Doses Of Ouabain Research Articles

Na +/K +-ATPase inhibition by ouabain induces CaMKII-dependent apoptosis in adult rat cardiac myocytes

The positive inotropic effect produced by Na +/K +-ATPase inhibition has been used for the treatment of heart failure for over 200 years. Recently, administration of toxic doses of ouabain has been shown to induce cardiac myocyte apoptosis. However, whether prolonged administration of non-toxic doses of ouabain can also promote cardiac myocyte cell death has never been explored. The aim of this study was to assess whether non-toxic doses of ouabain can induce myocyte apoptosis and if so, to examine the underlying mechanisms. For this purpose, cardiac myocytes from rat and cat, two species with different sensitivity to digitalis, were cultured for 24 h in the presence or absence of 2 µM (rat) and 25 nm–2 µM ouabain (cat). Cell viability and apoptosis assays showed that ouabain produced, in the rat, a 43 ± 5% decrease in cell viability due to apoptosis (enhanced caspase-3 activity, increased Bax/Bcl-2 and TUNEL-positive nuclei) and necrosis (LDH release and trypan blue staining). Similar results were obtained with 25 nM ouabain in the cat. Ouabain-induced reduction in cell viability was prevented by the NCX inhibitor KB-R7943 and by the CaMKII inhibitors, KN93 and AIP. Furthermore, CaMKII overexpression exacerbated ouabain-induced cell mortality which in contrast was reduced in transgenic mice with chronic CaMKII inhibition. However, KN93 failed to affect ouabain-induced inotropy. In addition, whereas ERK½ inhibition with PD-98059 had no effect on cell mortality, PI3K inhibition with wortmannin, exacerbated myocyte death. We conclude that ouabain triggers an apoptotic cascade that involves NCX and CaMKII as a downstream effector. Ouabain simultaneously activates an antiapoptotic cascade involving PI3K/AKT which is however, insufficient to completely repress apoptosis. The finding that KN93 prevents ouabain-induced apoptosis without affecting inotropy suggests the potential use of CaMKII inhibitors as an adjunct to digitalis treatment for cardiovascular disease.

Activation of Akt signaling in rat brain by intracerebroventricular injection of ouabain: A rat model for mania

Intracerebroventricular (ICV) injection of ouabain, a specific Na–K ATPase inhibitor, induces behavioral changes in rats resembling the manic phenotypes of bipolar disorder. The binding of ouabain to the Na–K ATPase affects signal events in vitro including Akt, a possible molecular target of mood disorders. However, the effects of ouabain on Akt in the brain need further clarification. In this study, we investigated changes in the phosphorylation state of Akt in the rat brain after ICV injection of ouabain. Consistent with our previous report, the locomotor activity of rats within 30 min after ouabain ICV injection changed according to the dose with higher doses of ouabain, 0.5 and 1 mM, inducing significant hyperactivity. In addition, ouabain administration induced a dose-dependent increase in the immunoreactivity of p-Akt (Ser473) in the frontal cortex, striatum, and hippocampus after 30 min, and reached statistical significance with 1 mM of ouabain. Phosphorylation of GSK-3β (Ser9), FOXO1 (Ser256), and eNOS (Ser1177), which are downstream molecules of Akt, was also increased in a dose-dependent manner within the same brain regions. Moreover, hyperactivity was seen for 8 h after a single 1 mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3β (Ser9), FOXO1 (Ser256), and eNOS (Ser1177) was also observed in the cortex, striatum, and hippocampus. Thus, intrabrain injection of ouabain induces activation of Akt signaling accompanied by hyperactivity, suggesting the possible role of Akt in ouabain rat model of mania.

Preconditioning by Subinotropic Doses of Ouabain in the Langendorff perfused Rabbit Heart

