Ouabain-induced Contraction Research Articles

Sympathetic nerves and the endothelium influence the vasoconstrictor effect of low concentrations of ouabain in pressurized small arteries

We hypothesized that in salt-dependent forms of hypertension, endogenous ouabain acts on arterial smooth muscle to cause enhanced vasoconstriction. Here, we tested for the involvement of the arterial endothelium and perivascular sympathetic nerve terminals in ouabain-induced vasoconstriction. Segments of rat mesenteric or renal interlobar arteries were pressurized to 70 mmHg at 37 degrees C and exposed to ouabain (10(-11)-10(-7) M). Removal of the endothelium enhanced ouabain-induced vasoconstriction by as much as twofold (at an ouabain concentration of 10(-9) M). A component of the ouabain-induced vasoconstriction is due to the enhanced spontaneous release of norepinephrine (NE) from nerve terminals in the arterial wall. The alpha(1)-adrenoceptor blocker prazosin (10(-6) M) decreased ouabain-induced vasoconstrictions by as much as 50%. However, neither the contraction induced by sympathetic nerve activity (SNA) nor the NE release evoked by SNA (measured directly by carbon fiber amperometry) was increased by ouabain (<10(-7) M). Nevertheless, the converse case was true: after brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75-fold). This effect may be mediated by neuropeptide Y and Y(1) receptors on smooth muscle. In arteries lacking the endothelium and exposed to prazosin, ouabain (10(-11) M and greater) caused vasoconstriction, indicating a direct effect of very "low" concentrations of ouabain on arterial smooth muscle. In conclusion, in intact arteries, the endothelium opposes ouabain (10(-11)-10(-7)M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10(-7)M) does not enhance SNA-mediated contractions, but SNA enhances ouabain-induced contractions. The effects of endogenous ouabain may be accentuated in forms of hypertension that involve sympathetic nerve hyperactivity and/or endothelial dysfunction.

Involvement of rho kinase in the ouabain-induced contractions of the rat renal arteries

Agonist and depolarization-induced vascular smooth muscle contractions include the activation of rho/rho kinase pathway. However, there are no reports addressing the question whether this pathway is involved in ouabain-induced vascular smooth muscle contractions. Therefore, in this study, the possible participation of the rho/rho kinase pathway in ouabain-induced contractions was evaluated in rat renal arteries. Effects of rho kinase inhibitors (fasudil and Y-27632) on ouabain-induced contractions, and phosphorylation of myosin binding subunits (MYPT/MBS85) of myosin phosphatase were determined using isolated tissue and Western blot experiments, respectively. Fasudil and Y-27632 inhibited ouabain-induced contractions in a concentration-dependent manner. The phosphorylation of MYPT was not altered by ouabain. However, ouabain significantly increased MBS85 phosphorylation of myosin phosphatase. The phosphorylation of both subunits of myosin phosphatase was inhibited by Y-27632. These results indicate that activation of rho kinase and the subsequent phosphorylation of MBS85 are involved in ouabain-induced contraction of rat renal arteries. This mechanism may be important in essential hypertension with elevated endogenous ouabain levels.

Contribution of Na+-K+ pump and KIR currents to extracellular pH-dependent changes of contractility in rat superior mesenteric artery

We compared the branches and trunk of rat superior mesenteric artery (SMA) with respect to extracellular pH (pHo)-dependent changes in vascular contractility. Decreases in pHo from 7.8 to 6.4 significantly reduced apparent affinity (pD2) to norepinephrine (NE) and maximal contraction by NE, which were more prominent in larger-diameter arteries. On the other hand, decreases in pHo significantly reduced Ba2+-sensitive K+-induced relaxation (which was evoked by elevation of extracellular K+ concentration from 6 to 12 mM) in the first branch and inhibited inwardly rectifying K+ (K(IR)) currents in cultured smooth muscle cells (SMCs) of SMA. RT-PCR revealed transcripts for Kir2.1 in the SMCs. Real-time PCR analysis revealed 6.1-, 3.3-, and 2.2-fold increases in the Kir2.1 mRNA-to-beta-actin mRNA ratios of SMCs of the third, second, and first branches, respectively, vs. the corresponding relative levels of trunk SMCs. The magnitudes of K+-induced relaxation were significantly greater in smaller-diameter arteries, and there was a strong correlation between the transcript levels of Kir2.1 and K+-induced relaxation. A decrease in pHo reduced ouabain-sensitive K+-induced relaxation and ouabain-induced contraction. A decrease in pHo from 7.4 to 6.4 depolarized membrane potential of the cultured SMCs. From these results, we conclude that an increase in pHo activates K(IR) currents and the Na+ -K+ pump, which then reduces vascular contractility. Inasmuch as K(IR) channel densities are significantly greater in smaller-diameter arteries, the reduction in vascular contractility on increasing pHo is more pronounced in smaller-diameter arteries.

