Estrogen Receptors and Effects of Estrogen on Membrane Electrical Properties of Coronary Vascular Smooth Muscle DAVID R. HARDER AND PATRICIA B. COULSON Department of Physiology, East Tennessee State Uniuersity, College of Medicine, Johnson City, Tennessee 37601
ABSTRACT The effect of estrogen stimulation in vitro on the electrical properties of vascular smooth muscle (VSM), and the concentration of estrogen receptors in VSM were measured in isolated coronary arteries. Microelectrode measurements of the dog coronary artery membrane potential (Em)showed quiescent values of -51 millivolts (mV) and an input resistance kin)of 10 megohms. Addition of diethylstilbestrol (DES) a t M hyperpolarized the membrane to -64 mV and reduced input resistance (Tin) to 5 megohms within 15 minutes. Extrapolation of the Em vs. log [Kl0 curve to zero potential gave similar values of [Kli of around 170 mM in both normal and DES treated muscles suggesting that the DES induced hyperpolarization is not due to increased Na-K pump activity. The 0.5% ethanol vehicle alone had no effect on the membrane potentials. Tetraethylammonium ion (TEA) induced action potentials in the previously quiescent tissue. When DES was applied in the presence of TEA, the membrane potential increased and the action potentials were abolished. Scatchard analysis of the estrogen receptor binding demonstrated both a high and a low affinity receptor for estrogen in the VSM. These data indicate that DES hyperpolarizes the VSM cells by a mechanism other than an increased Na-K pump activity. The mechanism of this increased Emmay be due to factors which increase K' conductance either mediated directly through estrogen interaction with its cytosolic receptors or through some unidentified second mechanism Infusion of estrogens has been demonstrat- the electrical properties of uterine smooth ed to increase blood flow in certain vascular muscle have been studied (Daniel and Lodge, beds of man, sheep, rats and other animal spe- '73; Spaziani, '751, but no reports have yet apcies (Altura and Altura, '77a,b). Addition of peared concerning the effects of estrogens on estradiol-17-/3 to isolated rat portal vein inhib- the electrical properties of VSM. its spontaneous contractions (McCalden, '75). It is the purpose of the present study to exHowever, other reports have indicated t h a t amine the effects of estrogens on various elecestrogens may maintain or enhance vascular trical properties of the VSM of canine corotone in cerebral arteries of adult women (Ga- nary arteries, and to determine the cytosolic brielson and Grietz, '70). The mechanisms re- estrogen receptor concentrations in VSM. sponsible for the effects of estrogen on vascuMATERIALS AND METHODS lar smooth muscle (VSM) are not well underVascular smooth muscle preparation stood, nor has the existence of receptors for Mongrel dogs were anesthetized with estrogens in VSM been demonstrated. Study of the direct effects of estrogens on VSM is sodium pentobarbitol, and the heart removed. complicated by the findings that the respon- The left main coronary artery was cannulated, siveness of blood vessels to other vasoactive and the coronary vasculature was perfused agents (e.g., angiotensin, epinephrine, seroto- with cold (22") oxygenated Ringer solution nin) are enhanced by their presence (Altura Received Feb. 7, '79. Accepted Apr. 4, '79. 'This work was supported by VA Grant lA(74)11~-430100. and Altura, '77b). The effects of estrogens on J. CELL. PHYSIOL. (1979) 100: 375-382
375
376
DAVID R. HARDER AND PATRICIA B. COULSON
while arteries were dissected from the myocardium. The isolated vessels were then stripped of fat and connective tissue. The vessel segments were slit open to form a sheet, allowing microelectrode penetration from the intimal surface of the vessel. These arterial strips were placed in a non-recirculating organ bath (10 ml volume) maintained a t 37". The isolated segments were continually suffused with a physiological salt solution containing (in mM): 141 Na+, 4.7 K+, 2.5 Ca++, 0.76 Mg", 124 C1-, 1.7 H2P0,, 25 HC03-, and 11 glucose. All solutions were aerated with 95%0 2 - 5 %C 0 2 (pH of 7.3-7.4). Tetraethylammonium chloride (10-15 mM) was added to induce excitability both in response to electrical stimulation and spontaneous (Harder et al., '79). The synthetic estrogen, diethylstilbestrol (DES), was dissolved in ethanol and added to the organ bath at final concentrations of to 1 0 - 5 ~ The . highest final concentration of ethanol in the organ bath was 0.5%. Vehicle controls were carried out by adding 0.5%ethanol to the organ bath without DES. Emvs log [KI, curves were generated by elevating K + in the suffusion solution keeping the sum of "a], 1K1, constant.
+
Electrical measurements Membrane potentials (Em) were recorded with glass microelectrodes filled with 3M KC1 and having tip resistances of 30-80 M a . The microelectrodes were mounted on a hydraulic micromanipulator allowing impalement of single VSM cells. Ag: AgCl half-cells were used. The recording preamplifier was a Dagan #8500 (Minneapolis, Minnesota) which possessed capacitance neutralization and a n internal bridge circuit. The bridge was balanced with the microelectrode outside the cell, and then the cell was impaled. The input resistance (rJ was determined by applying rectangular current pulses (I,) of varying intensities through the microelectrode and recording the associated voltage changes (AEm).The slope of the steady-state current vs. voltage (Av/AIJ curve through the origin was taken as the value or rln. The arterial strips were stimulated extracellularly by rectangular current pulses (50- to 100-msec duration) applied through platinum plate electrodes (field stimulation). Estrogen receptor assay Tissue wet weights were obtained and samples homogenized (100-400 mg/ml) with a polytron PT,, tissue disintegrator (Brink-
mann) in 10 mM Tris [Tris (hydroxymethyl) amino methane, pH 7-91, 1.5 mM EDTA (ethylene diamine tetraacetic acetate disodium), 15 mM monothioglycerol, 20% glycerol, final pH 7.5 a t 0". Samples were centrifuged for 60 minutes a t 105,000 x g and the aqueous fraction removed. The supernatant was pretreated with Norite charcoal (0.05%final concentration) for five minutes to remove endogenous steriods and rapidly centrifuged a t 2,000 rpm for ten minutes to pellet the charcoal. The cytoplasmic receptor fraction was then incubated (100 p1) for four hours at 0" in the presence of 3H-estradiol (S.A. = 143 Ci/mmol, 0.02-2.0 x 1 0 - 6 ~New , England Nuclear) with or without unlabeled diethylstilbesterol at 100 X higher concentrations. The unbound steriod was removed a t the end of the incubation with a second treatment of charcoal (0.25% final concentration) for 10 minutes. Supernatant fractions containing the bound 3H-steroidswere counted in 4 ml scintillation cocktail (4 gm omnifluor, 300 ml triton X-100, 700 ml toluene) in a Beckman LS 9000 scintillation counter. Quench correction was obtained by the Compton edge method using a I 3 T s external standard. Results were corrected to give moles of estradiol bound and then normalized to fmoles/mg soluble protein. The nonspecifically bound estradiol (in the presence of 100-fold higher concentrations of unlabelled steroid) was subtracted from the total steriod bound to give specifically bound estradiol. The soluble proteins were measured by the Folin-phenol assay (Lowry et al., '51) following a carboxymethylation treatment (Ross and Schatz, '73) to remove interference with reducing agents. RESULTS
Resting membrane properties Isolated strips of canine coronary arteries were impaled with microelectrodes to record the transmembrane potentials. In all but several cases the Emand rin were measured in the same cell (same impalement) since a stable Em had to be recorded before rin could be measured. This was the case in both normal and DES treated cells. The changes in rin and/or Emwere measured by impaling different cells from a given arterial strip. The values of resting membrane potential (Em)and input resistance (rin) are summarized (table 1).The Em measured in the vascular smooth muscle (VSM) of the coronary arteries was - 51 -t 0.9 mV (s.e.m.1. Addition of DES to low5M) hyperpolarized the VSM cells in a dose depen-
ESTROGEN RECEPTORS IN VASCULAR SMOOTH MUSCLE
377
Resting Potential v s IDES]
[DESI (MI Fig. 1 Illustration of the effects of the dose dependency of DES on the resting potential (Em)in the smooth muscle of coronary arteries of the dog. Each point represents the mean 1sem for five to eight cells sampled in 2 coronary arteries. Maximum hyperpolarization occurred at 5 X 10% DES.
