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THE EFFECTS OF CATECHOLAMINES ON THE SMOOTH MUSCLE CELL MEMBRANE OF.THE RAT PORTAL VEIN IN VARIOUS IONIC SOLUTIONS YUTAKATAKATA Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan (Received 5 March 1979)
Abstract-l. Noradrenaline (Norad; > 100 ng/ml) depolarized the membrane and increased the spike frequency of the smooth muscle of portal vein in both normotensive and hypertensive rats (NR and SHR). 2. Isoprenaline (Isop; IOOng/ml) suppressed the spike generation without hyperpolarization of the membrane. Repetitively applied Isop produced the desensitization to B-receptor but not to u-receptor, thus causing excitatory action on the membrane by appearance of a-excitatory action. 3. Norad also produced desensitization to a-receptor, thus causing reduction in the membrane activity but not below the control level. 4. Desensitization to Isop or Norad was observed in both strains with the same extent. 5. Excess Ba-Krebs solution depolarized and excess Sr-Krebs solution hyperpolarized the membrane in both strains. 6. Norad increased the spike frequency in Ba-Krebs solution but prolonged the duration of a burst discharge (plateau formation) in Sr-Krebs solution in both strains. However, in excess Ca-Krebs solution the membrane was hyperpolarized in NR but not in SHR. 7. The action of Isop was enhanced in excess Ca-Krebs solution, slight suppressed in Sr-Krebs solution and markedly suppressed in Ba-Krebs solution. Changes in [Nalo or [K& markedly modified the membrane potential and membrane activity. 8. However, catecholamine actions were still preserved in both strains. 9. The results obtained from the present experiments were concluded that catecholamine actions were intensely modified by the changes in divalent cations than in monovalent cation, and no remarkable difference in catecholamine actions on’ the muscle membrane of portal vein under various ionic environments was observed in both strains.
Johansson et al., 1967; Somlyo & Somlyo, 1968; Su et
INTRODUCTION
al, 1964).
Smooth muscle cell membrane in some vascular tissues generates spontaneous rhythmic spike gener-
ations in vitro (Somlyo, 1975; Speden, 1970). A typical vascular bed where this spontaneous spike activity occur is the portal vein, and the shapes and fiythms of the spikes recorded from the single cell. of the guinea-pig, rat and rabbit have been described in detail (Golenhofen et al., 1973; ito & Kuriyama, 1971; Ito et al., 1978; Kitamura et al., 1976). Even the shapes of the spike and the membrane potential were much the same in both guinea-pig and rat tissues yet the underlying mechanism generating the membrane potential is reported to be not the same. For example, in vascular muscles of the rat portal vein the contribution of the active ion transport mechanism was less (Kuriyama & Suzuki, 1978), but in the guinea-pig portal vein, at least one third of the membrane potential was found to be closely related to the active ion pump mechanism (Kuriyama et al., 1971). Catecholamines produce either vasoconstriction or vasodilation on the isolated vascular tissues, and these actions depend primarily on the type of catecholamines and the vascular tissues (Casteels et al., 1977;
The sensitivity to catecholamines to the vascular bed differed slightly in normotensive (NR) and spontaneously hypertensive rat (SHR), i.e. the smooth muscle cell membrane is more depolarized by noradrenaline (Norad) in SHR than that in NR (Kuriyama & Suzuki, 1978). Furthermore, it has been reported that the artery of the SHR shows specific responses to [Sr],, as substitutes for [Cal0 and produces contraction, while these responses are not observed in NR (Bohr, 1974; Shibata et al., 1973). The sensitivities of venous muscle from hypertensive animals to vasoconstrictor and vasodilator agonists have been investigated using mechanographical method, and the response was either unchanged, enhanced or decreased when compared with veins from normotensive animals (Bevan et al., 1974; Greenberg & Bohr, 1975; Hallblck et al., 1971; Levy, 1973). The present experiments were attempts to determine the effects of catecholamines on the membrane activity of venous smooth muscle. Catecholamine actions on the membrane activity of NR were recorded in various ionic environments, and the effects of catecholamines were also compared in tissues excised from NR and SHR.
531
YU~AKA
532 MATERIALS
AND METHODS
Normotensive rats (NR), Wistar King A and spontaneously hypertensive rats (SHR), Okamoto and Aoki strain (Okamoto & Aoki, 1963) were used. These animals were supplied by the Nippon Rat Supply Co. Ltd. The average weights of 4-5 month old SHR and NR of either sex were about 180 and 25Og, respectively. The systolic blood pressures measured from tail ranged from 160 to 195 mmHg in SHR and from 110 to 120 mmHg in NR. The rats were stunned and bled. The connective tissue was carefully removed under a binocular microscope in Krebs solution at room temperature. The portal vein (1012mm in length and 1.5mm in width) was cut along the longitudinal axis, and the tissue was mounted in an organ bath of 2 ml in capacity and the temperature was maintained at 35-36°C. The solution was continuously pumped by a thermostatically controlled perfusion pump and was perfused at a rate of 3 ml/min. A microelectrode made with glass capillary filled with 3 M KCl, was inserted into the muscle cell from the outer surface of the portal vein. Measurements of the membrane activity were started after 6090min superfusion in an organ bath of 2 ml in capacity. A modified Krebs solution (Biilbring, 1955) of the following composition was used (mM): Na+ 137.4, K+ 5.9, Mg ‘+ 1.2, Cazf 2.5, HCO; 15.5, H,PO; 1.2, Cl- 134.0 and glucose, 11.5. This solution was aerated with 97% 0, and 3% CO2 and pH was adjusted to 7.2. In low [K],, solution, KC1 was replaced with an equimolar NaCl, and further reduction in [K],, KHP04 with equimolar NaHPO,. In low [NaJa solution, NaCl was replaced by LiCl, Tris or choline, and Na-free solution was prepared by replacement of NaHCO, with 5.9mM KHCOj and KC1 was removed pH was adjusted by Tris-(hydroxymethyl-aminometham)-buffer (Tris-buffer).
TAKATA
In Na-deficient or Na-free choline solution, 100 ng/ml atropine was added. Na-free (Tris) solution was prepared by removal of NaCl and NaHCO, by Tris. To obtain various concentrations of Ca-. Sr- or BaKrebs solution, [Cal,, was varied from Ca-free solution to 7.5 mM Ca solution. In high Ca-solution, the tonicity was not adjusted. To prepare Sr- or Ba-Krebs solution. various concentrations of Sr or Ba were added in Ca-free Krebs solution, Drugs used in these experiments were I-noradrenaline (norad, Merck), phentolamine (Regitine, CIBA-Geigy), phenoxyhenzamine (Nikken), isoprenaline (isop., Merck) and ouabain (Takeda). Drugs were dissolved in the perfusion solution and final concentrations of the drugs expressed as g/ml.
