Administration Of Ouabain Research Articles

Role of the Na/Ca exchanger in arrhythmias in compensated hypertrophy.

Sudden, presumably arrhythmic, death is common in heart failure patients. Although total mortality is highest in end-stage failure, the fraction of sudden death in total mortality is higher in the early stages. In each of these stages various, not necessarily identical, ionic mechanisms may contribute to arrhythmogenesis. Dogs with chronic complete atrioventricular block (6-8 weeks) have an increased risk for arrhythmias and sudden death and have compensated biventricular hypertrophy. In this animal model, Ca(2+) release from the sarcoplasmic reticulum (SR) is not reduced. For low frequencies of stimulation, the SR Ca(2+) content is increased, related to a higher activity of the Na/Ca exchanger. Spontaneous Ca(2+) release induces inward Na/Ca exchange current, which can lead to delayed afterdepolarizations (DADs) triggering a new action potential. Such arrhythmogenic DADs and ectopic beats also can be observed in vivo during monophasic action potential recording. They appear after pacing protocols, and/or administration of ouabain, which result in contractile potentiation, suggestive of a enhanced sarcoplasmic reticulum Ca(2+) content. Other arrhythmogenic mechanisms related to increased dispersion of repolarization also can be identified in vivo. Downregulation of delayed K(+) currents is an important factor in prolongation of action potentials. In conclusion, in this animal model of compensated hypertrophy, Ca(2+) handling is different from end-stage heart failure. It is possible that arrhythmogenic mechanisms related to a higher Ca(2+) load contribute to the high incidence of sudden death in stages of compensated hypertrophy before overt heart failure. However, more than one ionic remodeling process is likely to be present, and different cellular mechanisms of arrhythmias can coexist.

Disruption of vagal efferent axon and nerve terminal function in the postischemic myocardium.

Despite the importance of vagal control over the ventricle, little is known regarding vagal efferent conduction and nerve terminal function in the postischemic myocardium. To elucidate postischemic changes in the cardiac vagal efferent neuronal function, we measured myocardial interstitial acetylcholine (ACh) levels by using in vivo cardiac microdialysis and examined the ACh responses to electrical stimulation of the vagi or local administration of ouabain in anesthetized cats. Sixty-minute occlusions of the left anterior descending coronary artery (LAD) followed by 60-min reperfusion abolished electrical stimulation-induced ACh release (20.4 +/- 3.9 vs. 0.9 +/- 0.4 nmol/l; means +/- SE, P < 0.01). In different groups of animals, 60-min LAD occlusion followed by 60-min reperfusion decreased but did not completely abolish ouabain-induced release of ACh (9.2 +/- 1.8 vs. 3.9 +/- 0.7 nmol/l; P < 0.05). These results indicate that function of the vagal efferent axon was completely interrupted, whereas the local ACh release was partially suppressed in the postischemic myocardium. The postischemic disruption of vagal efferent neuronal function might exert deleterious effects on cardiac regulation.

Alterations in phenylephrine-induced contractions and the vascular expression of Na+,K+-ATPase in ouabain-induced hypertension.