*
TABLE I
Summary of membrane potential and input resistance measurements and P ~ r J P ~ r a t iin o sarterial smooth muscle fSEM Em (mV)
Condition
Control DES ( l O - e ~ ) ETOH (0.5%) TEA (10mM) TEA (10mM) DES(lO-'M)
+
rin (megohms)
(n)
10.420.5 5.120.4 '
-48-tO.7
-
20/5 24/5 13/3 2015
-5521.2
-
1215
-51kO.9 -64f1.3 -5421.5
'
' Significantly different from control at P < 0.05.
*TEA + DES significantly different from TEA, or DES alone at P
< 0.05.
'Numerator refers to the number of cells impaled, denominator gives number of different preparations.
dent manner (fig. 1).Minimal hyperpolarization occurred at about 5 X M (n = 5 arteries) DES and reached a maximal hyperpolarization of 20 mV (-71 f2.0 mV) a t 5 X M DES. No significant changes in Em were observed until 15-20 minutes after addition of DES. TEA (10 mM) did not significantly decrease Em (48 f 0.7 mV). DES, added in the presence of TEA increased the Emwith respect to TEA alone (55 1.2 mV) but resulted in a Em of less magnitude than that obtained with DES alone (64 f 1.3) (table 1). The rin measured as the slope of the steady-state volta g e h r r e n t curve through the origin, averaged 10.4 f 0.5 M a , and decreased to 5.1 f 0.5 M a in the presence of DES M) (fig. 2). Again no effect on rincould be observed until 15to 20 minutes after addition of DES. Ad-
*
dition of 10 mM TEA has previously been shown not to significantly increase rin in canine coronary arteries (Harder et al., '79). Increasing TEA to 15 mM caused an increase in rin to around 15 megohms, and decreased the Emto 44 1.1mV. DES was added using ethanol as a vehicle in a manner similar to other in vitro estrogen studies (Pietras and Szego, '751. The Emmeasured in the presence of 0.5%ethanol (the highest final concentration of ethanol to which the muscle was exposed) was not significantly changed (table 1).
*
E , us. log [KI, curve The resting potentials were measured at different external K' concentrations, and the resultant Emvs. log [Kl0curve is shown (fig. 3). The results were obtained by impaling different cells at each [KI,. It can be seen in figure 3 that at physiologic concentrations Em is determined by factors other than [Klo,and that the departure from the Nernst equation for a membrane selective for K' &e., 60 mV/ decade) is less marked in the DES treated cells. These changes and the decrease in rin could be accounted for by an increased gK in the presence of DES although it is possible that the conductances for other ions may also be changed. Extrapolation of the curves obtained in the control situation and in the presence of DES M) to zero potential gave very similar values of [Kli to around 170 mM (fig. 3). In low (0.5 mM) [Kl0 the VSM cells depolarized to 39 mV both in DES treated and control muscles.
378
DAVID R. HARDER AND PATRICIA B. COULSON
A V(mVJ
I+~O Voltage vs. Current
1’””
c+’
Depolarizing
-10
- 20 - 30 - 40 Fig. 2 Steady-state voltage vs. applied current (I,) relationship for smooth muscle cells of canine coronary arteries both in control Ringer solution and in solution containing DES MI.Each point represents the change in Em (AV) in response to rectangular hyperpolarizing or depolarizing current pulses applied through the recording microelectrode. The vertical bars represent the f sem for at least six cells in three different preparations. The slope through the origin (zero applied current) gives the input resistance. Note that DES decreased the slope, and therefore the input resistance.
Effects of DES on the TEA induced action potential The VSM cells in the coronary arteries were quiescent with stable resting potentials. TEA (10 mM) allowed the production of action potential upon electrical stimulation (fig. 4A,B) presumably due to inhibition of K’ conductance (It0 et al., ’70). TEA induction of action potentials is discussed in a previous communication (Harder et al., ’79). Addition M) abolished the action potenof DES tials induced by field stimulation in the presence of TEA (10mM) with a time lag of 15- to 20 minutes in five preparations (fig. 4 0 . Spontaneous action potentials were produced
upon raising TEA to 15 mM (fig. 5A). Addition M) hyperpolarized the coronary of DES VSM membrane and increased the amplitude of the action potentials within 15 minutes (fig. 5B). Continual exposure t o DES hyperpolarized the VSM membrane further and abolished the action potential generation except for an occasional spike of reduced amplitude (fig. 5 0 .
Estrogen receptors in the vascular smooth muscle Tissues were incubated with varying doses of 3H-estradiolin the presence and absence of unlabeled competitive hormone (diethylstil-
379
ESTROGEN RECEPTORS IN VASCULAR SMOOTH MUSCLE
Resting Potential vs. log
- 70
I!,
15
- 60 Em
(md
- 50 -40
-30
-20
-10
I
I
0
0.5
1.0
4.0
I
I
8.0 16 [K],(mM)
I
,
32
64
1
120 200
Fig. 3 Resting potential (Em) as a function of external K + concentration ( [KIJ (log scale) for vascular smooth muscle of canine coronary artery. The closed circles represent control measurements, the open circles represent Em measured in the presence of DES MI. The vertical bars represent the 2 sem for the number of cells indicated over each bar; the data for each point were collected from three to five muscles. The curve extrapolated t o zero potential gives an estimated internal K' concentration of about 170 mM for both the control curve and t h e curve measured in t he presence of DES.