Mechanism of ouabain‐induced contractions in guinea‐pig tracheal rings

The aim of the present study was to analyse the mechanism that underlies the force development induced by ouabain (ED(100) = 100 micromol/L) in guinea-pig tracheal rings. The dose-response curve showed that concentrations of ouabain above 100 micromol/L evoked smaller contractions. Ouabain, at 100 micromol/L, produced two long-lasting consecutive transient contractions. The peak of the first contraction was 750 +/- 75 mg, whereas the peak of the second contraction was 280 +/- 46 mg. Both contractions induced by ouabain were dependent on extracellular Ca(2+). Consistent with this, verapamil (10 micromol/L) inhibited the first and second contractions by 77 and 59%, respectively. 3,4-Dichlorobenzamil (20 micromol/L) inhibited the first and second contractions by 68 and 97%, respectively. Simultaneous exposure to 15 mmol/L sodium solution and 100 micromol/L ouabain evoked only one transient contraction, larger (987 +/- 135 mg) than either of the ouabain-induced contractions. Inhibition of the sarcoendoplasmic reticulum Ca-ATPase with cyclopiazonic acid potentiated the first and second ouabain-induced contractions by 47 and 300%, respectively. Atropine (1 micromol/L) inhibited the first and second contractions by 44 and 76%, respectively. In conclusion, the results of the present study are relevant to the understanding of the mechanisms by which ouabain (100 micromol/L) contracts guinea-pig tracheal rings. At the muscular level, oubain induces Ca(2+) influx through L-type Ca(2+) channels and the reverse mode of the sodium-calcium exchanger. At the nerve terminals, ouabain promotes the release of acetylcholine secondary to the increase in Ca(2+) influx mediated by the reverse mode of the sodium-calcium exchanger.

Effects of canrenone on aorta and right ventricle of the rat.

Canrenone is a major active metabolite of spironolactone and, in addition to the antimineralocorticoid effect, shares with the parent compound the action as a partial agonist with respect to ouabain on the Na+-K+ ATPase. We have investigated whether canrenone, through its action on Na+-K+ ATPase, reverses rat aorta contractions induced by ouabain and has vasorelaxant properties unrelated to its interaction with ouabain. Contractile responses of endothelium-deprived aorta to 1 mM ouabain, 0.1 microM phenylephrine, 10 microM serotonin, and 60 mM K+ were relaxed by canrenone (50-250 microM), with maximum inhibitions of 85.3%, 55.3%, 56.7%, and 64.2%, respectively. Canrenone shifted to the right the concentration-response curve for Ca2+ in depolarized aorta and did not affect the response to 10 mM caffeine. In rat right ventricular strips driven at 0.1 Hz, canrenone exerted negative inotropic effect. The relaxation of ouabain-induced contraction may be due, at least in part, to an interaction between canrenone and ouabain on the Na+-K+ ATPase. Inhibition of calcium entry through calcium channels either in aorta or ventricles is the most parsimonious hypothesis of mechanism underlying the effect of canrenone on contractile responses of rat aorta to agonists and high K+ and the negative inotropic effect on ventricular strips.

O–249 - Effects of L-NMMA on ouabain-induced contraction of heart

O–249 - Effects of L-NMMA on ouabain-induced contraction of heart

Effect of ageing and hypertension on endothelial modulation of ouabain-induced contraction and sodium pump activity in the rat aorta.