Hypertension development, phenylephrine-induced contraction and Na(+),K(+)-ATPase functional activity and protein expression in aorta (AO), tail (TA) and superior mesenteric (SMA) arteries from ouabain- (25 microg day(-1), s.c., 5 weeks) and vehicle-treated rats were evaluated. Ouabain treatment increased systolic blood pressure (127+/-1 vs 160+/-2 mmHg, n=24, 35; P<0.001) while the maximum response to phenylephrine was reduced (P<0.01) in AO (102.8+/-3.9 vs 67.1+/-10.1% of KCl response, n=12, 9) and SMA (82.5+/-7.5 vs 52.2+/-5.8%, n=12, 9). Endothelium removal potentiated the phenylephrine response to a greater extent in segments from ouabain-treated rats. Thus, differences of area under the concentration-response curves (dAUC) in endothelium-denuded and intact segments for control and ouabain-treated rats were, respectively: AO, 56.6+/-9.6 vs 198.3+/-18.3 (n=9, 7); SMA, 85.5+/-15.4 vs 165.4+/-24.8 (n=6, 6); TA, 13.0+/-6.1 vs 39.5+/-10.4% of the corresponding control AUC (n=6, 6); P<0.05. The relaxation to KCl (1 - 10 mM) was similar in segments from both groups. Compared to controls, the inhibition of 0.1 mM ouabain on KCl relaxation was greater in AO (dAUC: 64.8+/-4.6 vs 84.0+/-5.1%, n=11, 14; P<0.05), similar in SMA (dAUC: 39.1+/-3.9 vs 43.3+/-7.8%, n=6, 7; P>0.05) and smaller in TA (dAUC: 62.1+/-5.5 vs 41.4+/-8.2%, n=12, 13; P<0.05) in ouabain-treated rats. Protein expression of both alpha(1) and alpha(2) isoforms of Na(+),K(+)-ATPase was augmented in AO, unmodified in SMA and reduced in TA from ouabain-treated rats. These results suggest that chronic administration of ouabain induces hypertension and regional vascular alterations, the latter possibly as a consequence of the hypertension.

In vivo assessment of acetylcholine-releasing function at cardiac vagal nerve terminals.

We examined whether the ACh concentration measured by cardiac microdialysis provided information on left ventricular ACh levels under a variety of vagal stimulatory and modulatory conditions in anesthetized cats. Local administration of KCl (n = 5) and ouabain (n = 7) significantly increased the ACh concentration in the dialysate to 4.3 +/- 0.8 and 7.3 +/- 1.3 nmol/l, respectively, from the baseline value of 0.6 +/- 0.5 nmol/l. Intravenous administration of phenylbiguanide (n = 5) and phenylephrine (n = 6) significantly increased the ACh concentration to 5.4 +/- 0.9 and 6.0 +/- 1.5 nmol/l, respectively, suggesting that the Bezold-Jarisch and arterial baroreceptor reflexes affected myocardial ACh levels. Modulation of vagal nerve terminal function by local administration of tetrodotoxin (n = 6), hemicholinium-3 (n = 6), and vesamicol (n = 5) significantly suppressed the electrical stimulation-induced ACh release from 20.4 +/- 3.9 to 0.6 +/- 0.1, 7.2 +/- 1.9, and 2.7 +/- 0.6 nmol/l, respectively. Increasing the heart rate from 120 to 200 beats/min significantly reduced the myocardial ACh levels during electrical vagal stimulation, suggesting a heart rate-dependent washout of ACh. We conclude that ACh concentration measured by cardiac microdialysis provides information regarding ACh release and disposition under a variety of pathophysiological conditions in vivo.

Direct inhibitory effect of digitalis on progesterone release from rat granulosa cells.

Digoxin (10(-7) - 10(-5) M) or digitoxin (10(-7) - 10(-5) M) decreased the basal and human chorionic gonadotropin (hCG)-stimulated release of progesterone from rat granulosa cells. Digoxin (10(-5) M) or digitoxin (10(-5) M) attenuated the stimulatory effects of forskolin and 8-bromo-cyclic 3' : 5'-adenosine monophosphate (8-Br-cAMP) on progesterone release from rat granulosa cells. Digoxin (10(-5) M) or digitoxin (10(-5) M) inhibited cytochrome P450 side chain cleavage enzyme (cytochrome P450(scc)) activity (conversion of 25-hydroxyl cholesterol to pregnenolone) in rat granulosa cells but did not influence the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Neither progesterone production nor P450scc activity in rat granulosa cells was altered by the administration of ouabain. Digoxin (10(-5) M) or digitoxin (10(-5) M), but not ouabain, decreased the expression of P450scc and steroidogenic acute regulatory (StAR) protein in rat granulosa cells. The present results suggest that digoxin and digitoxin decrease the progesterone release by granulosa cells via a Na(+),K(+)-ATPase-independent mechanism involving the inhibition of post-cyclic AMP pathway, cytochrome P450scc and StAR protein functions.

Pressor and vascular effects of cardiac glycosides.