RESULTS
Eflects
of catecholamines
in normal
Krebs
solution
Membrane potentials measured from the smooth muscle cells of portal vein of NR and SHR were -47.8 + 1.6 mV (n = 50) and -48.3 f 1.7 mV, SD (n = 50).
respectively, and no difference was evident between the tissues. The spontaneous activity appeared as burst discharges between the quiescent periods. Norad (10 ng/ml) depolarized the membrane and increased the frequency of burst discharges. Increased concentration of Norad (1 pg/ml) produced marked depolarization and a depolarization block of spike generation was seen in tissues from both strains. The effects of Isop on the membrane activity of portal vein in SHR were observed. As shown in Fig. 1, Isop (long/ml) reduced the spike generation without any
a
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50 mV Phentolamint 10’7qlml l wash 1 min Fig. 1. Effects of isoprenaline on the membrane activities of the smooth muscle cell of portal vein in SHR; (d) effects of 1 pg/ml isoprenaline at the first trial. (e) at the third trial. (f) pretreated with 100 pg/ml phentolamine at the sixth trial of 1 pg/ml isoprenaline. (c)-(f) are recorded from the same cell.
Catecholamines actions on rat portal vein
533
remarkable changes in the membrane potential (a and b). By application with 1 pg/ml Isop the spike generation was completely suppressed (d), However, when
0% 0 NR
1 lg/ml Isop was repetitively applied to the tissue with intervals of every 15 min, the spike frequency was increased (in e, at the third trial). This excitatory action of Isop (1 pg/ml) was completely blocked by pretreatment with 100 ng/ml phentolamine (in f, at the sixth trial) or by phenoxybenzamine (1 pg/ml). Repetitively applied Norad (100 ng/ml) also produced the desensitization of the muscle membrane, however, the frequency of discharges was not reduced below the control. These results suggested that Isop possesses adrenergic a- and p-actions but Norad almost a-action for the smooth muscle cell membrane of portal vein, and these drug actions are not modified by appearance of hypertension. E&cts
ofcatecholamines
in difirent
dionlent ion con-
mining solutions
Fig. 2. Effects of various concentrations of [Cal0 or [Sr], on the membrane potential of portal vein in NR and SHR. Vertical bars indicate 2 x SD, n = 15.
Figure 2 shows the effects of [Cal0 and [Sr],, on the membrane potential of portal vein in NR and SHR. In 7.5 mM [Ca],, the membrane was hyperpolarized in mortal vein of NR but the hvneroolarization was not observed in SHR. On the other hand, inthe creased concentrations of [Sr], hyperpolarized muscle membrane in both strains. The slope of the
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Fig. 3. Effects of noradrenaline and isoprenaline on the membrane activity of portal vein in SHR in high Ca2+-Krebs solution (7.5 mM [Ca&). (a) and (b) effects of noradrenaline. (c) and (d) effects of first trial of 1pg/ml isoprenaline. (e) at the third trial of 1 &ml isoprenaline. (f) at the sixth trial of 1 pg/ml isoprenaline. (a). (b) and (c)-(f) are recorded from the same cell, respectively. The effects of catecholamine were observed after 20 min perfusion in excess [Cal, solution.
YUTAKA
534
TAKATA
Isop was produced by repetitive applications e.g. at the third trial of Isop with intervals of every 15 min, the membrane activity reappeared rapidly (e) and at the sixth trial the inhibitory action of Isop on the membrane activity almost ceased (f). However, no increase in the spike frequency was observed. Figure 4 shows the effects of Norad on the membrane activity of portal vein of SHR in Sr-Krebs solution. In Sr-Krebs solution (2.5 mM) the frequency of train discharges appeared between the silent periods was reduced and during the spike generation the depolarization was sustained. Norad (1 pg/ml) slightly increased the frequency and significantly prolonged the duration of burst discharges (c), and in some cells Norad showed a plateau formation. In Sr-solution Isop reduced the frequency of burst discharges without any remarkable changes in the membrane potential. The desensitization of the muscle membrane to Isop was also observed in Sr-Krebs solution, however, compensatory a-adrenergic excitatory action was not observed in both strains. Figure 5 shows the effects’ of Norad on the membrane activity of portal vein of SHR in Ba-Krebs solution. In 1.25 mM Ba-Krebs solution, the membrane was depolarized to -43 _t 2.8 mV (n = 20) and increased the spike frequency (a). Norad (long/ml) depolarized the membrane further to -40 + 1.9 mV (n = 20) and modified the spike configuration. At
membrane hyperpolarization produced by a IO-fold increase in [Cal, or [Sr],, plotted by logarithmic scale (0.25-2.5 mM) was 12 or 14 mV in NR, respectively. Changes in the membrane potential of both tissues in low [Sr]e were much the same as those observed in various [Cal,. By application with [Bale, a larger depolarization of the membrane was observed in SHR than that of NR. For example, 2.5 mM [Ba],, depolarized the membrane from -48.5 f 1.4mV to -34.1 f 2.4 mV (n = 15) in SHR, and from -47.8 + 2.2 mV to -41.5 + 2.2 mV (n = 15) in NR. The depolarization block of spike generation appeared in 2.5 mM [Bale in the tissue of SHR but it appeared in 5 mM [Bale in the tissue of NR. Figure 3 shows the effects of Norad and Isop on the membrane activity of portal vein in SHR in the presence of 7.5 mM [Ca]e. Excess [Cal0 reduced the frequency of burst discharges and numbers of the spike in a train discharges. Application of Norad (100 ng/ml) increased the frequency of burst qdischarges and depolarized the membrane (a). Increased concentration of Norad (1 pg/ml) produced depolarization block of the spike (b). The action of Isop was markedly potentiated (d) i.e. the first trial of 1 pg/ml Isop suppressed the spike activity more than 6min after the tissue was washed with Krebs solution (c and d). Desensitization of smooth muscle membrane to
l
Norad. 10-gglmt
-_ l
wash
Fig. 4. Effects of various concentrations (lo-s, lo-’ and IOF g/ml) of noradrenaline on the membrane activity of portal vein of SHR in Sr-Krebs solution. (d)-(f); membrane activity recorded before application of noradrenaline. (d)‘-(f)‘; during application of noradrenaline, respectively. (a) and (d’); lo-* g/ml noradrenaline (b) and (e’); lo-‘g/ml noradrenaline (c) and (f); 10e6 g/ml noradrenaline (a)-(f) are recorded from the same cell.
