperpolarizing current pulse. Fig. 3 shows the exponential fit of the relaxation phase of the membrane potential response that is shown in Fig. 2. This close fit is predicted by the equivalent circuit and indicates that the cell membrane properties are passive in this voltage range. Membrane action potentials. Under conditions of 3mmollL calcium, action potentials could be elicited with depolarizing current pulses or after a hyperpolarizing pulse (Fig. 4). The cell membrane voltage responses are pure expressions of membrane ion channel activity only under conditions in which the holding current is zero. Therefore analysis of action potentials was limited to those that were elicited by hyperpolarizing
Electrophysiology of human uterine myometrium
Volume 164 Number 4
1135
A 20 0
>E
-20 -40
~
~
-60
~
0
>
-80 -100
.. 40 PAIr
A
.
t
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1.0
3.0
2.0
4.0
Time (sec)
B
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~
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'0
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~ >
-80 -100
."..,."..~
20PAln......___......
8
o
40..1-_, ........
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~ 4.0
lime (sec)
Fig. 4. Action potentials in the current-clamp experiment. A, Bathing solution I (3 mmollL calcium, TEA +). A 40 pA hyperpolarizing current pulse is applied for 750 msec. B, Same cell, with bathing solution changed to solution 2 (3 mmol/L calcium, 60 mmollL TEA +).
o mmollL
current pulses (anodal break excitation) or action potentials that occurred after depolarizing current pulses. Without the use of blocking agents, the maximum depolarization that was observed in six cells was - 17 m V (Fig. 4, A), and the maximal rate of voltage rise was 1.0 ± 0.2 V I sec for that cell. This voltage rise was the result of inward current that charged the membrane capacitance and is described by the relation i = em x dV/dt. Thus, with theuseofl f.LF/cm2,the corresponding current density is 1.0 f.LA/cm 2 • With exchange of bathing solution so that TEA + (tetraethylammonium ion) (60 mmollL) was substituted for sodium, the action potential duration lengthened, and the maximum depolarization increased to + 20 m V
(Fig. 4, B). However, the maximal rate of voltage rise for the action potential remained essentially unchanged at 0.79 V I sec. For three cells an average of 0.8 ± 0.2 V Isec was observed. Action potential generation results from a complex interplay of ion channel opening and closing in response to membrane potential changes, calcium ion concentrations, and other parameters that affect membrane excitability properties. To help delineate the contribu tion of the calcium channel to the excitability properties of the membrane, whole-cell voltage clamp analysis was undertaken. Voltage clamp analysis. In 3 mmollL calcium under voltage-clamp conditions (Fig. 5), these cells demon-
1136 Young, Smith, and Anderson
>E ~
April 1991 Am J Obstet Gynecol
20 -80 -100
2.0 1.5 1.0 ;(
..s
0.5
c
~
:;
0
0
-0.5 -1.0
o
200
400
600
time (msec)
Fig. 5. Whole-cell voltage-clamp experiment. Current responses to stepped voltages. Bathing solution I (3 mmollL calcium, 0 mmollL TEA +). Holding potential is - 80 mY.
2.0 1.S
<" .E..
E 1.0 ~
0
0.5
a
-100
20 I -80
40
I -60
60
I
-40
I
I
80
100
I
-20
I
120 (msec)
I
0
I
20 (mV)
Fig. 6. Current responses to voltage ramp. Bathing solution 2 (3 mmollL calcium, 60 mmollL TEA +). Data were obtained from same cell as in Fig. I.
strated complex currents. Outward currents in excess of 1 nA dominated inward currents, which made quantitative analysis of inward currents difficult. To isolate the inward calcium currents for study, outward currents were reduced by the use of TEA + (60 mmol/L) in the bathing solution. 10. 11 The effect of TEA + (60 mmoll L) on human uterine myocytes is most easily observed in the voltage-ramp experiment (compare Fig. 6 with Fig. 1). Between -60 and - 20 m V, similar inward currents occur in both the absence (Fig. I) and the presence (Fig. 6) of TEA + . At potentials above - 20 m V, outward current rises rapidly, as shown in Fig. 1. This outward current is inhib-
ited by TEA + (Fig. 6) and is consistent with blocking of potassium channels by TEA + . Current responses of the cell to stepped voltages with the TEA + (60 mmoll L) bathing solution a re shown in Fig. 7. With the reduction in outward currents, inward currents are more easily observed. The resulting current-vollage curve (Fig. 8) of the peak inward current demonstrates that activation of the inward current begins at - 60 m V and maximal inward current occurs at -40 mY. The value of 275 pA for maximal inward current corresponds to a current density of 1.1 J.LA/cm 2 (measured membrane capacitance was 240 picofarads for this cell). This is in good agreement with
Electrophysiology of human uterine myometrium
Volume 164
l'\umbcr 4
>
E
1137
20 -80 -100
0.5 0.4 0.3 0.2
<" c
0.1
C
0
~
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0
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I 0
100
200
300
Time (msec)
Fig. 7. Current responses to stepped voltages. Bathing solution 2 (3 mmollL calcium , 60 mmol/L TEA +). Holding potential and voltage protocol are identical to those in Fig. 5.