For the past two decades, it has generally been accepted ('Blaustein hypothesis') that cardiac glycosides such as ouabain and digoxin increase the sodium and calcium content of smooth muscle cells, so inducing arterial vasoconstriction and a rise in blood pressure. Recent data from an experimental study we carried out led us to question this assumption. A retrospective literature survey covering 20 years and including animal and human studies was performed. Representative results are presented. Contradictory effects of cardiac glycosides on blood pressure and vasculature have been described. Increased, decreased or unaltered blood-pressure values have been observed following administration of the glycosides ouabain, digoxin and digitoxin. Moreover, vasoconstricting as well as vasodilating effects of cardiac glycosides have been demonstrated. Several recent studies show that cardiac glycosides such as digoxin and digitoxin can lead to a reduction of at least diastolic blood pressure. A slight vasodilation of resistance vessels followed by a fall in diastolic blood pressure could be a contributing factor for the beneficial effects of cardiac glycosides in patients with congestive heart failure. This vasodilation may be caused by central (neurohumoral) effects of digitalis glycosides.

Pressor and vascular effects of cardiac glycosides

Background: For the past two decades, it has generally been accepted ('Blaustein hypothesis') that cardiac glycosides such as ouabain and digoxin increase the sodium and calcium content of smooth muscle cells, so inducing arterial vasoconstriction and a rise in blood pressure. Recent data from an experimental study we carried out led us to question this assumption. Design: A retrospective literature survey covering 20 years and including animal and human studies was performed. Representative results are presented. Results: Contradictory effects of cardiac glycosides on blood pressure and vasculature have been described. Increased, decreased or unaltered blood-pressure values have been observed following administration of the glycosides ouabain, digoxin and digitoxin. Moreover, vasoconstricting as well as vasodilating effects of cardiac glycosides have been demonstrated. Several recent studies show that cardiac glycosides such as digoxin and digitoxin can lead to a reduction of at least diastolic blood pressure. Conclusion: A slight vasodilation of resistance vessels followed by a fall in diastolic blood pressure could be a contributing factor for the beneficial effects of cardiac glycosides in patients with congestive heart failure. This vasodilation may be caused by central (neurohumoral) effects of digitalis glycosides.

Acute pressor actions of ouabain do not enhance the actions of phenylephrine or norepinephrine in anesthetized rats.

The inhibition of high-affinity isoforms of the Na+,K+-ATPase by nanomolar levels of ouabain has been proposed to enhance the actions of vasoconstrictor agents that act via a Ca+2-dependent mechanism. The present study tested this hypothesis by evaluating the effects of ouabain (6 and 18 microg/kg, i.v.) on the vasopressor actions of phenylephrine and norepinephrine in anesthetized, reflex-blocked rats. In separate groups of animals, dose-response curves for increases in diastolic pressure produced by phenylephrine were generated after the administration of saline (control), ouabain (18 microg/kg), L-omega-N-nitro arginine methyl ester (L-NAME, 3 micromol/kg) and angiotensin II (15 ng/kg per min). Treatment with ouabain (18 microg/kg) produced an increase in diastolic pressure of 19+/-3 mm Hg but did not significantly alter the potency or maximal response produced by phenylephrine. In contrast, treatment with angiotensin II and L-NAME, agents known to enhance the actions of alpha-adrenoceptor agonists, increased the potency of phenylephrine. In animals in which the pressor actions of norepinephrine were evaluated before and after the administration of ouabain (6 microg/kg), ouabain did not alter the pressor response to norepinephrine. Blockade of alpha-adrenoceptors with phentolamine was found to attenuate as well as partially reverse the increase in diastolic pressure produced by ouabain. These observations suggest that ouabain produces a pressor response by actions on sympathetic nerve endings as well as on vascular smooth muscle and that these actions do not alter the sensitivity to phenylephrine or norepinephrine.