-
Catecholamines actions on rat portal vein
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Fig. 5. Effects of noradrenaline on the membrane activity of portal vein of SHR in Ba-Krebs solution; (1.25mM [Ba]J. (a) Ca-Krebs solution (2.5mM) was replaced with Ba-Krebs solution at dot. (e) Ba-Krebs solution was replaced wi!h Ca-Krebs solution at dot. (a)-(c) were recorded continuously. Intervals between (c) and (d), and (d) and (e) were 5 and 12 min. respectively. I pgglml, Norad produced the depolarization block of .the spike (d). The muscle membrane of portal vein in NR showed much the same response to Norad as those observed in SHR. Isop (< 100 ng/ml) did not show any effects in BaKrebs solutian, and 1 pg/ml Isop only slightly reduced the spike frequency. EJects of catecholamines in various monovalent cation containing solutions In 1.25 mM [K],,, the muscle membrane of portal vein in NR was slightly hyperpolariaed (from -48.7 k 2.9mV to -51.3 f 2.0mV, n F 15), the spike amplitude was enlarged and the frequency of spike generation was increased (2.4 times the control). When [K& was reduced to below 0.59 mM, the membrane potential remained the same as that observed in Krebs solution, but the spike frequency was increased (2.1 times the control). Prolonged treatment with K-deficient solution (0.59 mM) more than 30 min, the spike frequency was reduced (0.6 times in 0.59 mM [K10 without any remarkable changes in the spike amplitude. In K-free solution, the membrane was -48.1 + 2.3 mV depolarized from .to -39.2 f 2.6 mV (n = 20) and the frequency and amplitude of spikes were markedly suppressed. By replacement of K-free solution with Krebs solution, the membrane was transiently hyperpolarized to -64.8 f 2.4 mV (n = 10) and it was gradually depolarized to the resting membrane potential level (the
-48.3 + 2.1 mV, n = 20). Similar mean value changes in the membrane potential of the portal vein were observed in SHR during and after treatment with K-free solution. The effects of ouabain (1 pg/ml) on the membrane potential during and after treatment with K-free solution in the rat portal vein were compared with those obtained in the guinea-pig portal vein. As shown in Fig. 6, ouabain (1 pg/ml) slightly depolarized the membrane in Krebs solution in NR, but it depolarized the membrane and reduced the amplitude and frequency of the spike in the guinea-pig (a and d). When the tissue was perfused by ouabain containing K-free solution more than 20 min, the membrane was depolarized in both tissues (-36 mV) in NR and -29 mV in the guinea-pig). Whathe tissue was superfused with ouabain containing Krebs solution following pretreatment with ouabain containing K-free solution, marked differences in the membrane potential of portal vein were observed between both species. The grade of hyperpolarization was not affected in the rat by either treatment with Krebs or ouabain containing krebs solution (- 68 mV in Krebs solution and - 63 mV in ouabain containing solution), however, in the guinea-pig the membrane was hyperpolarized to -85 mV in Krebs solution, and to -52mV in ouabain containing solution (c and 1).This means that the membrane potential of the guinea-pig portal vein is closely related to the active ion pump mechanism than the
YUTAKA TAKATA
536 a
l
. ouabain 10-6g/d
wash
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ouabain lO*g/ml
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______._ l
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l
ouabain lg*g/ml
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I min Fig. 6. Effects of ouabain and [K],, removal on the membrane potential and membrane activity of portal vein of the rat and guinea-pig; (aHc); the rat portal vein (NR). (dHf); the guinea-pig portal vein. (a) and (d); effects of ouabain (I pg/ml). (b) and (e); recovery process following pretreatment with K-free solution. (c) and (f); Recovery process after pretreatment with ouabain (1 pg/ml) containing K-free solution.
i l
K-free
b l
Norad. 10_7g/ml
l
wash
c./ l
d
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-
50 mV 0 Isop. to-7g1ml
I . wash 1 min
Fig. 7. Effects of noradrenaline and isoprenaline on the membrane activity of NR in K-free solution; (a) effects of K-free solution. (b) and (c) effects of noradrenaline in K-free solution. (d) effects of isoprenaline in K-free solution. (aHd) are recorded from the same cell. The drug action was examined after 30 min superfusion with K-free solution.
Catecholamines actions on rat portal vein
rat portal vein. However, no difference was appearent between NR and SHR under the above procedure. Figure 7 shows the effects of Norad (100ng/ml and 1 pg/ml) and Isop (lOOng/ml) on the membrane potential and spike activity recorded from the tissue of NR in K-free solution. Norad (100 ng/ml) depolarized the membrane from -4lmV to -34mV (-53 mV in Krebs solution) and the spike was reappeared (b). Increased concentration of Norad (1 pg/ml) depolarized the membrane further to - 20 mV and produced the depolarization block of spike generation (c). By washing the tissue with K-free solution, the spike generation was ceased and the repolarization of the membrane required longer time than that observed in Krebs solution (c). Isop (100 ng/ml) showed larger hyperpolarization of the membrane (9 mV) in K-free solution than that in’ Krebs solution (d). To prepare Na-deficient or Na-free solution, choline-cl, LiCl or Tris-Cl was prepared. Choline containing solutions (68 mM, 15.5 mM Na or Na-free) consistently depolarized the membrane and increased the spike frequency of the portal vein in both strains. In Na-free choline solution, the muscle membrane of in NR was depolarized from the tissue - 49. I + 2.4 mV to -40.4 4 2.8 mV (n = 15) and the spike frequency was increased (1.6 times the control). In Na-free Li solution, the muscle membrane of portal vein in NR was depolarized from -49.4 f 2.1 mV
537
to - 32.9 + 2.4 mV (n = 20) and the spike generation was ceased. By washing the tissue with Krebs solution, the membrane was transiently hyperpolarized and this change in the membrane potential was not affected by pretreatment with ouabain (1 pg/ml). Figure 8 shows the effects of catecholamines on the membrane potential of pdrtal vein in NR in Na-free Li solution. In Na-free Li solution, the generation of spike was mostly blocked (a). Isop (100 ng/ml) hyperpolarized the membrane (-36.5 + 2.1 mV, n = 15) and Norad (1 pg/ml) depolarized the membrane (mean value -27.1 + 2.9 mV, n = 16). During depolarization period, spikes were reappeared transiently. In Na-free Tris solution, the muscle membrane of portal vein in NR was slightly hyperpolarized (4 mV) and then gradually depolarized to above the control observed in Krebs solution (from value -49.3 f 3.1 mV, n = 10 to -42.2 k 2.9 mV, n = 15). Norad (1 pgcglml)showed smaller depolarization of the membrane in Tris solution (-36.2 + 3.1 mV, n = IO) than that observed in Li-solution. Figure 9 shows the effects of Norad and Isop on the membrane activity recorded from portal vein of NR in Na-free Tris solution. Norad (lOOng/ml) generated the spike from the quiescent membrane without depolarization of the membrane, and increased concentration of Norad (pg/ml) produced the depolarization block of the spike generation (c). Isop (100 ng/ml) hyperpolarized the membrane (7 mV, n = 4) in Na-free Tris solution.
a l
b,..
( sub&. with Li )
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!
I I
. wash
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I
C
. Norad. lo*g!ml
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I
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Fig. 8. Effects of noradrenaline and isoprenaline on the membrane potential and membrane activity of oortal vein in NR in Na-free ILi) solution: (a): effects of rNal,, removal. (b);.effects of isoprenaline in k-free solution. (c); effects oindradrenal& in Na-free &luti& (d); effects. of replacement of Na-free solution with Krebs solution. All records are obtained from the same cell. The drug effect was examined after 30 min superfusion with Na-free’solution.
50 mV
YUTAKA
TAKATA
I_ l
Na-free ( sub&.
with
I
Tris)
b
0 Norad. 10% g/ml
l
wash
d 0 Isop. 10-7glml
lwash
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I
-_ l
wash
50 mV
art
I
min
Fig. 9. Effects of catecholamines on the membrane potential and membrane activity of portal vein in NR in Na-free (Tris) solution. (a); effects of substitution for [Na& with Tris. (b) and (c); effects of noradrenaline in Na-free solution. (d); effects of isoprenaline in Na-free solution. (e); effects of replacement of Na-free (Tris) solution with Krebs solution. The drug action was examined after 30 min superfusion with Na-free solution.