To investigate the endothelial modulation of the rat thoracic aorta sodium-potassium ATPase activity and its possible alteration by ageing and hypertension. Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) aged 5 weeks, 3, 6, 12 and 18 months were anaesthetized. Their aortae were dissected and divided into cylindrical segments and vasomotor responses obtained in segments with or without endothelium were recorded on a polygraph. Endothelium removal increased ouabain responses in young (aged 3 and 6 months) WKY rat aortic segments, but reduced those obtained in old (aged 18 months) WKY rat segments and in segments from SHR aged 3, 6 and 12 months. An enhancement of contractions in response to ouabain with age was observed in both strains, but it occurred at earlier ages in vessels from SHR. At ages 3 and 6 months, responses to ouabain were higher in intact vessels from SHR than they were in those from WKY, but were lower in endothelium-denuded vessels. In aortic segments from 6-month-old WKY rats and SHR, ouabain reduced or abolished potassium-induced relaxations in segments with or without endothelium, respectively. In vessels from SHR and WKY rats aged 18 months, ouabain abolished potassium-induced relaxations both in intact and in endothelium-denuded vessels. Endothelium of WKY rat aorta might release a factor in response to ouabain that modulates its contraction negatively. Endothelium of SHR and old WKY rat aorta releases a contracting factor that modulates ouabain contractions positively. Thus, the inhibitory effect of endothelium on contractions induced by ouabain might be lost as a consequence both of hypertension and of age, being replaced by an endothelium-dependent contracting factor that facilitates ouabain responses.

Endothelial modulation of ouabain-induced contraction and sodium pump activity in aortas of normotensive Wistar-Kyoto and spontaneously hypertensive rats.

The influence of vascular endothelium on ouabain-induced contractions and sodium pump activity in aortic segments of Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR) was analyzed. De-endothelialization increased and reduced ouabain-induced contractions in WKY and SHR segments, respectively. The effects of de-endothelialization were not reproduced by pretreatment of the segments with NG-nitro-L-arginine methyl ester, indomethacin, or 5, 8, 11, 14-eicosatetraenoic acid, acetyl salicylic acid, dazoxiben, phosphoramidon, BQ-123, or superoxide dismutase. Bioassay experiments suggest that ouabain releases a diffusible factor from endothelial cells that inhibits or facilitates digitalis-induced contractions in WKY and SHR segments, respectively. In a potassium-free solution, potassium-induced relaxation in segments of both strains was abolished by ouabain in de-endothelialized aortas and reduced in intact ones. Ouabain-sensitive 86Rb+ uptake was significantly reduced by de-endothelialization both in WKY and in SHR. These results suggest that the vascular endothelium of WKY and SHR aortas releases a diffusible factor that stimulates the sodium pump and/or protects it from ouabain blockade. Ouabain also releases a diffusible endothelium-derived factor in SHR aortas that facilitates ouabain-induced contractions.

Neurogenic component of ouabain-evoked contractions is modulated by the endothelium.

The influence of endothelium on the neurogenic component of ouabain-induced contractions in isolated perfused guinea pig carotid arteries was analyzed. Ouabain (0.1 mumol/L to 0.1 mmol/L) evoked concentration-dependent increases of perfusion pressure. Phentolamine (0.3 to 10 mumol/L) and prazosin (30 nmol/L to 10 mumol/L) (nonselective antagonist of alpha-adrenergic receptors and selective antagonist of alpha 1-adrenergic receptors, respectively) induced a concentration-dependent relaxation in segments precontracted with ouabain (0.1 mmol/L). When the arteries were preincubated with those blockers (both at 3 mumol/L) or the animals were pretreated with reserpine, the contractions to the glycoside were diminished, indicating that they are partially mediated by norepinephrine release from adrenergic nerve endings. De-endothelialization abolished the effect of adrenergic blockade on ouabain-induced contractions. On the other hand, de-endothelialization did not modify significantly the effect of the adrenergic blockade on norepinephrine-induced contractions. The nitric oxide blocker oxyhemoglobin, at concentrations (10 mumol/L) that abolished endothelium-dependent relaxations induced by 3 mumol/L acetylcholine; or the cyclooxygenase blocker indomethacin (10 mumol/L) did not modify the relaxation caused by phentolamine. In bioassay experiments, 30 mumol/L phentolamine induced a relaxation on the ouabain-elicited contraction only when the glycoside was added through a donor segment with endothelium. Ouabain-induced tritiated norepinephrine release was significantly reduced by the removal of endothelium but not by 1 mumol/L oxyhemoglobin or 1 mumol/L indomethacin. These results suggest that the endothelium modulates the neurogenic component involved in contractions evoked by the glycoside by a diffusible factor (or factors) whose nature is unknown; however, the factor is neither nitric oxide nor a cyclooxygenase-related compound.