0.79 f.l.A / cm 2 , which was observed from the action po-
tential of that cell. Voltage inactivation of the inward current was performed by application of varying conditioning potentials for 250 msec followed immediately by a constant test pulse to - 40 m V. A plot of the current responses (0 the test pulse versus conditioning potentials yields the Hodgkin-Huxley inactivation curve" (Fig. 9) with V(I12) == -54.9 mV and k = 4.5 mv. Comment
The experiments that are described here were performed on freshly dispersed pregnant human uterine smooth muscle cells. These cells readily formed gigaohm seals with patch pipettes and demonstrated reproducible passive and active properties under wholecell voltage and current-clamp conditions. These experiments were performed on cells that were obtained from the lower uterine segment. Although the ideal tissue to study would be fundal tissue, those surgical specimens are difficult to obtain . Some elements of membrane excitability, such as channel density or receptor density, would be expected to vary depending on the site. However, the major features of membrane excitablity are the expression and regulation of ionspecific channels and are likely to be similar whether they originate in the fundus or the lower uterine segment. Membrane resistivity. With the cell surface area as determined by capacitance measurement, membrane
400
o + 200
+ +
o
•
•
•
•
+ + o
+
+
o
+
o o
·200
o o
o
·400 +-~.-~-r~--.~~r-"'-~-'--'--'--r~--.~ · 120 ·100 · 80 · 60 · 40 · 20 0 20 40
voltage (mV)
Fig. 8. Current-voltage relationship o f data In Fig. 7 (3 mmollL calcium, 60 mmoliL TEA +). D, Peak inward current: + , current measured at 290 msec. The apparent reversal of inward current is artifact of decay of residual outward current.
resistivity in excess of 10 5 n-cm 2 in 3 mmollL calcium was observed . The relatively large range among the six cells that are reported on here (from 0.8 to 2.25 x 10' n-cm Z) is in part explained by difficulties in the determination of accurate measurements of very large resistances. Although pipette seal resistances were > 5 cn, when I to 2 cn was measured, as much as a third of the current that was passed could be shunted by leakage of current from the electrode to [he bath. Thus
1138 Young, Smith, and Anderson
April 1991 Am J Obstet Gynecol
o
o
0.8
o
o
o
o
e 0.6 ><
X
0.4
0.2
o
o -100
-80
-60
-40
-20
millivolts Fig. 9. Hodgkin-Huxley inactivation curves of peak inward current. Data were obtained from same cell that was used to obtain data shown in Figs. 7 and 8. Best fit of data (solid line) yields V 112 = - 54.5 mY, K = 4.5 mY.
measured values of membrane resistivities should be interpreted as a lower limit. In addition , any significant cell damage during the isolation procedure would likely reduce measured membrane resistivity values. These values are similar to those that have been reported for visceral iO . 12 and vascular I S. 11 smooth muscle cells. The importance of the unit membrane passive properties in relation to the active membrane properties , hormonal stimulation, site of biopsy, previous labor, or other clinical parameters awaits systematic study. Inward current-the voltage-activated calcium channel. In agreement with many other smooth muscle systems, the inward currents that were observed in human myometrium are the expression of voltageactivated calcium channels. I The contribution of sodium currents" to the action potentials is probably not significant since replacement of half of the bathing solution sodium with TEA + increased rather than decreased the overshoot of the action potential (- 17 to + 20 m V). In addition, there was no significant change in the maximal rate of voltage rise of the action potential spike (1.0 vs 0.8 ± 0.2 V/sec) . Further voltageclamp studies in the 2 to to msec time frame will be required to determine whether sodium currents contribute to the action potential to any significant degree. Evidence for two types of calcium channels was recently reported.' In this work, the voltage inactivation of the inward current (Fig. 8) was well fit by the
Hodgkin-Huxley curve" with rapidly developing inactivation (k = 4.5 m V), which implies that the different types of calcium channels have similar, if not identical, inactivation characteristics. In the cells that were studied in this experiment, there were only minor differences among the currentvoltage relationships and the rate of decay of the inward current transients. Inward current was studied under ionic conditions that were as close to normal as possible. Specifically, a physiologic calcium concentration (3 mmol/L) was used to avoid artifactual changes of the current-voltage relationships that are noted in solutions with elevated calcium concentrations. 