Calcium waves in frog melanotrophs are generated by intracellular inactivation of TTX-sensitive membrane Na + channel

Two models of plasma membrane oscillators may explain the regulation of calcium homeostasis in frog melanotrophs. In the majority (70%) of cells a high frequency and small amplitude fluctuations characterize the spontaneous calcium level. In the 30% of remaining cells a low frequency and high amplitude oscillations were observed. Utilization of EGTA, U73122 and ryanodine suggested that calcium homeostasis in frog melanotrophs is dependent on extra- but not on intracellular calcium pools. EGTA was able to block calcium oscillations and to decrease basal calcium level in non-oscillatory cells. ω-Conotoxin, N-type calcium channels antagonist, stopped calcium oscillations but not modified calcium level in non-oscillatory cells. Nifedipine, antagonist of l-type calcium channels, had no effect either on calcium waves formation or on basal level of calcium in non-oscillatory cells. ω-Conotoxin and nifedipine were able to decrease the spontaneous α-MSH release from whole NILs while only ω-conotoxin had inhibitory effect on hormonal output from dispersed melanotrophs. Nickel (Ni 2+) provoked dose-dependent effect. At 2 mM concentration Ni 2+ blocked either calcium oscillations or α-MSH release. In contrast, a 0.5 mM concentration had stimulatory effect on both the phenomenons. Similarly, mibefradil (antagonist of T-type calcium channel), was able to induce an increase in [Ca 2+] i after modification of calcium fluctuations in non-oscillatory cells. Utilization of veratridine and TTX, agonist and antagonist of Na channels, respectively, indicated that mobilization of extracellular sodium, by TTX-sensitive and TTX-resistant Na channels, stimulates a hormonal output resulting from increase of [Ca 2+] i. In the presence of TTX, veratridine was able to generate a calcium oscillations, which were also observed after inactivation of TTX-sensitive channel. Bepridil (antagonist of Na–Na exchange of the Na +/Ca 2+ exchanger) and Na-free medium had powerful effect on increase of [Ca 2+] i. The same observations obtained after administration of ouabain, antagonist of Na +/K + dependent ATPase, confirmed dependence of calcium homeostasis on sodium distribution. Furthermore, dibutyryl-cAMP induced calcium oscillations suggesting implication of intracellular phosphorylation in the generation of calcium waves. Taken together, our results suggest that each type of calcium homeostasis is controlled by different mechanisms. Calcium fluctuations may be ascribed to the high frequency activity of T-type calcium channel, TTX-sensitive and TTX-resistant sodium channels. Calcium oscillations may be generated by the destabilization of the steady-state Na +/Ca 2+ gradient provoked by intracellular inactivation of TTX-sensitive Na channel. This ionic unbalance would increase Ca–Ca exchange of Na +/Ca 2+ exchanger, which by local depolarization promotes opening of N-type calcium channel responsible for calcium wave. In both types of homeostasis, the calcium and sodium overload is avoided by opening of K + voltage- and Ca-dependent channels, and by increase in activities of Na +/K + ATPase and forward mode of Na +/Ca 2+ exchanger.

Neuronal Fos-like immunoreactivity in ouabain-induced hypertension

In normotensive Wistar rats, systemic administration of exogenous ouabain for 10 days or more induces hypertension, presumably through central mechanisms. To identify which neuronal populations may be involved, we assessed Fos-like immunoreactivity (FLI) using an antibody that recognizes the protein products of the fos family comprising Fos, Fos B, Fra 1 and Fra 2, thus enabling detection of chronic neuronal activation. Young Wistar rats received s.c. infusions of either ouabain (50 μg/day) or saline for 7 or 14 days. At the end of the experimental period, mean arterial pressure (MAP) was assessed. In a separate set of rats FLI was detected immunohistochemically and quantified in cardiovascular and osmo-regulating centers. Resting MAP in ouabain-treated rats was significantly higher than in control rats at 14 but not at 7 days (125±4 vs. 101±6, P<0.05 and 102±4 vs. 98±6 (not significant), respectively). Within the supraoptic nucleus, ouabain induced significant increases in FLI compared with control rats at 14 days (9±2 vs. 2±2, P<0.05) but not at 7 days. Within the locus ceruleus, FLI was only detectable in rats that received ouabain infusions for 14 days but not in other groups of rats. Ouabain treatment did not induce significant changes in FLI within other areas. These results demonstrate that chronic s.c. ouabain infusion only increases neuronal FLI in the supraoptic nucleus and locus ceruleus where increases in FLI parallel the increase in blood pressure.