Endothelial role in ouabain-induced contractions in guinea pig carotid arteries.

The influence of endothelium on the direct contractile effects of ouabain in vascular smooth muscle was analyzed in isolated perfused guinea pig carotid arteries. After blocking the neurogenic component of the glycoside contraction with alpha-adrenergic receptor blocking drugs or treating the animals with reserpine, ouabain-induced contractions were markedly reduced in vessels with intact endothelium. However, removal of the vascular endothelium from reserpinized carotid arteries resulted in ouabain-induced contractions similar to those observed in control arteries. These effects were not mimicked by the inhibitor of nitric oxide NG-monomethyl L-arginine or by the cyclooxygenase blocker indomethacin. Bioassay experiments suggested that these endothelial effects are mediated by diffusible factors. Uptake of 86Rb to measure sodium pump activity was significantly reduced by removal of the endothelium. These results suggest the existence of an inhibitory modulation by the endothelium of contractions induced by ouabain, likely mediated by a diffusible factor (or factors) released from these cells. The nature of this substance is unknown, but it is neither related to prostaglandins nor a nitric oxide-related compound. Its mechanism of action could be the stimulation of vascular sodium pump activity, the antagonism of the pump's inhibition by ouabain, or both.

Endothelial modulation of the ouabain-induced contraction in human placental vessels.

Ouabain (1 x 10(-7)-3 x 10(-5) M) elicited concentration-dependent vasoconstriction in human placental arteries and veins. These responses, but not those produced by 10(-6) M 5-hydroxytryptamine, were increased after the removal of vascular endothelium. In placental arteries, the respective blockade of cyclooxygenase or lipoxygenase with indomethacin of 5,8,11,15-eicosatetraynoic acid as well as the inactivation of nitric oxide with phenidone or oxyhemoglobin and the inhibition of nitric oxide synthesis with NG-monomethyl L-arginine (all at 10(-5) M) did not mimic the effects of endothelial denudation on ouabain-evoked contractions. Bioassay experiments suggested that the above-mentioned endothelial effects are mediated by diffusible factors. 86Rb+ uptake, a method to measure sodium pump activity, was significantly reduced by the removal of endothelium. These results suggest the existence of an inhibitory modulation by the endothelium of the contractions induced by ouabain, likely mediated by a diffusible factor(s) released from these cells. The nature of this substance is unknown but is not related to prostaglandins or leukotrienes, and neither is it a nitric oxide-related compound. Its mechanism of action could be stimulating the activity of vascular sodium pump and/or antagonizing its inhibition by ouabain.

Na +/Ca 2+ exchange mediation in the ouabain-induced contraction in human placental vessels

1. 1. Ouabain induced concentration-dependent contractions in segments of human placental arteries and veins, which were practically abolished in a Ca 2+-free medium and not modified by the calcium antagonist nifedipine or the calcium agonist Bay K 8644. 2. 2. Ouabain (10 −4 M) elicited a time-dependent enhancement of the 45Ca 2+ uptake, which remained equal in presence of nifedipine or Bay K 8644. 3. 3. The Na +/Ca 2+ exchange blocker amiloride reduced both the contractions and the 45Ca 2+ uptake induced by ouabain, whereas the Na ionophore monensin produced a parallel shift to the left of the concentration-response curve to ouabain. 4. 4. These results suggest that ouabain-induced contractions in these vessels are dependent on the extracellular Ca 2+, which mainly enters into the cell through the Na +/Ca 2+ exchange system.