12 Importance of the resting potential. The beginning of the inward current activation (- 60 m V) is near the half-maximal inactivation ( - 54.9 m V). Thus this voltage range is crucial to the excitability of the cell membrane. The relatively short duration ofthe conditioning pulse (250 msec) in the inactivation protocol was chosen to ensure that inactivation of the calcium currents would occur on a time frame that was shorter than the time frame of slow-wave depolarization. I6 At resting membt'ane potentials that are more negative than - 60 m V, no calcium ions flow into the cell, but calcium channels would be activated on slight depolarization. At resting potentials in which most of the calcium channels are inactivated (around - 50 m V and more positive), no calcium ions flow into the cell and no action potentials would be generated regardless of further
Electrophysiology of human uterine myometrium
Volume 164 Number 4
depolarization. Thus a major controlling factor of the excitability of the membrane is the resting potential. Propagation of the action potential through a syncytium. The ability of an intact uterus to undergo a coordinated contraction presumably requires propagation of the action potential from the fundus to the lower uterine segment. The propagation mechanism likely involves the action potential of one myocyte that causes depolarization of adjacent cells and leads to a spread of action potentials. 17 This would require myometrial cells that are at the leading edge of the action potential to be at a resting potential more negative than -60 mY; otherwise, propagation would cease. The presence of gap junctions l7 between adjacent cells allows for electrical communication and tends to equate the membrane potentials of the cells. With significant numbers of gap junctions, an electrical syncytium is established, within which all of cells are near the same resting membrane potential. Thus we believe that gap junctions not only aid in the transmission of the action potential from cell to cell but are required to establish membrane potentials along a path of propagation that is sufficiently negative to prevent calcium channel inactivation. We thank Warren Smith for his assistance equivalent circuit analysis.
III
the
REFERENCES 1. Parkington HC, Coleman HA. Ionic mechanisms underlying action potentials in myometrium. Clin Exp Pharmacol Physiol 1988; 15:657-65. 2. lino M. Calcium-induced calcium release mechanism in guinea pig taenia caeci. J Gen Physiol 1989;94:363-83. 3. Anwer K, Sanborn BM. Changes in intracellular free calcium in isolated myometrial cells: role of extracellular and intracellular calcium and possible involvement of guanine nucleotide-sensitive proteins. Endocrinology 1989; 124: 17-23.
1139
4. Williams DA, Fay FS. Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin 2. Am J Physiol 1986;250(Cell Physiol 19):C779-91. 5. Inoue Y, Nakao K, Okabe K, et al. Some electrical properties of human pregnant myometrium. AMJ OBSTET GyNECOL 1990;162:1090-8. 6. Marty A, Neher E. Tight seal whole-cell recording. In: Sakmann B, Neher E, eds. Single channel recording. New York: Plenum Press, 1983 chap 7. 7. Pressman EK, Tucker JAJr, Anderson NCJr, Young RC. Morphologic and electro physiologic characterization of isolated pregnant human myometrial cells. AM J OBSTE1' GYNECOL 1988;159:1273-9. 8. Hodgkin AL, Huxley AF. The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J Physiol 1952; 116:497-506. 9. Singer JJ, Walsh JV Jr. Passive properties of the membrane of single freshly isolated smooth muscle cells. Am J Physiol 1980;239(Cell Physiol 8):CI53-61. 10. Benham CD, Bolton TB, Lang RJ, Takewaki T. The mechanism of action of Ba'+ and TEA on single Ca'+activated K + channels in arterial and intestinal smooth muscle cell membranes. PRugers Arch 1985;403: 120-7. 11. Stanfield PRo Tetraethylammonium ions and the potassium permeability of excitable cells. Rev Physiol Biochem PharmacoI1983;97:1-67. 12. Yamamoto Y, Hu SL, Kao CY. Inward current in single smooth muscle cells of the guinea pig taenia coli. J Gen Physiol 1989;93:521-50. 13. Bolton TB, Lang RJ, Takewaki T, Benham CD. Patch and whole-cell voltage-clamp studies on single smooth muscle cells. J Cardiovasc Pharmacol 1986;8(suppl 8):S20-4. 14. Bolton TB, Lang RJ, Takewaki T, Benham CD. Patch and whole-cell voltage clamp of single mamalian visceral and vascular smooth muscle cells. Experientia 1985;41:88794. 15. Ohya Y, Sperelakis N. Fast Na+ and slow Ca'+ channels in single uterine muscle cells from pregnant rats. Am J Physiol 1989;257(Cell Physiol 26):C408-12. 16. Kawarabayashi T, Kishikawa T, Sugimori H. Effect of oxytocin on spontaneous and mechanical activities in pregnant human myometrium. AM J OBS1'ET GYNECOL 1986;155:671-6. 17. Cole WC, Garfield RE, Kirkaldy JS. Gap junctions and direct intercellular communication between rat uterine smooth muscle cells. Am J Physiol 1985;249(Cell Physiol 18):C20-31.