Open field is more sensitive than automated activity monitor in documenting ouabain-induced hyperlocomotion in the development of an animal model for bipolar illness

1. 1. Intracerebroventricular (ICV) administration of ouabain to rats induces motor hyper- and hypoactivity that have been hypothesized to model the mania and depression of bipolar illness, respectively. 2. 2. The extent of ouabain-induced change in activity may vary according to the test environment. 3. 3. To determine the degree of differential response to ICV ouabain in the open field and automated activity monitors, the authors examined a large number of animals (n = 40) in both environments. 4. 4. ICV ouabain produced a four-fold increase in open field activity versus ICV artificial cerebrospinal fluid (aCSF) (mean ± SD: 258.7 ± 316.61 vs. 84.8 ± 86.16 squares traversed) (t = 2.648, P = 0.011). but did not alter horizontal activity in automated activity monitors (8193.5 ± 4902.52 vs. 7088.47 ± 3046.85 beam interruptions) (t = 0.847, P = 0.4). This increase in activity persisted for at least one week (161.0 ± 186.35 for ICV ouabain vs. 46.1 ± 47.46 for ICV aCSF. P = 0.065). 5. 5. Open field is superior to automated activity monitors in capturing ouabain- induced hyperlocomotion response.

EFFECTS OF OUABAIN ON TENSION RESPONSE AND [ 3H]NORADRENALINE RELEASE IN HUMAN PROSTATE

The activity of Na+/K+ ATPase is a crucial factor in lots of functions of vascular and nonvascular smooth muscles. However, its role in muscle contractility of human prostate has not been well elucidated. In this context, we have examined the effect of ouabain on the contractile response and noradrenaline release in human prostate to clarify the role of cardiac glycoside in the pathophysiology of bladder outlet obstruction associated by benign prostatic enlargement. Human prostatic tissues (n = 12) were obtained at operation from males by open prostatectomy (n = 2) or transurethral resection of the prostate (n = 10). The effect of ouabain on muscle contraction and [3H]noradrenaline release was studied in human prostatic tissue. The action mechanism following the administration of ouabain was also examined. Ouabain concentration-dependently induced a gradual contraction in human prostate and this delayed contraction was significantly blocked by prazosin other than the other receptor antagonists. In parallel experiments, ouabain also caused a concentration-dependent gradual increase in [3H]noradrenaline release, which was extracellular Ca2+-dependent. Furthermore, ouabain-induced [3H]noradrenaline release increased, in a concentration-dependent manner, with increasing Na+ concentrations from 25 to 140 mM. Moreover, K+-free Krebs solution could mimic the [3H]noradrenaline release action to ouabain. Ouabain may induce an increase in tension response in human prostate, and this effect is due mainly to an increase in noradrenaline release via an effect on the Na+-dependent Ca2+ influx system.

Renal functional responses to centrally administered ouabain in anesthetized rabbits

The effects of intracerebroventricularly administered ouabain on renal function were studied in rabbits. Ouabain, when given intracerebroventricularly in doses ranging from 0.1 to 10 micrograms/kg, dose-dependently produced antidiuretic, antinatriuretic and antikaliuretic effects, along with decreases in renal perfusion and glomerular filtration rate. Systemic blood pressure also increased significantly in a dose-related fashion. No increase in free water reabsorption was noted. Intravenously administered ouabain (10 micrograms/kg) caused less pronounced antidiuresis and antinatriuresis, with the renal hemodynamics tending slightly to decrease. No significant increment of systemic blood pressure with intravenous ouabain was observed. In rabbits with one kidney denervated and the contralateral left intact, ouabain 10 micrograms/kg i.c.v. produced the typical renal effects on the intact kidney, while the denervated kidney responded with prominent natriuresis and diuresis, showing the maximal response in the second 10-min period after administration. Renal nerve activity increased significantly upon administration of ouabain 10 micrograms/kg i.c.v. and reached a peak at around 10 to 20 min after drug administration, a period in which the most remarkable antidiuresis and antinatriuresis is produced by i.c.v. ouabain. These observations suggest that the antidiuresis and antinatriuresis elicited by centrally administered ouabain results mainly from the decrease of renal hemodynamics, which is caused by the increased renal nerve activity.