Calcium dependence of ouabain-induced contraction in aortas from spontaneously hypertensive rats

The calcium sensitivity of ouabain-induced contractions of aortic strips from spontaneously hypertensive rat (SHR) was examined using several drugs which affect Na + and Ca 2+ movements across the cell membrane, and the results were compared with those obtained with age-matched Wistar-Kyoto rat (WKY). The Ca 2+ concentration-response curves (10 −3 M ouabain-treated preparations) made with aortic strips from SHR lay to the left of those made with aortic strips from WKY (Ca EC 50 values: SHR, 0.51 ± 0.16 mM, n = 6; WKY, 1.23 ±0.41 mM, n = 7; P < 0.05). Amiloride (a Na + entry blocker) and nifedipine (a Ca 2+ entry blocker) attenuated the sensitivity to Ca 2+ of SHR and WKY aortic strips. With 2 × 10 −4 M amiloride, WKY vessels showed a 1.3-fold increase in the Ca EC 50 value and SHR a 2.1-fold increase. With 10 −6 M nifedipine. WKY vessels showed a 1.1-fold increase in the Ca EC 50 value and SHR a 1.5-fold increase. Addition of monensin (Na ionophore) produced a dose-dependent potentiation in ouabain-treated aorta from WKY, but not in ouabain-treated aorta from SHR. Addition of 1.5 × 10 −5 M A23187 (Ca ionophore) eliminated the difference between the Ca 2+-induced contractions in aortas from SHR and WKY. These results suggest that enhancement of Ca 2+ influx by Na +-Ca 2+ exchange and/or voltage-dependent Ca 2+ channels in vascular smooth muscle cell membranes may be an important factor in the difference between ouabain-induced contractions in aorta from SHR and WKY.

Effect of Sodium-Transport Inhibitors on Airway Smooth Muscle Contractility in Vitro

1. To determine whether alterations in membrane sodium transport in airway smooth muscle can alter its contractility, we studied the effect of ouabain (a Na+/K(+)-adenosine triphosphatase inhibitor) and amiloride on contractile responses in bovine trachea and human bronchial rings in a series of studies. 2. Ouabain (10(-6)-10(-4) mol/l) caused concentration-related contraction of bovine trachea with a maximum effect at 30 min; the mean increases in tension with 10(-6), 10(-5) and 10(-4) mol/l ouabain were 19, 27, and 32%, respectively, of the maximum response seen with 10(-3) mol/l histamine (n = 6). In human bronchial rings, ouabain (10(-5) mol/l) caused a mean contraction which was 40% of the maximum response to methacholine (n = 8). 3. Calcium-free fluid (plus ethylenediaminetetraacetic acid) and nifedipine (10(-5) mol/l) inhibited ouabain-induced contractions, suggesting that contraction was mediated in part by calcium entry via voltage-dependent calcium channels. Phentolamine (10(-5) mol/l) was without effect. 4. Ouabain (10(-5) mol/l) did not alter histamine responsiveness in bovine trachea or methacholine responsiveness in human bronchial rings. 5. Amiloride did not affect resting tone in bovine trachea but caused a concentration-dependent relaxation of bovine tracheal strips preconstricted with carbachol, 10(-3) mol/l amiloride relaxing strips completely over 15 minutes (n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of TYB 3823, a new antiarrhythmic agent, on mechanical responses in the vascular smooth muscles