Key words: Human uterine smooth muscle, electrophysiology, voltage-activated calcium channel Membrane excitability properties play an important role in the control of uterine contractility. I The rise in intracellular calcium that is necessary for smooth muscle contraction is a direct result of an action potential, which activates calcium ion flow across the surface membrane and possibly triggers release of intracellular calcium stores .•-. Single-channel calcium conductances have recently been observed 5 in human uterine tissue with the use of the patch clamp technique.6 The observation of two distinct conductance levels implied the existence of two types of calcium channels. We report here a characterization, including activation and inactivation properties, of the whole-cell calcium currents that were observed in human uterine smooth muscle cells freshly isolated from term pregnant women. Material and methods Preparation of cells. Cell isolation techniques were similar to those reported previously.7 Small strips of human myometrium (0.5 cm x 0.5 cm x 2.0 cm) were excised from the upper margin of the uterine incision through the lower uterine segment at the time of ceFrom the Division of Physiology, Department of Cell Biology, Duke University Medical Center, and the Department of Obstetrics and Gynecology, Medical University of South Carolina. This work was supported by grant HD-00827 from the National Institute of Child Health and Human Development. Received for publication March 5, 1990; revised September 14, 1990; accepted October 29, 1990. Reprint requests: Roger C. Young, MD, PhD, Department of Obstetrics and Gynecology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. 611 126410
1132
sarean delivery (Medical University of South Carolina Institutional Review Board Approval No. 3605). The tissue was transported and stored for up to 2 hours in 120 mmol/L sodium chloride, 5 mmol/L potassium chloride, 10 mmol/L HEPES (N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid) buffer at pH 7.2. The serosa and endometrium were dissected free and discarded. The myometrium was minced into 1 mm cubes and washed in calcium-free, magnesium-free Hank's balanced salt solution (Sigma Chemical Co., St. Louis). The washed myometrial pieces were placed into an enzyme solution that was composed of 0.4% crude collagenase (type CLS 3, Worthington, Freehold, N.].) in calcium-free magnesium-free Hank's balanced salt solution and agitated at 35° C for 20 minutes . The tissue was then placed into fresh enzyme solution under the same conditions for 30 minutes. The enzyme solution was then removed and the partially digested tissue was gently washed in 120 mmol/L sodium chloride, 5 mmol/L potassium chloride, 10 mmol/L HEPES buffer at pH 7.2. Free cells were liberated by repeated trituration of the tissue through a Pasteur pipette. These freshly dispersed cells are large, typically 150 to 300 JLm in length and 10 to 15 JLm in width. An aliquot of solution that contained free cells was then placed into the recording chamber. Most cells appeared to be elongated, with length-to-width ratios of approximately 20: 1, and exhibited tapered ends. Cells with rounded ends or low length-to-width ratios were not considered appropriate for study. Recording. The myometrial cells were allowed to settle and adhere to the glass bottom of the recording chamber. The recording chamber was then flooded
Electrophysiology of human uterine myometrium
Volume 164 I\umber 4
1133
2.0 1.5 -
« .s
E 1.0
~ ::I t.l
0.5 0
o I -100
20 I -80
40
I
60
80
I -60
-40
I -20
I
I
100
I 0
120 (msec) I 20 (mV)
Fig. 1. Whole-cell voltage-clamp experiment. Current responses to voltage ramp. Bathing solution I (:\ mmollL calci um . 0 mmol / L TEA +j. Holding potential is - 100 mY.