A
Control
6 TEA
(IOmM)
C + DES (15rnin)
H
0.2 s e c
Fig. 4 Induction of excitability by tetraethylammonium ion (TEA") in the normally inexcitable canine coronary artery, and inhibition of the action potential 15 minutes after addition of DES. A. Control showing a resting potential of about -54 mV and lack of response t o external electrical stimulation (one shock artifact shown). B. Record taken from same cell as in " A ' five minutes after addition of TEA (10 mM), illustrating a n action potential in response t o electrical stimulation. C. Record showing abolition of the TEA action potential 15 minutes after addition of DES M). Voltage calibration in C and time calibration in B apply to all panels.
besterol) in order to identify the ability of t h e tissue t o specifically bind estradiol. The bound/free ratios were calculated and the data evaluated by the method of Scatchard (Clark and Peck, '78). Bovine uterine tissue (fig. 6A) shows a negative slope with a binding affinity of Ka = 4.096 x lo9M-' and a receptor concentration of 158.9 fm/mg protein and represents a positive control tissue. Canine vascular smooth muscle (fig. 6B) shows a
break in the slope of the line suggesting more than one binding component present. The two slopes represent a high binding affinity of Ka = 3.6132 x lo9 M-' with a receptor concentration of 33.3 fm/mg protein and a lower affinity component of Ka = 4.001 x 10' M - ' with a receptor concentration of 205.5 fm/mg protein. Canine heart muscle (fig. 6C) has a slope close to zero indicating few or no specific estrogen receptors present.
380
DAVID R. HARDER AND PATRICIA B. COULSON
w 2 sec
Fig. 5 Hyperpolarization and inhibition of spontaneous action potentials induced by tetraethylammonium ion (TEA) in the presence of DES. A. Control action potentials induced by 15 mM TEA having an amplitude of 40-46mV and a frequency of 1.5lhec. B. Hyperpolarization and increase in amplitude of the spontaneous action potentials to 60-65mV beginning a t about 15 minutes after addition of DES ( 1 0 - 6 M ) with no change in frequency. C. Nearly complete inhibition of the spontaneous TEA induced action potentials with only a n occasional MI. Voltage and time calibrations in C apply undershooting spike 20 minutes after addition of DES throughout. All recordings were taken from the same cell. TABLE 2
Specific estrogen receptor concentrations Moles x 10-15/mg soluble protein
Coronary artery, adult canine Coronary artery, adult female Coronaryartery, adult male Uterus, adult bovine (non-preg.) Heart, canine Aorta, fetal bovine Aorta, adult bovine
20.96 ( 2 4.32) 21.02 ( 2 6.74) 22.10(2 8.32) 230.97 (210.05) 3.13(2 0.67) 147.40(212.20) 37.70(2 4.15)
9 3 5 4 4 4 4
The results are expressed as the mean values (moles x lO-'Vmg soluble protein) 2 standard error of the mean, n = number of separate tissue determinations.
Small tissue fragments from isolated coronary arteries (50-150mg each) were assayed for specific estrogen receptor concentrations by a dextran coated charcoal batch assay and normalized per mg soluble protein (table 2). The positive control tissue (Bovine uterus) gave 230.97 fmoledmg protein and the negative control tissue (canine heart) gave 3.134 fmoledmg when measured in the same dextran coated charcoal assay. Coronary artery tissue from adult dogs of mixed sex gave 20.96 fmoledmg 2 4.32 s.e.m. There was no significant difference when t h e samples were reevaluated according to sex (table 2); however, the age of the animal and the stage of the female reproducitve cycle were not available for further correlation and this may have obscured any differences present. The highest values for estrogen binding were obtained in fetal bovine aorta, 147.4 fm/mg, compared to adult bovine aorta values of 37.70 fm/mg. These values were significantly different (p L 0.010). Further studies on early development of VSM cell sensitivity to steroids would be of
considerable interest. Cold DES was used as the competitive inhibitor to distinguish specific and non-specific estrogen receptor binding. The uptake seen in the fetal tissues was probably not attributable to the presence of alpha fetoprotein since DES will not compete with estradiol for binding to AFP (Toft and Tomasi, '78). DISCUSSION
The present studies were designed to investigate the direct in vitro effects of estrogen on some biophysical properties of canine coronary vascular smooth muscle (VSM). Addition of DES to the suffusion solution caused a dose dependent hyperpolarization of the VSM membrane. Similar hyperpolarizations have been demonstrated in uterine smooth muscle (Spaziani, '75; Goto and Csapo, '59; Kuriyama and Csapo, '61). The mechanism for this hyperpolarization in uterine smooth muscle is not yet clear. However, there is some data indicating that it may be due to an increase in K' conductance (gK) (Altura and Altura, '77b). Our data, demonstrating that the departure from the Nernst potential for a K' selective membrane is less marked in t h e DES treated muscles suggest that gK is increased in the presence of DES. Also, the finding that extrapolation to zero potential gave statistically similar values of [Kli further supports the hypothesis that gK is increased in the presence of DES. If the hyperpolarization was due to an increased Na-K pump activity, then t h e extrapolated value of [Kli would be greater in the presence of DES. Also, inhibition of NaK ATPase in low [KI, (0.5mM) caused an identical depolarization both in the control situation and in the presence of DES. The shape of the Emvs. log [KI, curve in the untreated ca-
ESTROGEN RECEPTORS IN VASCULAR SMOOTH MUSCLE
]
38 1
cent artery were induced upon addition of TEA. It has been shown t h a t TEA induces action potentials in smooth muscle by inhibiting gK (It0 e t al., '70; Kirkpatrick, '75). The induction of action potentials in coronary artery by TEA is more fully discussed in previous communications (Harder et al., '79; Belardinelli e t al., '79). The inhibition of these action potentials and the concomitant hyperpolarization in the coronary VSM is consistent with the hypothesis that DES increases I 2 4 6 8 g e However, the results of this study do not allow us to exclude other mechanisms such as a decreased Ca++influx (McCalden, '751, or 6. VASCULAR SMOOTH state whether the possible increase in gK is .04 MUSCLE, CANINE due to a direct effect of DES or by some other mechanism such as secondary changes in response to increased levels of internal Ca++. Specific estrogen receptors were identified (fig. 6) in vascular smooth muscle tissue, giving a concentration of 33.3 fmoledmg soluble .011 protein, and having an apparent high affinity constant, Ka = 3.6132 x lo9 M-'. These 1 values are quite comparable to the range of 2 4 8 8 binding constants for specific high affinity estrogen receptors which have been described C. HEART, CANINE in uterine smooth muscle by many other .04 workers (Baulieu, '76; Pavlik and Coulson, '76). Although the physiochemical properties .03. of the estrogen receptors in VSM appear similar to endometrial and myometrial uterine receptors, they occur in much lower concentrations and may well be chemically distinct (Martel and Psychoyos, '78). Recent studies have suggested that there may be multiple molecular forms for estrogen receptors (Erdos et al., '77). How the estrogen receptor in VSM 1 2 3 4 compares