Different effects of in vivo ouabain and digoxin on renal artery function and blood pressure in the rat.

To investigate vascular mechanisms in hypertension, we isolated renal arterial rings from rats with ouabain-dependent hypertension and studied their function. In rats infused with ouabain for 5 weeks, systolic and mean blood pressures (BP) were increased relative to controls. Contractions evoked by high KCl solutions were greater in rings from ouabain-infused rats whereas the threshold concentrations and EC50s for KCl and the peak caffeine contractures were not different. KCl contractures were not affected by 5 microM prazosin. Phenylephrine contractures were increased marginally in ouabain-infused rats, while acetylcholine-induced relaxation was normal. In vitro superfusion of rings with 10 nM ouabain or digoxin did not affect the measured parameters. Plasma ouabain, BP, and all evoked responses were normal one week following interruption of the ouabain infusion. In a second study, BP increased in ouabain (15 microg/kg/day, n= 23), but not digoxin (30 microg/kg/day, n=12), or vehicle-infused (n=16) rats. KCl contractures were greater in rings from ouabain-but decreased in rings from digoxin-infused rats, respectively and correlated with systolic and mean BP (r=0.69, n=30, p<0.005). Peak caffeine (25 mM) responses were similar but the area under the contraction was reduced in the vessels from ouabain-infused rats and correlated inversely with MBP (r=-0.47, n=33, p<0.02). We conclude that a voltage-dependent component of tone in the rat renal artery is reversibly and specifically augmented by in vivo administration of ouabain whereas it is diminished by in vivo digoxin. Vascular production of and response to nitric oxide does not appear to be impaired in the ouabain model. Alterations of intracellular Ca2+ storage and Ca2+ influx in response to in vivo ouabain may underlie the increase in renal vascular resistance and hypertension in this model. The opposite effects of ouabain and digoxin on the hemodynamic and vascular parameters in this study indicate that these agents have novel mechanisms of action in vivo that may not be mediated exclusively by sodium-potassium pumps.

Effects of ouabain and flecainide on CO 2-induced slowly adapting pulmonary stretch receptor inhibition in the rabbit

The inhibitory effect of CO 2 on slowly adapting pulmonary stretch receptors (SARs) was examined before and after administration of ouabain, a Na +7z.sbnd;K + ATPase inhibitor, and flecainide, a Na + channel blocker. The experiments were performed in anesthetized, artificially ventilated rabbits after vagus nerve section. CO 2 inhalation (maximal tracheal CO 2 concentration ranging from 9.2 % to 10.4 %) for about 60 sec decreased the receptor activity during both inflation and deflation. The magnitude of decreased SAR activity during deflation was greater than that seen during inflation. Administration of ouabain (25 μg/kg) initially stimulated SAR activities during inflation and deflation, and after 20 min, the SAR response was still kept excitatory in both inflation and deflation phases. Under these conditions, CO 2 inhalation inhibited SAR activities during inflation and deflation. Flecainide treatment (3 mg/kg) that abolished veratridine (30 μg/kg)-induced SAR excitation had no significant effect on the inhibitory responses of SAR activity to CO 2. These results suggest that the inhibitory effect of CO 2, occurs when ouabain results in intracellular Na + concentration ([Na +] i) increases in the SAR endings, and that CO 2-induced SAR inhibition may not be related to the reduction of influx of Na + through voltage-gated Na + channels.

Hypoxia reduces airway epithelial sodium transport in rats.