1. 1. TYB 3823 (3 × 10 −5M to 10 −3 M) but not lidocaine (10 −4 M) inhibited the contractile response to norepinephrine in rabbit aorta in a competitive manner. On the other hand, TYB 3823 had no effect on contractile responses to histamine, 5-hydroxytryptamine, high KCl and Ca 2+. 2. 2. In rabbit aorta, TYB 3823 (10 −5 M and 10 −4 M) but not lidocaine (10 −4 M) also antagonized the ouabain-induced contraction and this action was less potent than that of phentolamine (10 −6 M). 3. 3. In rabbit coronary arteries precontracted with KCl (40 mM), either TYB 3823 or lidocaine inhibited the isoproterenol-induced relaxation to the same levels. Propranolol at 10 −8 M shifted the relaxation curve for isoproterenol to the right and at 10 −6 M it almost completely prevented the relaxation. Further, the effect of a combined treatment with propranolol (10 −8 M) and TYB 3823 (3 × 10 −5M) was not different from that of a single treatment with propranolol (10 −8 M). 4. 4. In rabbit iliac arteries, TYB 3823 (3 × 10 −5M and 10 −4 M) but not lidocaine (10 −4 M) suppressed the contractile response to caffeine in a Ca 2+-free medium with EGTA and nifedipine. Nitroglycerin (10 −4 M) also inhibited the response but this action was less potent than that of TYB 3823. The combined treatment of the tissue with TYB 3823 and nitroglycerin (both 10 −4 M) further inhibited the responses to caffeine as compared to a single treatment with each drug. 5. 5. These results suggested that TYB 3823 may interfere with the responses due to the mobilization of intracellular Ca 2+ activated by caffeine and also has both α- and β-adrenoceptor blocking actions on the vascular smooth muscles.

Ouabain-induced contraction of vascular smooth muscle in spontaneously hypertensive rats and the effect of hydralazine

The effect of ouabain (10 −3 M) on contractile responses of SHR (spontaneously hypertensive rat) and WKY (Wistar-Kyoto rat) aortas and mesenteric arteries was studied. Ouabain addition caused a rapid contraction of aortic strips with a steeper rate of rise and a larger maximal force development in strips from SHR than WKY. This difference in contractile response is known to occur in the prehypertensive period of SHR (4-week-old). Phentolamine (10 −6 M) pretreatment had no effect on the ouabain-induced contraction but partially suppressed it in both SHR and WKY aortas when diltiazem (10 −5 M) was also added. The difference in the ouabain-induced contractions of SHR and WKY aortas was more apparent in the residual contraction during suppression by diltiazem. The 45Ca uptake in the presence of ouabain was significantly larger in the early period of incubation in SHR aorta than in WKY aorta. The ouabain-induced contraction of hydralazine-treated SHR aorta from the prehypertensive period was very similar to that of non-treated WKY aorta. These results suggested that the abnormality of the ouabain-induced contraction in SHR arterial smooth muscle could have arisen from an increased Ca 2+ movement due to Ca 2+ leakage when ouabain inhibited the Na +-pump in the membrane. This abnormality seems to start during the prehypertensive period and continue in the hypertensive stage.

Effects of ouabain on isolated cerebral and femoral arteries of the cat: a functional and biochemical study.

1. This study analyzes the mechanisms involved in the responses to ouabain in cat cerebral and femoral arteries and characterizes the electrogenic Na+ pump present in these vessels. The latter was accomplished by measurement of [3H]-ouabain binding to arterial membrane fractions, K+-elicited relaxation and ouabain-sensitive 86Rb+ uptake. 2. Ouabain induced transient contraction in cylindrical segments of cerebral arteries. This contraction was reduced by verapamil (3 X 10(-6) M) and Ca2+-removal from the medium but was not modified by phentolamine (3 X 10(-6) M) or pretreatment with reserpine. However, the contraction elicited by ouabain in femoral artery segments lasted longer, and was reduced by Ca2+-omission, phentolamine or reserpine, but remained unaffected by verapamil. 3. The immersion of the arteries in low-Na+ (25 mM) medium abolished the contraction caused by ouabain. 4. The exposure of the arteries to a K+-free medium induced a small transient increase in tension, and the subsequent application of K+ (7.5 mM) elicited a marked relaxation. This effect was greater in cerebral than in peripheral arteries, and was suppressed by ouabain (10(-4) M). 5. Scatchard analysis of the [3H]-ouabain binding to arterial membrane fractions suggested a single class of binding sites. The KD values for both kinds of arteries were of similar order, while the Bmax value was greater in cerebral than in femoral arteries. 6. Total and ouabain-sensitive 86Rb+ uptakes were greater in cerebral than in femoral vessels. 7. These results indicate that: (1) ouabain-induced contraction of cerebral arteries is due to a direct effect on vascular smooth muscle cells, while in femoral arteries it is due to noradrenaline release from adrenergic nerve terminals; and (2) the electrogenic Na+ pump activity is greater in cerebral than in peripheral arteries.