DISCUSSION
When the membrane potentials of the smooth muscle of portal vein in NR and SHR were compared in various ionic environments, excess [Cal0 hyperpolarized the membrane in NR but not in SHR. The Ba-solution more depolarized the membrane in SHR than that in NR, and the excess [Sr], hyperpolarized the membrane as the same extent in both strains. Excess [K-Jo, low [K&, low [Na& and low [Cal0 depolarized the membrane but no difference was observed in both strains. Therefore, differences of the membrane in both strains were only observed from the responses to high [Cal, and low or high [Ba&,. In the guinea-pig taenia coli, the effects of [Ba],, and [Sr], on the membrane potential and spike activity are considered to be due to suppression of the K-conductance (Sakamoto, 1971) and catecholamine actions are suppressed in the above solutions (Ohashi et al., 1967). In the smooth muscle cell of the rat portal vein, the response differed from the guinea-pig taenia coli. Because excess [Bale depolarized and excess [Srlo hyperpolarized the membrane. Norad (> 100 ng/ml) merkedly depolarized the membrane and increased the spike frequency in Ba-solution, while Norad depolarized the membrane and prolonged the depolarization during the spike generation (plateau formation) in Sr-solution. Isop markedly enhanced the inhibitory action on the membrane ao
tivity in excess Ca-solution, slightly suppressed in Srsolution and markedly suppressed in Ba-solution. However, the responses of the membrane to catecholamines in various divalent cations were much the same in both strains. Greenberg & Bohr (1975) concluded the mechanical response observed from portal veins of NR and SHR that threshold concentrations for responses to adrenaline, Norad, BAC12 and SrCl, were similar in both strains, however, in SHR high concentration of [Cal0 had a less depressant action on the response to the prostaglandins. The present experiments partly confirmed the above conclusion that catecholamines showed the. same actions on the smooth muscle cell membrane of portal vein of NR and SHR obtained in various divalent cation containing solutions. K-free and Na-free (Li or Tris) solution depolarized the membrane and suppressed the spike activity of muscle cells of the portal vein in both strains. Norad markedly depolarized the membrane in Na-free Li solution, K-free solution and slightly depolarized the membrane in Na-free Tris solution. Presumably, Norad increases the Li-permeability as it does on the Na-permeability. Furthermore, low or high concentration of Norad regenerated the spike from the quiescent membrane in Na-free or K-free solution with or without depolarization of the membrane, respectively. This means that the spike generation induced by Norad is partly due to depolarization of the
Catecholamines actions on rat portal vein membrane but the depolarization is not an essential mechanism. Additionally, the spike could be evoked
539
muscle cells of the rabbit main pulmonary artery. J. Physiol. 271, 63-79.
in Na-free solution in both strains, thus suggesting that the spike is mainly due to an increase in Cainflux. Isop suppressed the spike generation with or without hyperpolarization of the membrane in Krebs solution, but Isop consistently hyperpolarized the membrane in Na-free and K-free solutions. This result indicates that Isop possesses the property to hyperpolarize the membrane but this property is not an essential factor to suppress the spike generation. Desensitization of the inhibitorv /3-adrenoreceotor to repetitively applied Isop showeh ‘the excitatiob of the muscle membrane, but that of the excitatory r-adrenoreceptor to Norad did not produce the inhibition of the membrane activity. Presumably Isop possesses potent /?- and weak u-actions, but Norad solely cc-action or/with very weak /?-action to muscle membranes of the rat portal vein in both strains. By numerous numbers of articles concerning the drug action on the contraction-relaxation phenomena of vascular smooth muscles, remarkable regional and species differences of the vascular muscle activity are reported. The species differences in the vascular tissue were also apparent from the present experiment i.e. the membrane potential is largely contributed by Na-K active pump mechanism in the guinea-pig portal vein and the membrane potential is less contributed by the pump mechanism in the rat portal vein, even the membrane potentials measured from both species were much the same. In aorta of SHR but not in that of NR, Sr-containing solution generates the contraction without any
GOLENHOFEN K., HERMSTE~N N. & LAMMELE. (1973) Membrane potential and contraction of vascular smooth muscle (portal vein) during application of noradrenaline and high potassium, and selective inhibitory effects of iproveratril (verapamil). Microvascular Res. 5, 73-80. GREENBERG S. & BOHRD. F. (1975) Venous smooth muscle in hypertension (Enhanced contractility of portal veins from spontaneously hypertensive rats). Circ. Res. (Suppl. I), 3637, I 208-I 215. HALLBACKM., LUNDGRENY. & WEISSL. (1971) Reactivity to noradrenaline of aortic strips and portal veins from spontaneously hypertensive and normotensive rats. Acta
additional treatment (Bohr, 1974; Shibata et al., 1973). However, such a remarkable difference on the membrane property of the rat portal vein in both strains was not observed. The present results showed that the action of cate-
409-424. LEVY J. V. (1973) Contractile responses of isolated portal veins from normal and spontaneously . hypertensive rats. .. C/in. Res. 21. 237. _ OHASHIH.. NONOMLJRA Y. & OHGA A. (1967) Effects of
cholamines is modified under various ionic environments especially in the changes in divalent cations, however the responses to catecholamines are much the same in both strains. Therefore, if the difference in the membrane property of vascular tissues exists in both strains, it might appear dominantly on the muscle cell of the arterial system rather than the venous system from the nature and aetiology of a hypertension.
BEVANJ. A., BEVANR. D.. PECRAMB. L., PURDYR. E. & Su C. (1974) Increased rZsponsiveness of veins to adrenergic stimulation in experimental hypertension. Blood Vessels 1I, 241-244. BOHR D. F. (1974) Reactivity of vascular smooth muscle from normal and hvoertensive rats; effect of several cations. Fedn Proc. Fk?n Am, Sots exp. Biol. 33, 127-l 32. B~~LRRING E. (1955) Correlation between membrane ootentension
in smooth
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ITO Y., SUZUKI H. & KUR~YAMAH. (1978) Effects of sodium nitroprusside on smooth muscle cells of rabbit pulmonary artery and portal vein. J. Pharmac. exp. Thu. 207, In press. JOHAN~~ON B., JOHSSON O., AXELSON J. & WAHLSTR~M B. (1967) Electrical and mechanical characteristics lar smooth muscle response to norepinephrine proterenol. Circ. Res. il, 619-633.
of vascuand iso-
KITAMURAK.. SUZUKI H. & KURIYAMAH. (1976) Prostaglandin action on the main pulmonary artery and portal vein of the rabbit. Jap. J. Physiol. 26, 68 l-692. KURIYAMAH., OHSHIMAK. & SAKAMOTO Y. (1971) The membrane properties of the smooth muscle of the guinea-pig portal vein in isotonic and hypertonic solutions. J. Physiol. 217, 179-199, 1971. KURIYAMAH. & SUZUKIH. (1978) Electrical property and chemical sensitivity of the smooth muscle cell membrane of portal vein and pulmonary artery from normotensive and spontaneously hypertensive rats. J. Physiol. 285,
angiotensin, bradykinin and oxytocin on electrical and mechanical activities in the taenia coli of the guinea pig. Jap. J. Pharmac. 17, 247-257.
OKAMOTO N. & AOKI K. (1963) Development of a strain of spontaneously hypertensive rats. Jap. Circ. J. 27. 282-293.
SAKAMOTO Y. (1971) Electrical activity of guinea-pig taenia coli in calcium locke solution. Jap. J. Physiol. 21, 295-306.
SHIBATA S., KURAHASHIK. & KUCHIIM. (1973) A possible etiology of contractility impairment of vascular smooth muscle from spontaneously hypertensive rats. J. Pharmat. exp. Ther: 185, 406-417. -SOMLYO A. P. (1975) Vascular smooth muscle. In Cellular Pharmacology of ‘Excitable Tissues (Edited by Toshio
REFERENCES
tial, spike hischarge and Physiol. 128, 20&22 I.
physiol. stand. 81, 176-181.