Short exposure to low concentrations of digitalis drugs like ouabain protects the rat heart against ischemia/reperfusion injury through the activation of the Na/K-adenosine triphosphatase (ATPase)/Src receptor complex and subsequent stimulation of key intracellular cardioprotective signals. Rat Na/K-ATPase, however, is relatively insensitive to digitalis, and it is not known if similar results could be obtained in species with higher sensitivity. Thus, to determine whether ouabain pretreatment protects against ischemic injury and activates the Na/K-ATPase signaling cascade in a species with cardiac glycoside sensitivity comparable to humans, the present study was conducted in the rabbit model. In Langendorff perfused rabbit hearts, 20-minute exposure to 500-nM ouabain resulted in positive inotropy as evidenced by a significant increase in +dP/dt, and this increase was accompanied by the activation of several well-characterized downstream mediators of the cardiac Na/K-ATPase receptor pathway, including Src, Akt, ERK1/2, and protein kinase Cepsilon. A short (4 minutes) administration of a subinotropic dose of ouabain (100 nM) followed by an 8-minute washout before 30 minutes of global ischemia and 120 minutes of reperfusion resulted in protection against cell death, as evidenced by a significant decrease in infarct size. These data indicate that ouabain administration activates the Na/K-ATPase signaling cascade and protects against ischemic injury in a species with high cardiac Na/K-ATPase sensitivity.

Activity of the Na,K-ATPase α4 isoform is important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat spermatozoa

While the function of the ubiquitous Na,K-ATPase alpha1 subunit has been well documented, the role of the sperm-specific alpha4 isoform of this ion transporter is less known. We have explored the importance of alpha4 in rat sperm physiology by taking advantage of the high sensitivity of this isoform for the inhibitor ouabain. Using concentrations that selectively block alpha4 activity, we found ouabain to reduce not only sperm total motility, but also multiple parameters of sperm movement, including progressive motility, straight line, curvilinear, and average path velocities, lateral head displacement, beat cross frequency, and linearity. According to a direct role of alpha4 in Na(+) transport, ouabain inhibition of alpha4 increased [Na(+)](i) in the male gametes. In addition, interference of alpha4 activity with ouabain produced cell membrane depolarization, diminished pH, and increased [Ca(2)(+)](i) in spermatozoa. Inhibition of alpha4 was sufficient to cause all these effects and additional blockage of alpha1, the other Na,K-ATPase alpha isoform expressed in sperm, and higher doses of ouabain did not result in further changes in the cell parameters studied. These results show that alpha4 is the Na,K-ATPase isoform primarily involved in controlling the transmembrane Na(+) gradient in sperm, and that alpha4 activity is necessary for maintaining membrane potential, [Ca(2)(+)](i), and [H(+)](i) in the cells. The high dependence of sperm motility on membrane excitability, [Ca(2)(+)](i), and acid-base balance suggests that their regulation is the mechanism by which alpha4 maintains motility of the male gametes.

Effect of GBE50 on experimental arrhythmias

The action and mechanism of ginkgo leaf extract (EGB50) in fighting arrhythmia were studies. The animal model of arrhythmia was established by intravenous drip of aconitine and ouabain, and the standard microelectrode technique was used. In Oua model, threshold doses of ouabain induced VP, VT, VF, CA were observed. GBE50 significantly increased threshold doses of ouabain. In Aco model, threshold doses of Aco induced VP, VT, VF, CA were observed, GBE50 significantly increased threshold doses of Aco. Effects of GBE50 on delayed afterdepolarization and triggered activity induced by ouabain in guinea pig papillary muscles DADs and TA were markedly suppressed by GBE50. The amplitude and duration of DADs were reduced by GBE50 (50 mg x L(-1)) from (13.25 +/- 2.38) mV and (198.38 +/- 53.62) ms to (4.04 +/- 1.44) mV and (57.00 +/- 18.62) ms, respectively, and the induced time of DADs was prolonged from (12.37 +/- 2.26) to (23.00 +/- 4.00) min. TA was reduced from 87.5% to 16.67% (P<0.05, P<0.01). GBE50 (2,10 mmol x L(-1)) had significant therapeutic effects on DADs. The amplitude and duration of DADs were reduced to (10.41 +/- 3.06) mV, (8.92 +/- 2.68) mV and (128.33 +/- 18.91) ms, 103.33 +/- 20.64 ms (P<0.05, P <0. 01 vs control). GBE50 can fight arrhythmia following aconitine and ouabain. GBE50 has inhibitory effects on DADs and TA induced by ouabain and high Ca2+ in guinea pig papillary muscles.

Counter current transfer of beta-endorphin in the perihypophyseal cavernous sinus--carotid rete vascular complex of sheep.