to the receptors already described in BOUND ESTRADIOL the endometrium, myometrium, pituitary, hymoles x10-l~ pothalamus, or mammary gland, and how the Fig. 6 Scatchard analysis of the binding of 3H-estradiol levels of this VSM receptor may be controiled or modulated will be the subject of future into three separate tissues were performed. The specifically bound steroid (total bound minus nonspecifically bound in vestigations. the presence of 100-foldhigher unlabeled steroid) is plotted Recent observations show that a-fetoproagainst the bound/free (B/F) ratio. Each point represents tein (AFP) can bind estradiol-17-/3 in the rat the mean of four determinations. or mouse with high affinity and specificity. In the bovine or human tissue, AFP has little or nine coronary VSM agrees well with that in no affinity for estrogens (Swartz and Soloff, other vascular smooth muscles (Kuriyama et ' 7 4 , however, little is known about AFP interal., '71; Mekata and Niu, '72). The extrapo- ference in canine estrogen binding. DES has lated value of 170 mM [KIi is similar to the been demonstrated to compete for estrogen values obtained in tissue ion analysis (Schof- binding to the estrogen receptor but not to feniels, '69; Haljamae et al., '70). Thus, it ap- compete for the estrogen binding to AFP. (Rapears that in canine coronary smooth muscle danyi et al., '77; Toft and Tomasi, '78). Thus DES induces hyperpolarization by increasing the specific binding measured by dextran g K . Action potentials in this previously quiescoated charcoal with DES competition seen in .04
1 I
A. UTERUS, BOVINE
382
DAVID R. HARDER AND PATRICIA B. COULSON
this study for bovine fetal aorta is probably due to a specific estrogen receptor. It has been repeatedly suggested that sex hormones influence blood flow, blood pressure, and vascular tone by “indirect” routes involving circulating or local levels of histamine, acetylcholene, angiotensin, kinins, catecholamines or neurohypophyseal hormones (Altura and Altura, ’77a,b). The present studies were able to demonstrate in vitro direct electrical responses of VSM to sex hormone stimulation. The demonstration of the presence of estrogen receptors in the isolated cells suggests a “direct” influence of estrogen on the electrical properties of vascular smooth muscle. Extensive future studies measuring the effect of estrogen on the contractile properties of isolated arteries will need to be done to determine their affect on excitation-contraction coupling. LITERATURE CITED Altura, B. T., and B. M. Altura 1977a Factors affecting vascular responsiveness. In: Microcirculation. Vol. 11. G. Kaley and B. M. Altura, eds. University Park Press, Baltimore, pp. 547-615. Altura, B. M., and B. T. Altura 1977b Influence of sex hormones, oral contraceptives and pregnancy on vascular muscle and its reactivity. In: Factors Influencing Vascular Reactivity. 0. Carrier and S. Shibata, eds. IgakuShoin Press, New York, pp. 221-254. Baulieu, E. E. 1976 Steroid receptors and hormone receptivity: New approaches in pharmacology and therapeutics. In: Breast Cancer: Trends in Research and Treatment. J. C. Heuson et al., eds. Raven Press, New York, pp. 165-176. Batra. S.. and B. Bengtsson 1978 Effects of diethylstilbes. terol and ovarian steroids on the contractile responses and calcium movements in rat uterine muscle. J. Physiol., 276: 329-342. Belardinelli, L., D. Harder, N. Sperelakis, R. Rubio and R. M. Berne 1979 Cardiac glycoside stimulation of inward Ca” current in vascular smooth muscle of canine coronary artery. J. Pharmacol. and Exp. Therap., 209: 62-69. Clark, J. H., and E. J. Peck, Jr. 1978 Steroid hormone receptors: basic principles and measurement. In: Laboratory Methods for Hormone Action and Molecular Endocrinology. W. T. Schrader and B. W. O’Malley, eds. Baylor College of Medicine, Houston, Texas, 2: 1-45. Daniel, E. E., and S. Lodge 1973 Electrophysiology of myometrium. In: Uterine Contraction - Side Effects of Steroid Contraceptives. J. B. Josimovich, ed. Wiley-Interscience, New York, pp. 19-64. Erdos, T., R. Bessada and J. Fries 1977 Multiple molecular forms of the uterine estradiol receptor. In: Multiple Molecular Forms of Steroid Hormone Receptors. M. K. Agrawol, ed. ElsevierINorth Holland, pp. 113-128. Gabrielson, T., and T. Grietz 1970 Normal size of t he internal carotid, middle cerebral and anterior cerebral arteries. Acta Radio!., 10: 1-10,
Goto, M., and H. Csapo 1959 The effect of ovarian steroids on the membrane potential of uterine muscle. J. Gen. Physiol., 43: 455-466. Haljamae, H., B. Johansson, 0. Jonssonand H. Rdcket 1970 The distribution of sodium, potassium and chloride in the smooth muscle of the ra t portal vein. Acta Physiol. Scand., 78: 255-268. Harder, D. R., L. Belardinelli, N. Sperelakis, R. Rubio and R. M. Berne 1979 Effects of adenosine and nitroglycerin on the action potentials of large and small coronary arteries. Circulation Res., 44: 176-182. Ito, Y., H. Kuriyama and Y. Sakamoto 1970 Effects of tetraethylammonium chloride on the membrane activity of guinea pig stomach smooth muscle. J . Physiol., 211: 445-460. Kirkpatrick, C. T. 1975 Excitation and contraction in bovine tracheal smooth muscle. J. Physiol., 244: 263-281. Kuriyama, H., and A. Csapo 1961 A study of parturient uterus using the micro electrode technique. Endocrinol., 68: 1010-1025. Kuriyama, H., K. Ohshima and Y. Sakamoto 1971 The membrane properties of smooth muscle of the guinea pig portal vein in isotonic and hypertonic solutions. J. Physiol., 17: 179-199. Lowry, 0.H., N. H. Rosenbrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin-phenol reagent. J. Biol. Chem., 193: 265-271. Martel, D., and A. Psychoyos 1978 Progesterone induced estrogen receptors in the rat uterus. J. Endocrinol., 76: 145-154. McCalden, T. A. 1975 Theinhibitory action of oestradiol17-6 and progesterone on venous smooth muscle. British J. Pharmac., 53: 183-192. Mekata, F., and H. Niu 1972 Biophysical effects of adrenaline on the smooth muscle of the rabbit common carotid artery. J. Gen. Physiol., 59: 92.102. Pavlik, E. J., and P. B. Coulson 1976 Hydroxylapatite “batch” assay for estrogen receptors: increased sensitivity over present receptor assays. J. Steroid Biochem., 7: 357-368. Pietras, R. J., and C. M. Szego 1975 Steroid hormoneresponsive isolated endometrial cell. Endocrinol., 96: 946-954. Radanyi, C., C. Mercier-Bodard, C. Secco-Millet, E. E. Baulieu and H. Richard-Foy 1977 aFetoprotein is not a component of the estradiol receptor of ra t uterus. Proc. Nat’l. Acad. Sci., 74: 2269-2272. Ross, E., and G. Schatz 1973 Assay of protein in the presence of high concentrations of sulfhydryl compounds. Analytical Biochem., 54: 304-306. Schoffeniels, E. 1969 Ionic composition of the arterial wall. Angiologica, 6: 65-87. Spasiani, E. 1975 Accessory reproductive organs in mammals: Control of cell and tissue transport by sex hormones. Pharmacol. Rev., 27: 207-286. Spaziani, E., and C. M. Szego 1958 The influence of estradiol and cortisol on uterine histamine of the overiectomized rat. Endocrinol., 63: 669-678. Swartz, S. K., and M. S. Soloff 1974 The lack of estrogen binding by human afetoprtein. J. Clin. Endocrinol. Metabol., 39: 589-591. Toft, D. O., and T. B. Tomasi 1978 An immunological comparison between mouse afetoprotein and the uterine estrogen receptor. Proc. SOC.Exp. Biol. and Med., 157: 594-598.