ITO Y. & KURIYAMAH. (1971) Membrane properties of the smooth-muscle fibres of the guinea-pig portal vein. J.
muscle.
J.
CAS~EEL~R., KITAMURAK., KURIYAMAH. & SUZUKI H. (1977) Excitation-contraction coupling in the smooth
NARAHASHIT.), pp. 360-407. Thomas, Springfield, Illinois. SOMLYO A. V. & SOMLYO A. P. (1968) Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J. Phormoc. exp. Ther. 159, 12%145. SPEDENR. N. (1970) Excitation of vascular smooth muscle. In Smooth Muscle (Edited by BULBRINGE., BRADING A. F., JONES A. W. & TOMITAT.), pp. . . 129-145. Edward Arnold, London. Su C., BEVANJ. A. & URISILLOR. C. (1964) Electrical quiescence of pulmonary artery smooth muscle during sympathomimetic stimulation. Circ. Res. 15, 20-27.
I IOI-0531$0?.00 0
THE EFFECTS OF CATECHOLAMINES ON THE SMOOTH MUSCLE CELL MEMBRANE OF.THE RAT PORTAL VEIN IN VARIOUS IONIC SOLUTIONS YUTAKATAKATA Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan (Received 5 March 1979)
Abstract-l. Noradrenaline (Norad; > 100 ng/ml) depolarized the membrane and increased the spike frequency of the smooth muscle of portal vein in both normotensive and hypertensive rats (NR and SHR). 2. Isoprenaline (Isop; IOOng/ml) suppressed the spike generation without hyperpolarization of the membrane. Repetitively applied Isop produced the desensitization to B-receptor but not to u-receptor, thus causing excitatory action on the membrane by appearance of a-excitatory action. 3. Norad also produced desensitization to a-receptor, thus causing reduction in the membrane activity but not below the control level. 4. Desensitization to Isop or Norad was observed in both strains with the same extent. 5. Excess Ba-Krebs solution depolarized and excess Sr-Krebs solution hyperpolarized the membrane in both strains. 6. Norad increased the spike frequency in Ba-Krebs solution but prolonged the duration of a burst discharge (plateau formation) in Sr-Krebs solution in both strains. However, in excess Ca-Krebs solution the membrane was hyperpolarized in NR but not in SHR. 7. The action of Isop was enhanced in excess Ca-Krebs solution, slight suppressed in Sr-Krebs solution and markedly suppressed in Ba-Krebs solution. Changes in [Nalo or [K& markedly modified the membrane potential and membrane activity. 8. However, catecholamine actions were still preserved in both strains. 9. The results obtained from the present experiments were concluded that catecholamine actions were intensely modified by the changes in divalent cations than in monovalent cation, and no remarkable difference in catecholamine actions on’ the muscle membrane of portal vein under various ionic environments was observed in both strains.
Johansson et al., 1967; Somlyo & Somlyo, 1968; Su et
INTRODUCTION
al, 1964).
Smooth muscle cell membrane in some vascular tissues generates spontaneous rhythmic spike gener-
ations in vitro (Somlyo, 1975; Speden, 1970). A typical vascular bed where this spontaneous spike activity occur is the portal vein, and the shapes and fiythms of the spikes recorded from the single cell. of the guinea-pig, rat and rabbit have been described in detail (Golenhofen et al., 1973; ito & Kuriyama, 1971; Ito et al., 1978; Kitamura et al., 1976). Even the shapes of the spike and the membrane potential were much the same in both guinea-pig and rat tissues yet the underlying mechanism generating the membrane potential is reported to be not the same. For example, in vascular muscles of the rat portal vein the contribution of the active ion transport mechanism was less (Kuriyama & Suzuki, 1978), but in the guinea-pig portal vein, at least one third of the membrane potential was found to be closely related to the active ion pump mechanism (Kuriyama et al., 1971). Catecholamines produce either vasoconstriction or vasodilation on the isolated vascular tissues, and these actions depend primarily on the type of catecholamines and the vascular tissues (Casteels et al., 1977;
The sensitivity to catecholamines to the vascular bed differed slightly in normotensive (NR) and spontaneously hypertensive rat (SHR), i.e. the smooth muscle cell membrane is more depolarized by noradrenaline (Norad) in SHR than that in NR (Kuriyama & Suzuki, 1978). Furthermore, it has been reported that the artery of the SHR shows specific responses to [Sr],, as substitutes for [Cal0 and produces contraction, while these responses are not observed in NR (Bohr, 1974; Shibata et al., 1973). The sensitivities of venous muscle from hypertensive animals to vasoconstrictor and vasodilator agonists have been investigated using mechanographical method, and the response was either unchanged, enhanced or decreased when compared with veins from normotensive animals (Bevan et al., 1974; Greenberg & Bohr, 1975; Hallblck et al., 1971; Levy, 1973). The present experiments were attempts to determine the effects of catecholamines on the membrane activity of venous smooth muscle. Catecholamine actions on the membrane activity of NR were recorded in various ionic environments, and the effects of catecholamines were also compared in tissues excised from NR and SHR.
531
YU~AKA
532 MATERIALS
AND METHODS
Normotensive rats (NR), Wistar King A and spontaneously hypertensive rats (SHR), Okamoto and Aoki strain (Okamoto & Aoki, 1963) were used. These animals were supplied by the Nippon Rat Supply Co. Ltd. The average weights of 4-5 month old SHR and NR of either sex were about 180 and 25Og, respectively. The systolic blood pressures measured from tail ranged from 160 to 195 mmHg in SHR and from 110 to 120 mmHg in NR. The rats were stunned and bled. The connective tissue was carefully removed under a binocular microscope in Krebs solution at room temperature. The portal vein (1012mm in length and 1.5mm in width) was cut along the longitudinal axis, and the tissue was mounted in an organ bath of 2 ml in capacity and the temperature was maintained at 35-36°C. The solution was continuously pumped by a thermostatically controlled perfusion pump and was perfused at a rate of 3 ml/min. A microelectrode made with glass capillary filled with 3 M KCl, was inserted into the muscle cell from the outer surface of the portal vein. Measurements of the membrane activity were started after 6090min superfusion in an organ bath of 2 ml in capacity. A modified Krebs solution (Biilbring, 1955) of the following composition was used (mM): Na+ 137.4, K+ 5.9, Mg ‘+ 1.2, Cazf 2.5, HCO; 15.5, H,PO; 1.2, Cl- 134.0 and glucose, 11.5. This solution was aerated with 97% 0, and 3% CO2 and pH was adjusted to 7.2. In low [K],, solution, KC1 was replaced with an equimolar NaCl, and further reduction in [K],, KHP04 with equimolar NaHPO,. In low [NaJa solution, NaCl was replaced by LiCl, Tris or choline, and Na-free solution was prepared by replacement of NaHCO, with 5.9mM KHCOj and KC1 was removed pH was adjusted by Tris-(hydroxymethyl-aminometham)-buffer (Tris-buffer).