with an electrolyte bathing solution that was selected from the group of solutions in Table I. Cells were observed with a Zeiss Axiovert 35 inverted phase-contrast microscope (Carl Zeiss Inc., Thornwood . N.Y.). Electrodes were pulled on a horizontal puller (Flaming-Brown, Sutter Instruments , San Rafael, Calif.) with borosilicate capillary tubes. Electrode resistances ranged from 4 to 7 Mn when filled with electrolyte solution (solution 3, Table I) . Patch clamp recordings were performed with the Axopatch I-B amplifier (Axon Instruments, Foster City, Calif.). A 1 cn feedback resistor was used. and data were filtered through a four-pole Bessel filter that was set at 2 kHz cutoff. After formation of a gigaohm seal between the patch pipette and the cell membrane, intracellular access was accomplished with moderate suction on the pipette at a holding potential of - 60 m V under voltage-clamp conditions. Data were digitized with the Axolab J 100 data acquisition system (Axon Instruments) and analyzed with the p-CLAMP software (Axon Instruments) on a Compaq 286 computer (Com pac Computer Corp., Houston). All recordings were made at 20° to 25° C. Electrode capacitance and stray capacitance were electrically compensated; however, capacitive currents as a result of cell membrane capacitance were not compensated . Voltage-clamp data were obtained by application of voltage-step or voltage-ramp commands from the holding potential to test potentials while the membrane current was monitored. By standard convention, positive ions that flow into the cell are defined as inward current and are represented by a downward deflection of current. The magnitude and time course of membrane currents were stable over time (up to 1 hour). An interval of at leas t 2 seconds between test pulses was necessary for the cell to reequilibrate and to avoid inactivation of membrane currents. For data reported here ,
Table I. Composition of solutions in millimoles per liter Bath
Sodium chloride Potassium chloride Calcium chloride TEA + HEPES EGTA pH
Solution
11 Solution 2
120 5 3 0 10
60 5 3 60 10
7.2
7.2
Pipette -Solution 3
5 120 0.1 10 I
7.2
pH was adjusted to 7.2 :t 0.05 with sodium hydroxide for bathing solutions and with potassi um h ydrox ide for the pipette solution. EGTA . Eth yleneglycol-bis-(f:l-aminoeth yletherjN,N,N',N'-telraacetic acid.
5 seconds was generally allowed as a convenient interval between test pulses. Current-voltage curves were obtained by plotting observed currents against test potentials. Membrane currents were measured from the holding level, and unless specifically noted, were not corrected for leak. Da ta for inactivation curves were obtained with the double-pulse technique-a varying conditioning potential that was immediately followed by a constant test potential. These currents were corrected for leak before analysis. Curve-fitting of the inactivation curves was performed with Marquardt nonlinear regression with the aid of STATCRAPHICS software (STSC. Inc., Rockville, Md.). The equation fit was the Hodgkin-Huxley formulation 8 for inactivation: I;)I;n(max) = (l + exp[(V - Vo;)/kW' Fig. 1 demonstrates the use of a voltage ramp to determine membrane capacitance. At the beginning of the tracing the holding potential is - 100 m V. At time 0, a voltage ramp of 1.07 V Isec is begun and applied for 140 msec. Be-
1134 Young, Smith, and Anderson
April 1991 Am J Obstet Gynecol
-40 -60 >-80 E ~
~-100
(5
>
-120
o
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Time (sec)
Fig_ 2. Passive membrane potential response in the current clamp experiment. Bathing solution I (3 mmollL calcium, 0 mmollL TEA +). A small hyperpolarizing current is applied to the cell lO establish a resting membrane potential of -75 m V.
the voltage ramp between - 90 to - 60 m V through the relation Rm = IlE/lli. Unit membrane resistivities are membrane resistances that are corrected for cell surface area. After the voltage-clamp experiments, current-clamp experiments were performed. Membrane potentials at zero applied current varied between - 30 and -70 m V. Usually a small hyperpolarizing current was required to maintain the membrane potential in the - 70 to - 80 mV range.
-80 -85
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-90 -95
Q)
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-100 -105 -1 10 -115
Results
o
20
40
60
80
100
Time (msec)
Fig. 3. Best-fit single exponential function (solid line) of voltage relaxation from data in Fig. 2.