ABSTRACT The effect of estrogen stimulation in vitro on the electrical properties of vascular smooth muscle (VSM), and the concentration of estrogen receptors in VSM were measured in isolated coronary arteries. Microelectrode measurements of the dog coronary artery membrane potential (Em)showed quiescent values of -51 millivolts (mV) and an input resistance kin)of 10 megohms. Addition of diethylstilbestrol (DES) a t M hyperpolarized the membrane to -64 mV and reduced input resistance (Tin) to 5 megohms within 15 minutes. Extrapolation of the Em vs. log [Kl0 curve to zero potential gave similar values of [Kli of around 170 mM in both normal and DES treated muscles suggesting that the DES induced hyperpolarization is not due to increased Na-K pump activity. The 0.5% ethanol vehicle alone had no effect on the membrane potentials. Tetraethylammonium ion (TEA) induced action potentials in the previously quiescent tissue. When DES was applied in the presence of TEA, the membrane potential increased and the action potentials were abolished. Scatchard analysis of the estrogen receptor binding demonstrated both a high and a low affinity receptor for estrogen in the VSM. These data indicate that DES hyperpolarizes the VSM cells by a mechanism other than an increased Na-K pump activity. The mechanism of this increased Emmay be due to factors which increase K' conductance either mediated directly through estrogen interaction with its cytosolic receptors or through some unidentified second mechanism Infusion of estrogens has been demonstrat- the electrical properties of uterine smooth ed to increase blood flow in certain vascular muscle have been studied (Daniel and Lodge, beds of man, sheep, rats and other animal spe- '73; Spaziani, '751, but no reports have yet apcies (Altura and Altura, '77a,b). Addition of peared concerning the effects of estrogens on estradiol-17-/3 to isolated rat portal vein inhib- the electrical properties of VSM. its spontaneous contractions (McCalden, '75). It is the purpose of the present study to exHowever, other reports have indicated t h a t amine the effects of estrogens on various elecestrogens may maintain or enhance vascular trical properties of the VSM of canine corotone in cerebral arteries of adult women (Ga- nary arteries, and to determine the cytosolic brielson and Grietz, '70). The mechanisms re- estrogen receptor concentrations in VSM. sponsible for the effects of estrogen on vascuMATERIALS AND METHODS lar smooth muscle (VSM) are not well underVascular smooth muscle preparation stood, nor has the existence of receptors for Mongrel dogs were anesthetized with estrogens in VSM been demonstrated. Study of the direct effects of estrogens on VSM is sodium pentobarbitol, and the heart removed. complicated by the findings that the respon- The left main coronary artery was cannulated, siveness of blood vessels to other vasoactive and the coronary vasculature was perfused agents (e.g., angiotensin, epinephrine, seroto- with cold (22") oxygenated Ringer solution nin) are enhanced by their presence (Altura Received Feb. 7, '79. Accepted Apr. 4, '79. 'This work was supported by VA Grant lA(74)11~-430100. and Altura, '77b). The effects of estrogens on J. CELL. PHYSIOL. (1979) 100: 375-382
375
376
DAVID R. HARDER AND PATRICIA B. COULSON
while arteries were dissected from the myocardium. The isolated vessels were then stripped of fat and connective tissue. The vessel segments were slit open to form a sheet, allowing microelectrode penetration from the intimal surface of the vessel. These arterial strips were placed in a non-recirculating organ bath (10 ml volume) maintained a t 37". The isolated segments were continually suffused with a physiological salt solution containing (in mM): 141 Na+, 4.7 K+, 2.5 Ca++, 0.76 Mg", 124 C1-, 1.7 H2P0,, 25 HC03-, and 11 glucose. All solutions were aerated with 95%0 2 - 5 %C 0 2 (pH of 7.3-7.4). Tetraethylammonium chloride (10-15 mM) was added to induce excitability both in response to electrical stimulation and spontaneous (Harder et al., '79). The synthetic estrogen, diethylstilbestrol (DES), was dissolved in ethanol and added to the organ bath at final concentrations of to 1 0 - 5 ~ The . highest final concentration of ethanol in the organ bath was 0.5%. Vehicle controls were carried out by adding 0.5%ethanol to the organ bath without DES. Emvs log [KI, curves were generated by elevating K + in the suffusion solution keeping the sum of "a], 1K1, constant.
+
Electrical measurements Membrane potentials (Em) were recorded with glass microelectrodes filled with 3M KC1 and having tip resistances of 30-80 M a . The microelectrodes were mounted on a hydraulic micromanipulator allowing impalement of single VSM cells. Ag: AgCl half-cells were used. The recording preamplifier was a Dagan #8500 (Minneapolis, Minnesota) which possessed capacitance neutralization and a n internal bridge circuit. The bridge was balanced with the microelectrode outside the cell, and then the cell was impaled. The input resistance (rJ was determined by applying rectangular current pulses (I,) of varying intensities through the microelectrode and recording the associated voltage changes (AEm).The slope of the steady-state current vs. voltage (Av/AIJ curve through the origin was taken as the value or rln. The arterial strips were stimulated extracellularly by rectangular current pulses (50- to 100-msec duration) applied through platinum plate electrodes (field stimulation). Estrogen receptor assay Tissue wet weights were obtained and samples homogenized (100-400 mg/ml) with a polytron PT,, tissue disintegrator (Brink-
mann) in 10 mM Tris [Tris (hydroxymethyl) amino methane, pH 7-91, 1.5 mM EDTA (ethylene diamine tetraacetic acetate disodium), 15 mM monothioglycerol, 20% glycerol, final pH 7.5 a t 0". Samples were centrifuged for 60 minutes a t 105,000 x g and the aqueous fraction removed. The supernatant was pretreated with Norite charcoal (0.05%final concentration) for five minutes to remove endogenous steriods and rapidly centrifuged a t 2,000 rpm for ten minutes to pellet the charcoal. The cytoplasmic receptor fraction was then incubated (100 p1) for four hours at 0" in the presence of 3H-estradiol (S.A. = 143 Ci/mmol, 0.02-2.0 x 1 0 - 6 ~New , England Nuclear) with or without unlabeled diethylstilbesterol at 100 X higher concentrations. The unbound steriod was removed a t the end of the incubation with a second treatment of charcoal (0.25% final concentration) for 10 minutes. Supernatant fractions containing the bound 3H-steroidswere counted in 4 ml scintillation cocktail (4 gm omnifluor, 300 ml triton X-100, 700 ml toluene) in a Beckman LS 9000 scintillation counter. Quench correction was obtained by the Compton edge method using a I 3 T s external standard. Results were corrected to give moles of estradiol bound and then normalized to fmoles/mg soluble protein. The nonspecifically bound estradiol (in the presence of 100-fold higher concentrations of unlabelled steroid) was subtracted from the total steriod bound to give specifically bound estradiol. The soluble proteins were measured by the Folin-phenol assay (Lowry et al., '51) following a carboxymethylation treatment (Ross and Schatz, '73) to remove interference with reducing agents. RESULTS
Resting membrane properties Isolated strips of canine coronary arteries were impaled with microelectrodes to record the transmembrane potentials. In all but several cases the Emand rin were measured in the same cell (same impalement) since a stable Em had to be recorded before rin could be measured. This was the case in both normal and DES treated cells. The changes in rin and/or Emwere measured by impaling different cells from a given arterial strip. The values of resting membrane potential (Em)and input resistance (rin) are summarized (table 1).The Em measured in the vascular smooth muscle (VSM) of the coronary arteries was - 51 -t 0.9 mV (s.e.m.1. Addition of DES to low5M) hyperpolarized the VSM cells in a dose depen-
ESTROGEN RECEPTORS IN VASCULAR SMOOTH MUSCLE
377
Resting Potential v s IDES]
[DESI (MI Fig. 1 Illustration of the effects of the dose dependency of DES on the resting potential (Em)in the smooth muscle of coronary arteries of the dog. Each point represents the mean 1sem for five to eight cells sampled in 2 coronary arteries. Maximum hyperpolarization occurred at 5 X 10% DES.