TAKATA
In Na-deficient or Na-free choline solution, 100 ng/ml atropine was added. Na-free (Tris) solution was prepared by removal of NaCl and NaHCO, by Tris. To obtain various concentrations of Ca-. Sr- or BaKrebs solution, [Cal,, was varied from Ca-free solution to 7.5 mM Ca solution. In high Ca-solution, the tonicity was not adjusted. To prepare Sr- or Ba-Krebs solution. various concentrations of Sr or Ba were added in Ca-free Krebs solution, Drugs used in these experiments were I-noradrenaline (norad, Merck), phentolamine (Regitine, CIBA-Geigy), phenoxyhenzamine (Nikken), isoprenaline (isop., Merck) and ouabain (Takeda). Drugs were dissolved in the perfusion solution and final concentrations of the drugs expressed as g/ml.
RESULTS
Eflects
of catecholamines
in normal
Krebs
solution
Membrane potentials measured from the smooth muscle cells of portal vein of NR and SHR were -47.8 + 1.6 mV (n = 50) and -48.3 f 1.7 mV, SD (n = 50).
respectively, and no difference was evident between the tissues. The spontaneous activity appeared as burst discharges between the quiescent periods. Norad (10 ng/ml) depolarized the membrane and increased the frequency of burst discharges. Increased concentration of Norad (1 pg/ml) produced marked depolarization and a depolarization block of spike generation was seen in tissues from both strains. The effects of Isop on the membrane activity of portal vein in SHR were observed. As shown in Fig. 1, Isop (long/ml) reduced the spike generation without any
a
.
l Wuh
i0-9g/ml
hp.
b
l
l
hp.
hp.
lO*glml
. wash
a wash
lo-‘g/ml
d
l
~sap.1O+g/mI
*w&h
e ,,I,
‘1 \
‘I
lwliW l
f Pretreated
with
ISOP.
lo-gg/mt
Phentolamine
l
wash
10D7g1ml
50 mV Phentolamint 10’7qlml l wash 1 min Fig. 1. Effects of isoprenaline on the membrane activities of the smooth muscle cell of portal vein in SHR; (d) effects of 1 pg/ml isoprenaline at the first trial. (e) at the third trial. (f) pretreated with 100 pg/ml phentolamine at the sixth trial of 1 pg/ml isoprenaline. (c)-(f) are recorded from the same cell.
Catecholamines actions on rat portal vein
533
remarkable changes in the membrane potential (a and b). By application with 1 pg/ml Isop the spike generation was completely suppressed (d), However, when
0% 0 NR
1 lg/ml Isop was repetitively applied to the tissue with intervals of every 15 min, the spike frequency was increased (in e, at the third trial). This excitatory action of Isop (1 pg/ml) was completely blocked by pretreatment with 100 ng/ml phentolamine (in f, at the sixth trial) or by phenoxybenzamine (1 pg/ml). Repetitively applied Norad (100 ng/ml) also produced the desensitization of the muscle membrane, however, the frequency of discharges was not reduced below the control. These results suggested that Isop possesses adrenergic a- and p-actions but Norad almost a-action for the smooth muscle cell membrane of portal vein, and these drug actions are not modified by appearance of hypertension. E&cts
ofcatecholamines
in difirent
dionlent ion con-
mining solutions
Fig. 2. Effects of various concentrations of [Cal0 or [Sr], on the membrane potential of portal vein in NR and SHR. Vertical bars indicate 2 x SD, n = 15.
Figure 2 shows the effects of [Cal0 and [Sr],, on the membrane potential of portal vein in NR and SHR. In 7.5 mM [Ca],, the membrane was hyperpolarized in mortal vein of NR but the hvneroolarization was not observed in SHR. On the other hand, inthe creased concentrations of [Sr], hyperpolarized muscle membrane in both strains. The slope of the
a l
Norad. 10~7g1ml
c --J_.-4_ 0
l
wash
l wash
__’
.
Isop.
l
,
j
to-Qrd
lsep . IO~glml
I
-
Isop. lO*glml
l
l
wash
i_I
wash
I min
Fig. 3. Effects of noradrenaline and isoprenaline on the membrane activity of portal vein in SHR in high Ca2+-Krebs solution (7.5 mM [Ca&). (a) and (b) effects of noradrenaline. (c) and (d) effects of first trial of 1pg/ml isoprenaline. (e) at the third trial of 1 &ml isoprenaline. (f) at the sixth trial of 1 pg/ml isoprenaline. (a). (b) and (c)-(f) are recorded from the same cell, respectively. The effects of catecholamine were observed after 20 min perfusion in excess [Cal, solution.
YUTAKA
534
TAKATA
Isop was produced by repetitive applications e.g. at the third trial of Isop with intervals of every 15 min, the membrane activity reappeared rapidly (e) and at the sixth trial the inhibitory action of Isop on the membrane activity almost ceased (f). However, no increase in the spike frequency was observed. Figure 4 shows the effects of Norad on the membrane activity of portal vein of SHR in Sr-Krebs solution. In Sr-Krebs solution (2.5 mM) the frequency of train discharges appeared between the silent periods was reduced and during the spike generation the depolarization was sustained. Norad (1 pg/ml) slightly increased the frequency and significantly prolonged the duration of burst discharges (c), and in some cells Norad showed a plateau formation. In Sr-solution Isop reduced the frequency of burst discharges without any remarkable changes in the membrane potential. The desensitization of the muscle membrane to Isop was also observed in Sr-Krebs solution, however, compensatory a-adrenergic excitatory action was not observed in both strains. Figure 5 shows the effects’ of Norad on the membrane activity of portal vein of SHR in Ba-Krebs solution. In 1.25 mM Ba-Krebs solution, the membrane was depolarized to -43 _t 2.8 mV (n = 20) and increased the spike frequency (a). Norad (long/ml) depolarized the membrane further to -40 + 1.9 mV (n = 20) and modified the spike configuration. At
membrane hyperpolarization produced by a IO-fold increase in [Cal, or [Sr],, plotted by logarithmic scale (0.25-2.5 mM) was 12 or 14 mV in NR, respectively. Changes in the membrane potential of both tissues in low [Sr]e were much the same as those observed in various [Cal,. By application with [Bale, a larger depolarization of the membrane was observed in SHR than that of NR. For example, 2.5 mM [Ba],, depolarized the membrane from -48.5 f 1.4mV to -34.1 f 2.4 mV (n = 15) in SHR, and from -47.8 + 2.2 mV to -41.5 + 2.2 mV (n = 15) in NR. The depolarization block of spike generation appeared in 2.5 mM [Bale in the tissue of SHR but it appeared in 5 mM [Bale in the tissue of NR. Figure 3 shows the effects of Norad and Isop on the membrane activity of portal vein in SHR in the presence of 7.5 mM [Ca]e. Excess [Cal0 reduced the frequency of burst discharges and numbers of the spike in a train discharges. Application of Norad (100 ng/ml) increased the frequency of burst qdischarges and depolarized the membrane (a). Increased concentration of Norad (1 pg/ml) produced depolarization block of the spike (b). The action of Isop was markedly potentiated (d) i.e. the first trial of 1 pg/ml Isop suppressed the spike activity more than 6min after the tissue was washed with Krebs solution (c and d). Desensitization of smooth muscle membrane to
l
Norad. 10-gglmt
-_ l
wash
Fig. 4. Effects of various concentrations (lo-s, lo-’ and IOF g/ml) of noradrenaline on the membrane activity of portal vein of SHR in Sr-Krebs solution. (d)-(f); membrane activity recorded before application of noradrenaline. (d)‘-(f)‘; during application of noradrenaline, respectively. (a) and (d’); lo-* g/ml noradrenaline (b) and (e’); lo-‘g/ml noradrenaline (c) and (f); 10e6 g/ml noradrenaline (a)-(f) are recorded from the same cell.