tween - 100 and - 60 m V the response of the cell membrane is "passive" in that the electrical properties of the cell membrane can be described by a resistor (Rm) in parallel with a capacitor (Cm). The current response to the beginning of the voltage ramp is the charging of the membrane capacitance through the electrode resistance (Re) and is the current step that is seen at time 0 in Fig. I. The capacitance is calculated from the height of the current step (Ili) through the relation Ili = Cm x Il V lilt, where Il V lilt is the ramp rate (1.07 V I sec). For the purpose of approximation of cell membrane surface area, we assume a unit membrane capacitance of 1.0 microfarads per square centimeter, which is found in other smooth muscle preparations,9 and calculate the surface area from the capacitance measurement. Membrane resistances were calculated from the slope of the current response to
Passive membrane properties. Surface membrane capacitance and resistance measurements of freshly dispersed human uterine cells were obtained from voltage-ramp experiments (Fig. I). The surface area of six cells varied between 8.0 X 10" and 2.9 x 10-' cm 2 • Measured membrane resistance values varied between I and 2 eil for those cells. Calculated resistivity varied between 0.8 and 2.25 x 105 il-cm 2 • Fig. 2 shows results from a current-damp experiment and demonstrates membrane potential response to a hyperpolarizing current pulse. Fig. 3 shows the exponential fit of the relaxation phase of the membrane potential response that is shown in Fig. 2. This close fit is predicted by the equivalent circuit and indicates that the cell membrane properties are passive in this voltage range. Membrane action potentials. Under conditions of 3mmollL calcium, action potentials could be elicited with depolarizing current pulses or after a hyperpolarizing pulse (Fig. 4). The cell membrane voltage responses are pure expressions of membrane ion channel activity only under conditions in which the holding current is zero. Therefore analysis of action potentials was limited to those that were elicited by hyperpolarizing
Electrophysiology of human uterine myometrium
Volume 164 Number 4
1135
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Fig. 4. Action potentials in the current-clamp experiment. A, Bathing solution I (3 mmollL calcium, TEA +). A 40 pA hyperpolarizing current pulse is applied for 750 msec. B, Same cell, with bathing solution changed to solution 2 (3 mmol/L calcium, 60 mmollL TEA +).
o mmollL
current pulses (anodal break excitation) or action potentials that occurred after depolarizing current pulses. Without the use of blocking agents, the maximum depolarization that was observed in six cells was - 17 m V (Fig. 4, A), and the maximal rate of voltage rise was 1.0 ± 0.2 V I sec for that cell. This voltage rise was the result of inward current that charged the membrane capacitance and is described by the relation i = em x dV/dt. Thus, with theuseofl f.LF/cm2,the corresponding current density is 1.0 f.LA/cm 2 • With exchange of bathing solution so that TEA + (tetraethylammonium ion) (60 mmollL) was substituted for sodium, the action potential duration lengthened, and the maximum depolarization increased to + 20 m V
(Fig. 4, B). However, the maximal rate of voltage rise for the action potential remained essentially unchanged at 0.79 V I sec. For three cells an average of 0.8 ± 0.2 V Isec was observed. Action potential generation results from a complex interplay of ion channel opening and closing in response to membrane potential changes, calcium ion concentrations, and other parameters that affect membrane excitability properties. To help delineate the contribu tion of the calcium channel to the excitability properties of the membrane, whole-cell voltage clamp analysis was undertaken. Voltage clamp analysis. In 3 mmollL calcium under voltage-clamp conditions (Fig. 5), these cells demon-
1136 Young, Smith, and Anderson
>E ~
April 1991 Am J Obstet Gynecol
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time (msec)
Fig. 5. Whole-cell voltage-clamp experiment. Current responses to stepped voltages. Bathing solution I (3 mmollL calcium, 0 mmollL TEA +). Holding potential is - 80 mY.
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60
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80
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I
120 (msec)
I
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20 (mV)
Fig. 6. Current responses to voltage ramp. Bathing solution 2 (3 mmollL calcium, 60 mmollL TEA +). Data were obtained from same cell as in Fig. I.
strated complex currents. Outward currents in excess of 1 nA dominated inward currents, which made quantitative analysis of inward currents difficult. To isolate the inward calcium currents for study, outward currents were reduced by the use of TEA + (60 mmol/L) in the bathing solution. 10. 11 The effect of TEA + (60 mmoll L) on human uterine myocytes is most easily observed in the voltage-ramp experiment (compare Fig. 6 with Fig. 1). Between -60 and - 20 m V, similar inward currents occur in both the absence (Fig. I) and the presence (Fig. 6) of TEA + . At potentials above - 20 m V, outward current rises rapidly, as shown in Fig. 1. This outward current is inhib-
ited by TEA + (Fig. 6) and is consistent with blocking of potassium channels by TEA + . Current responses of the cell to stepped voltages with the TEA + (60 mmoll L) bathing solution a re shown in Fig. 7. With the reduction in outward currents, inward currents are more easily observed. The resulting current-vollage curve (Fig. 8) of the peak inward current demonstrates that activation of the inward current begins at - 60 m V and maximal inward current occurs at -40 mY. The value of 275 pA for maximal inward current corresponds to a current density of 1.1 J.LA/cm 2 (measured membrane capacitance was 240 picofarads for this cell). This is in good agreement with
Electrophysiology of human uterine myometrium
Volume 164
l'\umbcr 4
>
E
1137
20 -80 -100
0.5 0.4 0.3 0.2
<" c
0.1
C
0
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100
200
300
Time (msec)
Fig. 7. Current responses to stepped voltages. Bathing solution 2 (3 mmollL calcium , 60 mmol/L TEA +). Holding potential and voltage protocol are identical to those in Fig. 5.