*
TABLE I
Summary of membrane potential and input resistance measurements and P ~ r J P ~ r a t iin o sarterial smooth muscle fSEM Em (mV)
Condition
Control DES ( l O - e ~ ) ETOH (0.5%) TEA (10mM) TEA (10mM) DES(lO-'M)
+
rin (megohms)
(n)
10.420.5 5.120.4 '
-48-tO.7
-
20/5 24/5 13/3 2015
-5521.2
-
1215
-51kO.9 -64f1.3 -5421.5
'
' Significantly different from control at P < 0.05.
*TEA + DES significantly different from TEA, or DES alone at P
< 0.05.
'Numerator refers to the number of cells impaled, denominator gives number of different preparations.
dent manner (fig. 1).Minimal hyperpolarization occurred at about 5 X M (n = 5 arteries) DES and reached a maximal hyperpolarization of 20 mV (-71 f2.0 mV) a t 5 X M DES. No significant changes in Em were observed until 15-20 minutes after addition of DES. TEA (10 mM) did not significantly decrease Em (48 f 0.7 mV). DES, added in the presence of TEA increased the Emwith respect to TEA alone (55 1.2 mV) but resulted in a Em of less magnitude than that obtained with DES alone (64 f 1.3) (table 1). The rin measured as the slope of the steady-state volta g e h r r e n t curve through the origin, averaged 10.4 f 0.5 M a , and decreased to 5.1 f 0.5 M a in the presence of DES M) (fig. 2). Again no effect on rincould be observed until 15to 20 minutes after addition of DES. Ad-
*
dition of 10 mM TEA has previously been shown not to significantly increase rin in canine coronary arteries (Harder et al., '79). Increasing TEA to 15 mM caused an increase in rin to around 15 megohms, and decreased the Emto 44 1.1mV. DES was added using ethanol as a vehicle in a manner similar to other in vitro estrogen studies (Pietras and Szego, '751. The Emmeasured in the presence of 0.5%ethanol (the highest final concentration of ethanol to which the muscle was exposed) was not significantly changed (table 1).
*
E , us. log [KI, curve The resting potentials were measured at different external K' concentrations, and the resultant Emvs. log [Kl0curve is shown (fig. 3). The results were obtained by impaling different cells at each [KI,. It can be seen in figure 3 that at physiologic concentrations Em is determined by factors other than [Klo,and that the departure from the Nernst equation for a membrane selective for K' &e., 60 mV/ decade) is less marked in the DES treated cells. These changes and the decrease in rin could be accounted for by an increased gK in the presence of DES although it is possible that the conductances for other ions may also be changed. Extrapolation of the curves obtained in the control situation and in the presence of DES M) to zero potential gave very similar values of [Kli to around 170 mM (fig. 3). In low (0.5 mM) [Kl0 the VSM cells depolarized to 39 mV both in DES treated and control muscles.
378
DAVID R. HARDER AND PATRICIA B. COULSON
A V(mVJ
I+~O Voltage vs. Current
1’””
c+’
Depolarizing
-10
- 20 - 30 - 40 Fig. 2 Steady-state voltage vs. applied current (I,) relationship for smooth muscle cells of canine coronary arteries both in control Ringer solution and in solution containing DES MI.Each point represents the change in Em (AV) in response to rectangular hyperpolarizing or depolarizing current pulses applied through the recording microelectrode. The vertical bars represent the f sem for at least six cells in three different preparations. The slope through the origin (zero applied current) gives the input resistance. Note that DES decreased the slope, and therefore the input resistance.
Effects of DES on the TEA induced action potential The VSM cells in the coronary arteries were quiescent with stable resting potentials. TEA (10 mM) allowed the production of action potential upon electrical stimulation (fig. 4A,B) presumably due to inhibition of K’ conductance (It0 et al., ’70). TEA induction of action potentials is discussed in a previous communication (Harder et al., ’79). Addition M) abolished the action potenof DES tials induced by field stimulation in the presence of TEA (10mM) with a time lag of 15- to 20 minutes in five preparations (fig. 4 0 . Spontaneous action potentials were produced
upon raising TEA to 15 mM (fig. 5A). Addition M) hyperpolarized the coronary of DES VSM membrane and increased the amplitude of the action potentials within 15 minutes (fig. 5B). Continual exposure t o DES hyperpolarized the VSM membrane further and abolished the action potential generation except for an occasional spike of reduced amplitude (fig. 5 0 .
Estrogen receptors in the vascular smooth muscle Tissues were incubated with varying doses of 3H-estradiolin the presence and absence of unlabeled competitive hormone (diethylstil-
379
ESTROGEN RECEPTORS IN VASCULAR SMOOTH MUSCLE
Resting Potential vs. log
- 70
I!,
15
- 60 Em
(md
- 50 -40
-30
-20
-10
I
I
0
0.5
1.0
4.0
I
I
8.0 16 [K],(mM)
I
,
32
64
1
120 200
Fig. 3 Resting potential (Em) as a function of external K + concentration ( [KIJ (log scale) for vascular smooth muscle of canine coronary artery. The closed circles represent control measurements, the open circles represent Em measured in the presence of DES MI. The vertical bars represent the 2 sem for the number of cells indicated over each bar; the data for each point were collected from three to five muscles. The curve extrapolated t o zero potential gives an estimated internal K' concentration of about 170 mM for both the control curve and t h e curve measured in t he presence of DES.