-
Catecholamines actions on rat portal vein
a
__ ......,
ll~.-.-.-.---“..-r-
_._...__II._.._““-
.-
0
Ra 1.25mb-i
. wash
l
. wash
Norad. 10-7g/ml
d l
Norad. 10-6glml
l
wash
50 mV
e . wash
out
1 min
Fig. 5. Effects of noradrenaline on the membrane activity of portal vein of SHR in Ba-Krebs solution; (1.25mM [Ba]J. (a) Ca-Krebs solution (2.5mM) was replaced with Ba-Krebs solution at dot. (e) Ba-Krebs solution was replaced wi!h Ca-Krebs solution at dot. (a)-(c) were recorded continuously. Intervals between (c) and (d), and (d) and (e) were 5 and 12 min. respectively. I pgglml, Norad produced the depolarization block of .the spike (d). The muscle membrane of portal vein in NR showed much the same response to Norad as those observed in SHR. Isop (< 100 ng/ml) did not show any effects in BaKrebs solutian, and 1 pg/ml Isop only slightly reduced the spike frequency. EJects of catecholamines in various monovalent cation containing solutions In 1.25 mM [K],,, the muscle membrane of portal vein in NR was slightly hyperpolariaed (from -48.7 k 2.9mV to -51.3 f 2.0mV, n F 15), the spike amplitude was enlarged and the frequency of spike generation was increased (2.4 times the control). When [K& was reduced to below 0.59 mM, the membrane potential remained the same as that observed in Krebs solution, but the spike frequency was increased (2.1 times the control). Prolonged treatment with K-deficient solution (0.59 mM) more than 30 min, the spike frequency was reduced (0.6 times in 0.59 mM [K10 without any remarkable changes in the spike amplitude. In K-free solution, the membrane was -48.1 + 2.3 mV depolarized from .to -39.2 f 2.6 mV (n = 20) and the frequency and amplitude of spikes were markedly suppressed. By replacement of K-free solution with Krebs solution, the membrane was transiently hyperpolarized to -64.8 f 2.4 mV (n = 10) and it was gradually depolarized to the resting membrane potential level (the
-48.3 + 2.1 mV, n = 20). Similar mean value changes in the membrane potential of the portal vein were observed in SHR during and after treatment with K-free solution. The effects of ouabain (1 pg/ml) on the membrane potential during and after treatment with K-free solution in the rat portal vein were compared with those obtained in the guinea-pig portal vein. As shown in Fig. 6, ouabain (1 pg/ml) slightly depolarized the membrane in Krebs solution in NR, but it depolarized the membrane and reduced the amplitude and frequency of the spike in the guinea-pig (a and d). When the tissue was perfused by ouabain containing K-free solution more than 20 min, the membrane was depolarized in both tissues (-36 mV) in NR and -29 mV in the guinea-pig). Whathe tissue was superfused with ouabain containing Krebs solution following pretreatment with ouabain containing K-free solution, marked differences in the membrane potential of portal vein were observed between both species. The grade of hyperpolarization was not affected in the rat by either treatment with Krebs or ouabain containing krebs solution (- 68 mV in Krebs solution and - 63 mV in ouabain containing solution), however, in the guinea-pig the membrane was hyperpolarized to -85 mV in Krebs solution, and to -52mV in ouabain containing solution (c and 1).This means that the membrane potential of the guinea-pig portal vein is closely related to the active ion pump mechanism than the
YUTAKA TAKATA
536 a
l
. ouabain 10-6g/d
wash
b. Prelreated wilh K-free
c. Pretreated with K-free.
l
ouabain lO*g/ml
wash( Krebs + wabaln
Pretreated
ouatain tOg/mt wrth K-free
t+treated
with K-free.
l
lO*gIml
)
ouabain lO-6glmt
I 50 mV I
______._ l
wash ( Krcbs
l
ouabain lg*g/ml
)
I min Fig. 6. Effects of ouabain and [K],, removal on the membrane potential and membrane activity of portal vein of the rat and guinea-pig; (aHc); the rat portal vein (NR). (dHf); the guinea-pig portal vein. (a) and (d); effects of ouabain (I pg/ml). (b) and (e); recovery process following pretreatment with K-free solution. (c) and (f); Recovery process after pretreatment with ouabain (1 pg/ml) containing K-free solution.
i l
K-free
b l
Norad. 10_7g/ml
l
wash
c./ l
d
Norad. lodglmt
. wash
-
50 mV 0 Isop. to-7g1ml
I . wash 1 min
Fig. 7. Effects of noradrenaline and isoprenaline on the membrane activity of NR in K-free solution; (a) effects of K-free solution. (b) and (c) effects of noradrenaline in K-free solution. (d) effects of isoprenaline in K-free solution. (aHd) are recorded from the same cell. The drug action was examined after 30 min superfusion with K-free solution.
Catecholamines actions on rat portal vein
rat portal vein. However, no difference was appearent between NR and SHR under the above procedure. Figure 7 shows the effects of Norad (100ng/ml and 1 pg/ml) and Isop (lOOng/ml) on the membrane potential and spike activity recorded from the tissue of NR in K-free solution. Norad (100 ng/ml) depolarized the membrane from -4lmV to -34mV (-53 mV in Krebs solution) and the spike was reappeared (b). Increased concentration of Norad (1 pg/ml) depolarized the membrane further to - 20 mV and produced the depolarization block of spike generation (c). By washing the tissue with K-free solution, the spike generation was ceased and the repolarization of the membrane required longer time than that observed in Krebs solution (c). Isop (100 ng/ml) showed larger hyperpolarization of the membrane (9 mV) in K-free solution than that in’ Krebs solution (d). To prepare Na-deficient or Na-free solution, choline-cl, LiCl or Tris-Cl was prepared. Choline containing solutions (68 mM, 15.5 mM Na or Na-free) consistently depolarized the membrane and increased the spike frequency of the portal vein in both strains. In Na-free choline solution, the muscle membrane of in NR was depolarized from the tissue - 49. I + 2.4 mV to -40.4 4 2.8 mV (n = 15) and the spike frequency was increased (1.6 times the control). In Na-free Li solution, the muscle membrane of portal vein in NR was depolarized from -49.4 f 2.1 mV
537
to - 32.9 + 2.4 mV (n = 20) and the spike generation was ceased. By washing the tissue with Krebs solution, the membrane was transiently hyperpolarized and this change in the membrane potential was not affected by pretreatment with ouabain (1 pg/ml). Figure 8 shows the effects of catecholamines on the membrane potential of pdrtal vein in NR in Na-free Li solution. In Na-free Li solution, the generation of spike was mostly blocked (a). Isop (100 ng/ml) hyperpolarized the membrane (-36.5 + 2.1 mV, n = 15) and Norad (1 pg/ml) depolarized the membrane (mean value -27.1 + 2.9 mV, n = 16). During depolarization period, spikes were reappeared transiently. In Na-free Tris solution, the muscle membrane of portal vein in NR was slightly hyperpolarized (4 mV) and then gradually depolarized to above the control observed in Krebs solution (from value -49.3 f 3.1 mV, n = 10 to -42.2 k 2.9 mV, n = 15). Norad (1 pgcglml)showed smaller depolarization of the membrane in Tris solution (-36.2 + 3.1 mV, n = IO) than that observed in Li-solution. Figure 9 shows the effects of Norad and Isop on the membrane activity recorded from portal vein of NR in Na-free Tris solution. Norad (lOOng/ml) generated the spike from the quiescent membrane without depolarization of the membrane, and increased concentration of Norad (pg/ml) produced the depolarization block of the spike generation (c). Isop (100 ng/ml) hyperpolarized the membrane (7 mV, n = 4) in Na-free Tris solution.
a l
b,..