Ascent to high altitude leads to pulmonary edema formation in some individuals. Recent laboratory evidence supports the hypothesis that hypoxia may impair the function of the alveolar epithelium and thus augment edema accumulation via reduced clearance of lung liquid. We investigated the effect of hypobaric hypoxia on epithelial sodium transport in adult Sprague-Dawley rats by measuring the nasal transepithelial potential difference (PD) as an index of airway sodium transport. Baseline PDs were similar to those previously reported in other species. Administration of amiloride resulted in a significant fall in nasal PD, as did ouabain administration for 24 h (-27.8 vs. -18.8 mV; P = 0.001; n = 5 rats). Exposure to hypobaric hypoxia (0.5 atm) for 24 h caused a significant fall in nasal PD (-23.7 vs. -18.8 mV; P = 0.002; n = 15 rats), which was not additive to the changes in nasal PD produced by amiloride or ouabain. We conclude that subacute exposure to moderate hypobaric hypoxia can inhibit sodium transport by the airway epithelium in rats.

L-arginine restores the effect of ouabain on baroreceptor activity and prevents hypertension.

In spontaneously hypertensive rats, ouabain exerts an excitatory effect on baroreceptor nerve activity (BNA). The aim of this study was to determine the effects of ouabain on BNA in other experimental models of hypertension and its interaction with nitric oxide. Rats were made hypertensive using the procedures for N(omega)-nitro-L-arginine methyl ester (L-NAME), deoxycorticosterone acetate (DOCA) salt, and 2-kidney, 1 clip (2K1C) hypertension models. In these groups, systolic arterial pressure was 195+/-7, 149+/-6, and 148+/-4 mm Hg, respectively, compared with 110+/-4 mm Hg in normotensive rats. Acute ouabain administration had an excitatory effect on BNA in normotensive rats (37+/-4%), an inhibitory effect in L-NAME hypertensive rats (-60+/-7%), and no effect in DOCA-salt and 2K1C hypertensive rats. The effects of ouabain were not related to arterial pressure levels, and no excitatory effect on BNA was observed in prehypertensive DOCA-salt rats. Long-term administration of L-arginine (3 g x kg(-1) x day(-1)) prevented DOCA-salt (121+/-8 mm Hg) and 2K1C (104+/-4 mm Hg) hypertension, markedly attenuated L-NAME (130+/-9 mm Hg) hypertension, and restored the excitatory effect of ouabain on BNA in these groups to levels similar to the normotensive rats and their respective control groups. We conclude that ouabain has a diverse effect on BNA in experimental models of hypertension, and it can be normalized by L-arginine. The data also indicate that nitric oxide may play a pivotal role in mediating the excitatory effect of ouabain on BNA, and we speculate that a therapeutic combination of ouabain and L-arginine may be beneficial in secondary hypertension.

Brain renin-angiotensin system and ouabain-induced sympathetic hyperactivity and hypertension in Wistar rats.

In Dahl salt-sensitive rats on a high salt diet or normotensive rats with chronic central infusion of sodium, increased brain "ouabain" results in sympathetic hyperactivity and hypertension, possibly by activating the brain renin-angiotensin system. In the present study, we tested whether the hypertension caused by exogenous ouabain also depends on activation of brain renin-angiotensin system. In Wistar rats, ouabain (50 micrograms/d) was infused subcutaneously for 14 days with the use of osmotic minipumps. Concomitantly, in one group, the angiotensin II type 1 receptor blocker losartan (1 mg/kg per day) was infused intracerebroventricularly. On day 15, mean arterial pressure, heart rate, central venous pressure, and renal sympathetic nerve activity were recorded in conscious rats at rest and in response to air-jet stress, intracerebroventricular injection of the alpha(2)-agonist guanabenz (25 and 75 micrograms) or angiotensin II (30 ng), acute volume expansion, and ramp changes of blood pressure by +/-50 mm Hg with phenylephrine and nitroprusside. Compared with control rats, in rats treated with ouabain, resting mean arterial pressure was significantly increased (111+/-4 versus 93+/-3 mm Hg; P<0.05), and increases or decreases in mean arterial pressure, heart rate, and renal sympathetic nerve activity in response to air stress or guanabenz were enhanced significantly. These effects of ouabain were prevented when losartan was given concomitantly. Maximal slopes of arterial baroreflex control of renal sympathetic nerve activity and heart rate tended to be decreased in ouabain-treated versus control rats and were significantly increased in ouabain-treated rats with versus without losartan. No differences in cardiopulmonary baroreflex function were detected. It seems that by day 14 to 15, the central effect of ouabain on baroreflex control prevails over its peripheral sensitizing effect on baroreceptors, leading to a tendency of desensitization. These results indicate that chronic administration of ouabain activates the brain renin-angiotensin system, resulting in decreased sympathoinhibition and increased sympathoexcitation, impairment of baroreflex function, and hypertension.