0.79 f.l.A / cm 2 , which was observed from the action po-
tential of that cell. Voltage inactivation of the inward current was performed by application of varying conditioning potentials for 250 msec followed immediately by a constant test pulse to - 40 m V. A plot of the current responses (0 the test pulse versus conditioning potentials yields the Hodgkin-Huxley inactivation curve" (Fig. 9) with V(I12) == -54.9 mV and k = 4.5 mv. Comment
The experiments that are described here were performed on freshly dispersed pregnant human uterine smooth muscle cells. These cells readily formed gigaohm seals with patch pipettes and demonstrated reproducible passive and active properties under wholecell voltage and current-clamp conditions. These experiments were performed on cells that were obtained from the lower uterine segment. Although the ideal tissue to study would be fundal tissue, those surgical specimens are difficult to obtain . Some elements of membrane excitability, such as channel density or receptor density, would be expected to vary depending on the site. However, the major features of membrane excitablity are the expression and regulation of ionspecific channels and are likely to be similar whether they originate in the fundus or the lower uterine segment. Membrane resistivity. With the cell surface area as determined by capacitance measurement, membrane
400
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+ +
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•
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o o
·200
o o
o
·400 +-~.-~-r~--.~~r-"'-~-'--'--'--r~--.~ · 120 ·100 · 80 · 60 · 40 · 20 0 20 40
voltage (mV)
Fig. 8. Current-voltage relationship o f data In Fig. 7 (3 mmollL calcium, 60 mmoliL TEA +). D, Peak inward current: + , current measured at 290 msec. The apparent reversal of inward current is artifact of decay of residual outward current.
resistivity in excess of 10 5 n-cm 2 in 3 mmollL calcium was observed . The relatively large range among the six cells that are reported on here (from 0.8 to 2.25 x 10' n-cm Z) is in part explained by difficulties in the determination of accurate measurements of very large resistances. Although pipette seal resistances were > 5 cn, when I to 2 cn was measured, as much as a third of the current that was passed could be shunted by leakage of current from the electrode to [he bath. Thus
1138 Young, Smith, and Anderson
April 1991 Am J Obstet Gynecol
o
o
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o
o
o
o
e 0.6 ><
X
0.4
0.2
o
o -100
-80
-60
-40
-20
millivolts Fig. 9. Hodgkin-Huxley inactivation curves of peak inward current. Data were obtained from same cell that was used to obtain data shown in Figs. 7 and 8. Best fit of data (solid line) yields V 112 = - 54.5 mY, K = 4.5 mY.
measured values of membrane resistivities should be interpreted as a lower limit. In addition , any significant cell damage during the isolation procedure would likely reduce measured membrane resistivity values. These values are similar to those that have been reported for visceral iO . 12 and vascular I S. 11 smooth muscle cells. The importance of the unit membrane passive properties in relation to the active membrane properties , hormonal stimulation, site of biopsy, previous labor, or other clinical parameters awaits systematic study. Inward current-the voltage-activated calcium channel. In agreement with many other smooth muscle systems, the inward currents that were observed in human myometrium are the expression of voltageactivated calcium channels. I The contribution of sodium currents" to the action potentials is probably not significant since replacement of half of the bathing solution sodium with TEA + increased rather than decreased the overshoot of the action potential (- 17 to + 20 m V). In addition, there was no significant change in the maximal rate of voltage rise of the action potential spike (1.0 vs 0.8 ± 0.2 V/sec) . Further voltageclamp studies in the 2 to to msec time frame will be required to determine whether sodium currents contribute to the action potential to any significant degree. Evidence for two types of calcium channels was recently reported.' In this work, the voltage inactivation of the inward current (Fig. 8) was well fit by the