A
Control
6 TEA
(IOmM)
C + DES (15rnin)
H
0.2 s e c
Fig. 4 Induction of excitability by tetraethylammonium ion (TEA") in the normally inexcitable canine coronary artery, and inhibition of the action potential 15 minutes after addition of DES. A. Control showing a resting potential of about -54 mV and lack of response t o external electrical stimulation (one shock artifact shown). B. Record taken from same cell as in " A ' five minutes after addition of TEA (10 mM), illustrating a n action potential in response t o electrical stimulation. C. Record showing abolition of the TEA action potential 15 minutes after addition of DES M). Voltage calibration in C and time calibration in B apply to all panels.
besterol) in order to identify the ability of t h e tissue t o specifically bind estradiol. The bound/free ratios were calculated and the data evaluated by the method of Scatchard (Clark and Peck, '78). Bovine uterine tissue (fig. 6A) shows a negative slope with a binding affinity of Ka = 4.096 x lo9M-' and a receptor concentration of 158.9 fm/mg protein and represents a positive control tissue. Canine vascular smooth muscle (fig. 6B) shows a
break in the slope of the line suggesting more than one binding component present. The two slopes represent a high binding affinity of Ka = 3.6132 x lo9 M-' with a receptor concentration of 33.3 fm/mg protein and a lower affinity component of Ka = 4.001 x 10' M - ' with a receptor concentration of 205.5 fm/mg protein. Canine heart muscle (fig. 6C) has a slope close to zero indicating few or no specific estrogen receptors present.
380
DAVID R. HARDER AND PATRICIA B. COULSON
w 2 sec
Fig. 5 Hyperpolarization and inhibition of spontaneous action potentials induced by tetraethylammonium ion (TEA) in the presence of DES. A. Control action potentials induced by 15 mM TEA having an amplitude of 40-46mV and a frequency of 1.5lhec. B. Hyperpolarization and increase in amplitude of the spontaneous action potentials to 60-65mV beginning a t about 15 minutes after addition of DES ( 1 0 - 6 M ) with no change in frequency. C. Nearly complete inhibition of the spontaneous TEA induced action potentials with only a n occasional MI. Voltage and time calibrations in C apply undershooting spike 20 minutes after addition of DES throughout. All recordings were taken from the same cell. TABLE 2
Specific estrogen receptor concentrations Moles x 10-15/mg soluble protein
Coronary artery, adult canine Coronary artery, adult female Coronaryartery, adult male Uterus, adult bovine (non-preg.) Heart, canine Aorta, fetal bovine Aorta, adult bovine
20.96 ( 2 4.32) 21.02 ( 2 6.74) 22.10(2 8.32) 230.97 (210.05) 3.13(2 0.67) 147.40(212.20) 37.70(2 4.15)
9 3 5 4 4 4 4
The results are expressed as the mean values (moles x lO-'Vmg soluble protein) 2 standard error of the mean, n = number of separate tissue determinations.
Small tissue fragments from isolated coronary arteries (50-150mg each) were assayed for specific estrogen receptor concentrations by a dextran coated charcoal batch assay and normalized per mg soluble protein (table 2). The positive control tissue (Bovine uterus) gave 230.97 fmoledmg protein and the negative control tissue (canine heart) gave 3.134 fmoledmg when measured in the same dextran coated charcoal assay. Coronary artery tissue from adult dogs of mixed sex gave 20.96 fmoledmg 2 4.32 s.e.m. There was no significant difference when t h e samples were reevaluated according to sex (table 2); however, the age of the animal and the stage of the female reproducitve cycle were not available for further correlation and this may have obscured any differences present. The highest values for estrogen binding were obtained in fetal bovine aorta, 147.4 fm/mg, compared to adult bovine aorta values of 37.70 fm/mg. These values were significantly different (p L 0.010). Further studies on early development of VSM cell sensitivity to steroids would be of
considerable interest. Cold DES was used as the competitive inhibitor to distinguish specific and non-specific estrogen receptor binding. The uptake seen in the fetal tissues was probably not attributable to the presence of alpha fetoprotein since DES will not compete with estradiol for binding to AFP (Toft and Tomasi, '78). DISCUSSION
The present studies were designed to investigate the direct in vitro effects of estrogen on some biophysical properties of canine coronary vascular smooth muscle (VSM). Addition of DES to the suffusion solution caused a dose dependent hyperpolarization of the VSM membrane. Similar hyperpolarizations have been demonstrated in uterine smooth muscle (Spaziani, '75; Goto and Csapo, '59; Kuriyama and Csapo, '61). The mechanism for this hyperpolarization in uterine smooth muscle is not yet clear. However, there is some data indicating that it may be due to an increase in K' conductance (gK) (Altura and Altura, '77b). Our data, demonstrating that the departure from the Nernst potential for a K' selective membrane is less marked in t h e DES treated muscles suggest that gK is increased in the presence of DES. Also, the finding that extrapolation to zero potential gave statistically similar values of [Kli further supports the hypothesis that gK is increased in the presence of DES. If the hyperpolarization was due to an increased Na-K pump activity, then t h e extrapolated value of [Kli would be greater in the presence of DES. Also, inhibition of NaK ATPase in low [KI, (0.5mM) caused an identical depolarization both in the control situation and in the presence of DES. The shape of the Emvs. log [KI, curve in the untreated ca-
ESTROGEN RECEPTORS IN VASCULAR SMOOTH MUSCLE
]
38 1
cent artery were induced upon addition of TEA. It has been shown t h a t TEA induces action potentials in smooth muscle by inhibiting gK (It0 e t al., '70; Kirkpatrick, '75). The induction of action potentials in coronary artery by TEA is more fully discussed in previous communications (Harder et al., '79; Belardinelli e t al., '79). The inhibition of these action potentials and the concomitant hyperpolarization in the coronary VSM is consistent with the hypothesis that DES increases I 2 4 6 8 g e However, the results of this study do not allow us to exclude other mechanisms such as a decreased Ca++influx (McCalden, '751, or 6. VASCULAR SMOOTH state whether the possible increase in gK is .04 MUSCLE, CANINE due to a direct effect of DES or by some other mechanism such as secondary changes in response to increased levels of internal Ca++. Specific estrogen receptors were identified (fig. 6) in vascular smooth muscle tissue, giving a concentration of 33.3 fmoledmg soluble .011 protein, and having an apparent high affinity constant, Ka = 3.6132 x lo9 M-'. These 1 values are quite comparable to the range of 2 4 8 8 binding constants for specific high affinity estrogen receptors which have been described C. HEART, CANINE in uterine smooth muscle by many other .04 workers (Baulieu, '76; Pavlik and Coulson, '76). Although the physiochemical properties .03. of the estrogen receptors in VSM appear similar to endometrial and myometrial uterine receptors, they occur in much lower concentrations and may well be chemically distinct (Martel and Psychoyos, '78). Recent studies have suggested that there may be multiple molecular forms for estrogen receptors (Erdos et al., '77). How the estrogen receptor in VSM 1 2 3 4 compares to