Spinal Antinociceptive Action of Na+-K+Pump Inhibitor Ouabain and Its Interaction with Morphine and Lidocaine in Rats

The Na+,K+-adenosine triphosphatase is a ubiquitous enzyme system that maintains the ion gradient across the plasma membrane of a variety of cell types, including cells in the central nervous system. We investigated the antinociceptive effect of intrathecally administered ouabain and examined its potential interaction with spinal morphine and lidocaine. Using rats chronically implanted with lumbar intrathecal catheters, the ability of intrathecally administered ouabain, morphine, and lidocaine and of mixtures of ouabain-morphine and ouabain-lidocaine to alter tail-flick latency was examined. To characterize any interactions, isobolographic analysis was performed. The effects of pretreatment with intrathecally administered atropine or naloxone also were tested. Intrathecally administered ouabain (0.1-5.0 microg), morphine (0.2-10.0 microg), and lidocaine (25-300 microg) given alone produced significant dose- and time-dependent antinociception, but systemic administration of ouabain did not produce such an effect. The median effective dose (ED50) values for intrathecally administered ouabain, morphine, and lidocaine were 2.3, 5.0, and 227.0 microg, respectively. Isobolographic analysis exhibited a synergistic interaction after the coadministration of ouabain and morphine. With ouabain and lidocaine, there was no such evidence of synergism. Intrathecally administered atropine, but not naloxone, completely blocked the antinociceptive effect of ouabain and attenuated its interaction with spinally administered morphine. Intrathecally administered ouabain produces antinociception, at least in part, via an enhancement of cholinergic transmission in the spinal nociceptive processing system. The results of the interaction of ouabain with morphine and lidocaine suggest that modulation of Na+-,K+-electrochemical gradients and thus subsequent release of neurotransmitters in the spinal cord are likely to play important roles in the spinal antinociceptive effect of intrathecally administered ouabain.

Ouabain- and central sodium-induced hypertension depend on the ventral anteroventral third ventricle region.

To examine the role of the ventral anteroventral third ventricle (vAV3V) in the hypertension induced by chronic subcutaneous ouabain and intracerebroventricular hypertonic saline, neurons in this area were destroyed by microinjection of an excitotoxin, ibotenic acid. Sham-operated or lesioned Wistar rats were administered ouabain (50 microgram/day) or placebo for 3 wk from subcutaneously implanted controlled release pellets or artificial cerebrospinal fluid (CSF) or CSF containing 0.8 mol/l NaCl (5 microliter/h) infused intracerebroventricularly for 2 wk. At the end of the experiment, mean arterial pressure (MAP) and heart rate at rest and in response to ganglionic blockade by intravenous hexamethonium (30 mg/kg) were assessed. In rats infused with hypertonic saline, responses to air jet stress were also assessed. Baseline MAP in sham-operated rats receiving intracerebroventricular hypertonic saline or subcutaneous ouabain was significantly higher than in control rats (115 +/- 1 vs. 97 +/- 3 and 121 +/- 3 vs. 103 +/- 3 mmHg, respectively). vAV3V lesions abolished the increase in MAP elicited by chronic infusion of hypertonic saline or administration of ouabain. Sham-operated rats treated with hypertonic saline or ouabain exhibited significantly enhanced decreases in MAP to hexamethonium, but lesioned rats did not. Rats infused with hypertonic saline demonstrated enhanced responses to air jet stress that were similar in sham-operated and lesioned rats. These results demonstrate that neurons in the vAV3V are essential for the hypertension induced by intracerebroventricular hypertonic saline and subcutaneous ouabain, possibly by increasing sympathetic tone. Cardiovascular responses to air jet stress appear not to be mediated by the vAV3V.