Hodgkin-Huxley curve" with rapidly developing inactivation (k = 4.5 m V), which implies that the different types of calcium channels have similar, if not identical, inactivation characteristics. In the cells that were studied in this experiment, there were only minor differences among the currentvoltage relationships and the rate of decay of the inward current transients. Inward current was studied under ionic conditions that were as close to normal as possible. Specifically, a physiologic calcium concentration (3 mmol/L) was used to avoid artifactual changes of the current-voltage relationships that are noted in solutions with elevated calcium concentrations. 12 Importance of the resting potential. The beginning of the inward current activation (- 60 m V) is near the half-maximal inactivation ( - 54.9 m V). Thus this voltage range is crucial to the excitability of the cell membrane. The relatively short duration ofthe conditioning pulse (250 msec) in the inactivation protocol was chosen to ensure that inactivation of the calcium currents would occur on a time frame that was shorter than the time frame of slow-wave depolarization. I6 At resting membt'ane potentials that are more negative than - 60 m V, no calcium ions flow into the cell, but calcium channels would be activated on slight depolarization. At resting potentials in which most of the calcium channels are inactivated (around - 50 m V and more positive), no calcium ions flow into the cell and no action potentials would be generated regardless of further
Electrophysiology of human uterine myometrium
Volume 164 Number 4
depolarization. Thus a major controlling factor of the excitability of the membrane is the resting potential. Propagation of the action potential through a syncytium. The ability of an intact uterus to undergo a coordinated contraction presumably requires propagation of the action potential from the fundus to the lower uterine segment. The propagation mechanism likely involves the action potential of one myocyte that causes depolarization of adjacent cells and leads to a spread of action potentials. 17 This would require myometrial cells that are at the leading edge of the action potential to be at a resting potential more negative than -60 mY; otherwise, propagation would cease. The presence of gap junctions l7 between adjacent cells allows for electrical communication and tends to equate the membrane potentials of the cells. With significant numbers of gap junctions, an electrical syncytium is established, within which all of cells are near the same resting membrane potential. Thus we believe that gap junctions not only aid in the transmission of the action potential from cell to cell but are required to establish membrane potentials along a path of propagation that is sufficiently negative to prevent calcium channel inactivation. We thank Warren Smith for his assistance equivalent circuit analysis.
III
the
REFERENCES 1. Parkington HC, Coleman HA. Ionic mechanisms underlying action potentials in myometrium. Clin Exp Pharmacol Physiol 1988; 15:657-65. 2. lino M. Calcium-induced calcium release mechanism in guinea pig taenia caeci. J Gen Physiol 1989;94:363-83. 3. Anwer K, Sanborn BM. Changes in intracellular free calcium in isolated myometrial cells: role of extracellular and intracellular calcium and possible involvement of guanine nucleotide-sensitive proteins. Endocrinology 1989; 124: 17-23.
1139
4. Williams DA, Fay FS. Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin 2. Am J Physiol 1986;250(Cell Physiol 19):C779-91. 5. Inoue Y, Nakao K, Okabe K, et al. Some electrical properties of human pregnant myometrium. AMJ OBSTET GyNECOL 1990;162:1090-8. 6. Marty A, Neher E. Tight seal whole-cell recording. In: Sakmann B, Neher E, eds. Single channel recording. New York: Plenum Press, 1983 chap 7. 7. Pressman EK, Tucker JAJr, Anderson NCJr, Young RC. Morphologic and electro physiologic characterization of isolated pregnant human myometrial cells. AM J OBSTE1' GYNECOL 1988;159:1273-9. 8. Hodgkin AL, Huxley AF. The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J Physiol 1952; 116:497-506. 9. Singer JJ, Walsh JV Jr. Passive properties of the membrane of single freshly isolated smooth muscle cells. Am J Physiol 1980;239(Cell Physiol 8):CI53-61. 10. Benham CD, Bolton TB, Lang RJ, Takewaki T. The mechanism of action of Ba'+ and TEA on single Ca'+activated K + channels in arterial and intestinal smooth muscle cell membranes. PRugers Arch 1985;403: 120-7. 11. Stanfield PRo Tetraethylammonium ions and the potassium permeability of excitable cells. Rev Physiol Biochem PharmacoI1983;97:1-67. 12. Yamamoto Y, Hu SL, Kao CY. Inward current in single smooth muscle cells of the guinea pig taenia coli. J Gen Physiol 1989;93:521-50. 13. Bolton TB, Lang RJ, Takewaki T, Benham CD. Patch and whole-cell voltage-clamp studies on single smooth muscle cells. J Cardiovasc Pharmacol 1986;8(suppl 8):S20-4. 14. Bolton TB, Lang RJ, Takewaki T, Benham CD. Patch and whole-cell voltage clamp of single mamalian visceral and vascular smooth muscle cells. Experientia 1985;41:88794. 15. Ohya Y, Sperelakis N. Fast Na+ and slow Ca'+ channels in single uterine muscle cells from pregnant rats. Am J Physiol 1989;257(Cell Physiol 26):C408-12. 16. Kawarabayashi T, Kishikawa T, Sugimori H. Effect of oxytocin on spontaneous and mechanical activities in pregnant human myometrium. AM J OBS1'ET GYNECOL 1986;155:671-6. 17. Cole WC, Garfield RE, Kirkaldy JS. Gap junctions and direct intercellular communication between rat uterine smooth muscle cells. Am J Physiol 1985;249(Cell Physiol 18):C20-31.