the receptors already described in BOUND ESTRADIOL the endometrium, myometrium, pituitary, hymoles x10-l~ pothalamus, or mammary gland, and how the Fig. 6 Scatchard analysis of the binding of 3H-estradiol levels of this VSM receptor may be controiled or modulated will be the subject of future into three separate tissues were performed. The specifically bound steroid (total bound minus nonspecifically bound in vestigations. the presence of 100-foldhigher unlabeled steroid) is plotted Recent observations show that a-fetoproagainst the bound/free (B/F) ratio. Each point represents tein (AFP) can bind estradiol-17-/3 in the rat the mean of four determinations. or mouse with high affinity and specificity. In the bovine or human tissue, AFP has little or nine coronary VSM agrees well with that in no affinity for estrogens (Swartz and Soloff, other vascular smooth muscles (Kuriyama et ' 7 4 , however, little is known about AFP interal., '71; Mekata and Niu, '72). The extrapo- ference in canine estrogen binding. DES has lated value of 170 mM [KIi is similar to the been demonstrated to compete for estrogen values obtained in tissue ion analysis (Schof- binding to the estrogen receptor but not to feniels, '69; Haljamae et al., '70). Thus, it ap- compete for the estrogen binding to AFP. (Rapears that in canine coronary smooth muscle danyi et al., '77; Toft and Tomasi, '78). Thus DES induces hyperpolarization by increasing the specific binding measured by dextran g K . Action potentials in this previously quiescoated charcoal with DES competition seen in .04
1 I
A. UTERUS, BOVINE
382
DAVID R. HARDER AND PATRICIA B. COULSON
this study for bovine fetal aorta is probably due to a specific estrogen receptor. It has been repeatedly suggested that sex hormones influence blood flow, blood pressure, and vascular tone by “indirect” routes involving circulating or local levels of histamine, acetylcholene, angiotensin, kinins, catecholamines or neurohypophyseal hormones (Altura and Altura, ’77a,b). The present studies were able to demonstrate in vitro direct electrical responses of VSM to sex hormone stimulation. The demonstration of the presence of estrogen receptors in the isolated cells suggests a “direct” influence of estrogen on the electrical properties of vascular smooth muscle. Extensive future studies measuring the effect of estrogen on the contractile properties of isolated arteries will need to be done to determine their affect on excitation-contraction coupling. LITERATURE CITED Altura, B. T., and B. M. Altura 1977a Factors affecting vascular responsiveness. In: Microcirculation. Vol. 11. G. Kaley and B. M. Altura, eds. University Park Press, Baltimore, pp. 547-615. Altura, B. M., and B. T. Altura 1977b Influence of sex hormones, oral contraceptives and pregnancy on vascular muscle and its reactivity. In: Factors Influencing Vascular Reactivity. 0. Carrier and S. Shibata, eds. IgakuShoin Press, New York, pp. 221-254. Baulieu, E. E. 1976 Steroid receptors and hormone receptivity: New approaches in pharmacology and therapeutics. In: Breast Cancer: Trends in Research and Treatment. J. C. Heuson et al., eds. Raven Press, New York, pp. 165-176. Batra. S.. and B. Bengtsson 1978 Effects of diethylstilbes. terol and ovarian steroids on the contractile responses and calcium movements in rat uterine muscle. J. Physiol., 276: 329-342. Belardinelli, L., D. Harder, N. Sperelakis, R. Rubio and R. M. Berne 1979 Cardiac glycoside stimulation of inward Ca” current in vascular smooth muscle of canine coronary artery. J. Pharmacol. and Exp. Therap., 209: 62-69. Clark, J. H., and E. J. Peck, Jr. 1978 Steroid hormone receptors: basic principles and measurement. In: Laboratory Methods for Hormone Action and Molecular Endocrinology. W. T. Schrader and B. W. O’Malley, eds. Baylor College of Medicine, Houston, Texas, 2: 1-45. Daniel, E. E., and S. Lodge 1973 Electrophysiology of myometrium. In: Uterine Contraction - Side Effects of Steroid Contraceptives. J. B. Josimovich, ed. Wiley-Interscience, New York, pp. 19-64. Erdos, T., R. Bessada and J. Fries 1977 Multiple molecular forms of the uterine estradiol receptor. In: Multiple Molecular Forms of Steroid Hormone Receptors. M. K. Agrawol, ed. ElsevierINorth Holland, pp. 113-128. Gabrielson, T., and T. Grietz 1970 Normal size of t he internal carotid, middle cerebral and anterior cerebral arteries. Acta Radio!., 10: 1-10,
Goto, M., and H. Csapo 1959 The effect of ovarian steroids on the membrane potential of uterine muscle. J. Gen. Physiol., 43: 455-466. Haljamae, H., B. Johansson, 0. Jonssonand H. Rdcket 1970 The distribution of sodium, potassium and chloride in the smooth muscle of the ra t portal vein. Acta Physiol. Scand., 78: 255-268. Harder, D. R., L. Belardinelli, N. Sperelakis, R. Rubio and R. M. Berne 1979 Effects of adenosine and nitroglycerin on the action potentials of large and small coronary arteries. Circulation Res., 44: 176-182. Ito, Y., H. Kuriyama and Y. Sakamoto 1970 Effects of tetraethylammonium chloride on the membrane activity of guinea pig stomach smooth muscle. J . Physiol., 211: 445-460. Kirkpatrick, C. T. 1975 Excitation and contraction in bovine tracheal smooth muscle. J. Physiol., 244: 263-281. Kuriyama, H., and A. Csapo 1961 A study of parturient uterus using the micro electrode technique. Endocrinol., 68: 1010-1025. Kuriyama, H., K. Ohshima and Y. Sakamoto 1971 The membrane properties of smooth muscle of the guinea pig portal vein in isotonic and hypertonic solutions. J. Physiol., 17: 179-199. Lowry, 0.H., N. H. Rosenbrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the Folin-phenol reagent. J. Biol. Chem., 193: 265-271. Martel, D., and A. Psychoyos 1978 Progesterone induced estrogen receptors in the rat uterus. J. Endocrinol., 76: 145-154. McCalden, T. A. 1975 Theinhibitory action of oestradiol17-6 and progesterone on venous smooth muscle. British J. Pharmac., 53: 183-192. Mekata, F., and H. Niu 1972 Biophysical effects of adrenaline on the smooth muscle of the rabbit common carotid artery. J. Gen. Physiol., 59: 92.102. Pavlik, E. J., and P. B. Coulson 1976 Hydroxylapatite “batch” assay for estrogen receptors: increased sensitivity over present receptor assays. J. Steroid Biochem., 7: 357-368. Pietras, R. J., and C. M. Szego 1975 Steroid hormoneresponsive isolated endometrial cell. Endocrinol., 96: 946-954. Radanyi, C., C. Mercier-Bodard, C. Secco-Millet, E. E. Baulieu and H. Richard-Foy 1977 aFetoprotein is not a component of the estradiol receptor of ra t uterus. Proc. Nat’l. Acad. Sci., 74: 2269-2272. Ross, E., and G. Schatz 1973 Assay of protein in the presence of high concentrations of sulfhydryl compounds. Analytical Biochem., 54: 304-306. Schoffeniels, E. 1969 Ionic composition of the arterial wall. Angiologica, 6: 65-87. Spasiani, E. 1975 Accessory reproductive organs in mammals: Control of cell and tissue transport by sex hormones. Pharmacol. Rev., 27: 207-286. Spaziani, E., and C. M. Szego 1958 The influence of estradiol and cortisol on uterine histamine of the overiectomized rat. Endocrinol., 63: 669-678. Swartz, S. K., and M. S. Soloff 1974 The lack of estrogen binding by human afetoprtein. J. Clin. Endocrinol. Metabol., 39: 589-591. Toft, D. O., and T. B. Tomasi 1978 An immunological comparison between mouse afetoprotein and the uterine estrogen receptor. Proc. SOC.Exp. Biol. and Med., 157: 594-598.