Analysis of Ouabain-Induced Contractions in Isolated Coronary Arteries

Contractile responses to ouabain in helical strips of dog and monkey coronary arteries were investigated. Ouabain (5×10−8 to 5×10−6 M) caused a dose-related contraction in dog and monkey arteries; the response of monkey coronary arteries was significantly greater. In dog coronary arteries, contractile responses to high concentrations of ouabain were potentiated by treatment with propranolol. In the arteries contracted with ouabain, the addition of phentolamine caused a relaxation. Contractile responses of dog coronary arteries to ouabain were markedly suppressed by exposure to Ca2+-free media or by treatment with verapamil. Reduction of external concentration of K+ or lowering the temperature of bathing media did not selectively influence the ouabain-induced contraction. These results suggest that ouabain-induced contractions of dog coronary arteries are associated mainly with an increase in the Ca2+-influx, which does not result from an inhibition of the Na+, K+-activated ATPase nor from an activation of α adrenoceptors by noradrenaline released from adrenergic nerves. Ouabain in high concentrations seems to liberate noradrenaline from adrenergic nerves, which preferentially activates β adrenoceptors in dog coronary artery.

Studies on the calcium antagonistic action of tetrandrine: VIII. Antagonistic effects of tetrandrine on ouabain-induced contraction of coronary arterial strips.

The antagonistic effects of tetrandrine (Tet) on ouabain-induced contraction were studied in swine coronary arterial strips. Like verapamil (Ver), the contraction evoked by ouabain (Oub) 0.85 μM, the synergistic effect of Oub 85 nM and Ca++ 2–16 mM, and the response to electrical stimulation in the presence of Oub 85 nM were all inhibited by Tet (10 μM-1.2 mM), although these effects were less potent than those of Ver. All these pharmacological actions of Tet and Ver were reversed by the addition of extra Ca++ to the bathing medium. The results we obtained confirm that Tet is a Ca++-antagonist of plant origin.

Canine mesenteric artery Na+,K+-ATPase: vasopressor receptor for digitalis?

Localization and characterization studies of Na+,K+-ATPase in canine superior mesenteric artery were undertaken to examine the role of the Na+-K+ pump in the vasopressor response of cardiac glycosides. The enzymatic component of the membrane-bound Na+-K+ pump, Na+,K+-ATPase, was found by histochemical and cell fractionation techniques to be localized primarily in the sarcolemma of the smooth muscle cell in superior mesenteric artery. The enzyme could be enriched in microsomal and partially purified sarcolemma preparations of superior mesenteric artery and first-order arterial side branches. Binding of [3H]ouabain to arterial microsomes followed pseudo-first-order reaction kinetics. In the presence of magnesium plus ATP, sodium stimulates and potassium inhibits the rate of binding. Scatchard analysis indicated a single class of [3H]ouabain binding sites with a KD of 2-9 nM and a Bmax of 2.3-3.5 pmol/mg protein. Although the characteristics of [3H]ouabain binding to mesenteric artery microsomes resemble the characteristics of [3H]ouabain binding to purified Na+,K+-ATPase, the density or total number of Na+-K+ pump sites in mesenteric artery is small compared with either heart muscle or kidney parenchyma. In isolated mesenteric arterial strips, more than 80% of a 2.5 microM ouabain-induced contraction could be inhibited or reversed by alpha-adrenoceptor blockade with 1 microM phentolamine. These data indicate that although cardiac glycosides interact with specific receptor sites in smooth muscle of canine superior mesenteric artery, the direct vasoconstrictor effect, which may be related to this digitalis-smooth muscle Na+,K+-ATPase interaction, is meager and may be a reflection of the low density of Na+-K+ pump sites.