( sub&. with Li )
t&free
!
I I
. wash
0 Isop. 10’‘g/ml
I
C
. Norad. lo*g!ml
l
wash
d
“I” l
1
I’
I
JJJJ&Jj I min.
Fig. 8. Effects of noradrenaline and isoprenaline on the membrane potential and membrane activity of oortal vein in NR in Na-free ILi) solution: (a): effects of rNal,, removal. (b);.effects of isoprenaline in k-free solution. (c); effects oindradrenal& in Na-free &luti& (d); effects. of replacement of Na-free solution with Krebs solution. All records are obtained from the same cell. The drug effect was examined after 30 min superfusion with Na-free’solution.
50 mV
YUTAKA
TAKATA
I_ l
Na-free ( sub&.
with
I
Tris)
b
0 Norad. 10% g/ml
l
wash
d 0 Isop. 10-7glml
lwash
c
I
-_ l
wash
50 mV
art
I
min
Fig. 9. Effects of catecholamines on the membrane potential and membrane activity of portal vein in NR in Na-free (Tris) solution. (a); effects of substitution for [Na& with Tris. (b) and (c); effects of noradrenaline in Na-free solution. (d); effects of isoprenaline in Na-free solution. (e); effects of replacement of Na-free (Tris) solution with Krebs solution. The drug action was examined after 30 min superfusion with Na-free solution.
DISCUSSION
When the membrane potentials of the smooth muscle of portal vein in NR and SHR were compared in various ionic environments, excess [Cal0 hyperpolarized the membrane in NR but not in SHR. The Ba-solution more depolarized the membrane in SHR than that in NR, and the excess [Sr], hyperpolarized the membrane as the same extent in both strains. Excess [K-Jo, low [K&, low [Na& and low [Cal0 depolarized the membrane but no difference was observed in both strains. Therefore, differences of the membrane in both strains were only observed from the responses to high [Cal, and low or high [Ba&,. In the guinea-pig taenia coli, the effects of [Ba],, and [Sr], on the membrane potential and spike activity are considered to be due to suppression of the K-conductance (Sakamoto, 1971) and catecholamine actions are suppressed in the above solutions (Ohashi et al., 1967). In the smooth muscle cell of the rat portal vein, the response differed from the guinea-pig taenia coli. Because excess [Bale depolarized and excess [Srlo hyperpolarized the membrane. Norad (> 100 ng/ml) merkedly depolarized the membrane and increased the spike frequency in Ba-solution, while Norad depolarized the membrane and prolonged the depolarization during the spike generation (plateau formation) in Sr-solution. Isop markedly enhanced the inhibitory action on the membrane ao
tivity in excess Ca-solution, slightly suppressed in Srsolution and markedly suppressed in Ba-solution. However, the responses of the membrane to catecholamines in various divalent cations were much the same in both strains. Greenberg & Bohr (1975) concluded the mechanical response observed from portal veins of NR and SHR that threshold concentrations for responses to adrenaline, Norad, BAC12 and SrCl, were similar in both strains, however, in SHR high concentration of [Cal0 had a less depressant action on the response to the prostaglandins. The present experiments partly confirmed the above conclusion that catecholamines showed the. same actions on the smooth muscle cell membrane of portal vein of NR and SHR obtained in various divalent cation containing solutions. K-free and Na-free (Li or Tris) solution depolarized the membrane and suppressed the spike activity of muscle cells of the portal vein in both strains. Norad markedly depolarized the membrane in Na-free Li solution, K-free solution and slightly depolarized the membrane in Na-free Tris solution. Presumably, Norad increases the Li-permeability as it does on the Na-permeability. Furthermore, low or high concentration of Norad regenerated the spike from the quiescent membrane in Na-free or K-free solution with or without depolarization of the membrane, respectively. This means that the spike generation induced by Norad is partly due to depolarization of the
Catecholamines actions on rat portal vein membrane but the depolarization is not an essential mechanism. Additionally, the spike could be evoked
539
muscle cells of the rabbit main pulmonary artery. J. Physiol. 271, 63-79.
in Na-free solution in both strains, thus suggesting that the spike is mainly due to an increase in Cainflux. Isop suppressed the spike generation with or without hyperpolarization of the membrane in Krebs solution, but Isop consistently hyperpolarized the membrane in Na-free and K-free solutions. This result indicates that Isop possesses the property to hyperpolarize the membrane but this property is not an essential factor to suppress the spike generation. Desensitization of the inhibitorv /3-adrenoreceotor to repetitively applied Isop showeh ‘the excitatiob of the muscle membrane, but that of the excitatory r-adrenoreceptor to Norad did not produce the inhibition of the membrane activity. Presumably Isop possesses potent /?- and weak u-actions, but Norad solely cc-action or/with very weak /?-action to muscle membranes of the rat portal vein in both strains. By numerous numbers of articles concerning the drug action on the contraction-relaxation phenomena of vascular smooth muscles, remarkable regional and species differences of the vascular muscle activity are reported. The species differences in the vascular tissue were also apparent from the present experiment i.e. the membrane potential is largely contributed by Na-K active pump mechanism in the guinea-pig portal vein and the membrane potential is less contributed by the pump mechanism in the rat portal vein, even the membrane potentials measured from both species were much the same. In aorta of SHR but not in that of NR, Sr-containing solution generates the contraction without any
GOLENHOFEN K., HERMSTE~N N. & LAMMELE. (1973) Membrane potential and contraction of vascular smooth muscle (portal vein) during application of noradrenaline and high potassium, and selective inhibitory effects of iproveratril (verapamil). Microvascular Res. 5, 73-80. GREENBERG S. & BOHRD. F. (1975) Venous smooth muscle in hypertension (Enhanced contractility of portal veins from spontaneously hypertensive rats). Circ. Res. (Suppl. I), 3637, I 208-I 215. HALLBACKM., LUNDGRENY. & WEISSL. (1971) Reactivity to noradrenaline of aortic strips and portal veins from spontaneously hypertensive and normotensive rats. Acta
additional treatment (Bohr, 1974; Shibata et al., 1973). However, such a remarkable difference on the membrane property of the rat portal vein in both strains was not observed. The present results showed that the action of cate-
409-424. LEVY J. V. (1973) Contractile responses of isolated portal veins from normal and spontaneously . hypertensive rats. .. C/in. Res. 21. 237. _ OHASHIH.. NONOMLJRA Y. & OHGA A. (1967) Effects of
cholamines is modified under various ionic environments especially in the changes in divalent cations, however the responses to catecholamines are much the same in both strains. Therefore, if the difference in the membrane property of vascular tissues exists in both strains, it might appear dominantly on the muscle cell of the arterial system rather than the venous system from the nature and aetiology of a hypertension.
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