This study was performed to answer the question of whether counter current retrograde transfer of beta-endorphin in the perihypophyseal cavernous sinus-carotid rete vascular complex depends on the reproductive activity of sheep and if this transfer depends on membrane Na+ K+ ATP-ase blocking by ouabain. Sheep were anaesthetised and the jugular vein and the carotid artery were cannulated on both sides. Multielectrolitical liquids (Solfin, Polfa "Kutno", Poland): 500 ml of Solfin with heparin (25,000 IU), or Solfin with heparin and ouabain (Sigma, St. Louis, USA) in concentrations of 10(-5) or 10(-4) mol 1(-1) were infused into the brain through the carotid artery. Heparinized blood was collected through the carotid artery. After exsanguination, the head with the neck was removed. The isolated head was supplied with oxygenated, heated, autologous blood diluted with Solfin (4:1) without or with ouabain in concentration of 10(-5) or 10(-4) mol 1(-1). Blood pressure and temperature were measured throughout the duration of the experiment. During the experiment 125I-beta-endorphin (7.9 x 107 dpm) dissolved in 10 ml of Solfin was infused for 5 min (5 ml) into each cavernous sinus through the angularis oculi veins. Blood samples for radioactivity measurements were collected each min from the carotid rete (through the opposite carotid artery to the artery supplying the brain with arterial blood) and from both jugular veins. In all the experiments significant 125I-beta-endorphin radioactivity was found in arterial blood supplying the brain and hypophysis in the early luteal phase in sheep. No radioactivity was found (with the exception of one animal) in sheep during seasonal anoestrus. A blockage of Na+ K+ ATP-ase by ouabain administered during exsanguination and during head perfusion with dose of 10(-4) mol 1(-1) reduced beta-endorphin counter current transfer to arterial blood, but this effect was not evident with the dose of 10(-5) mol 1(-1). Increased blood pressure was observed in all the experiments with either dose of ouabain.

Intracerebroventricular Administration of Ouabain to Rats Changes the Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus

Ouabain exerts neurotoxic action and activates the population of NMDA receptors. Herein the effect of ouabain on the expression of NMDA subunits was evaluated. Adult Wistar rats were administered intracerebroventricularly with 0.1, 10 and 100 nmol ouabain or saline solution (control). Two days later, membranes of cerebral cortex and hippocampus were isolated. Western blots with antibodies for the NMDA receptor subunits: NR1; NR2A; NR2B; NR2C and NR2D were carried out. In cerebral cortex, NR2D subunit increased 30% with 10 nmol ouabain dose. With 100 nmol ouabain, NR1 and NR2D subunits enhanced 40 and 20%, respectively. In hippocampus, with the dose of 0.1 nmol ouabain, NR1 subunit enhanced roughly 50% whereas NR2B subunit decreased 30%. After administration of 10 nmol ouabain dose, NR2A, NR2B and NR2C subunits decreased 40, 50 and 30%, respectively. With the dose of 100 nmol of ouabain, NR1, NR2A and NR2B subunits diminished 10-20%. It is concluded that ouabain administration led to a differential regulation in the expression of NMDA subunits. These results may be correlated with the modulatory action of ouabain on NMDA receptor.

Mechanisms in the PVN mediating local and central sodium-induced hypertension in Wistar rats

Sympathoexcitatory and hypertensive responses to central infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF) are enhanced by aldosterone and mediated by mineralocorticoid receptors (MRs) and benzamil-blockable Na(+) influx, leading to "ouabain" release and ANG II type 1 (AT(1)) receptor stimulation. The present study evaluated the functional role of these mechanisms in the paraventricular nucleus (PVN). In conscious Wistar rats, Na(+)-rich aCSF was infused either directly into the PVN or intracerebroventricularly preceded by aldosterone and blockers. Infusion of Na(+)-rich aCSF in the PVN caused gradual increases in blood pressure (BP) and heart rate (HR). Aldosterone and a subpressor dose of ouabain in the PVN alone did not affect BP and HR but enhanced responses to Na(+). Eplerenone, benzamil, and "ouabain"-binding Fab fragments only blocked the enhancement by aldosterone, whereas losartan blocked all responses to Na(+)-rich aCSF in the PVN. Increases in BP and HR by intracerebroventricular infusion of Na(+)-rich aCSF were enhanced by aldosterone infused intracerebroventricularly, but not in the PVN. Telmisartan in the PVN again blocked all responses. In contrast, both eplerenone and benzamil in the PVN did not change the pressor responses to intracerebroventricular infusion of aldosterone and Na(+)-rich aCSF. These findings indicate that AT(1) receptors in the PVN mediate the responses to Na(+)-rich aCSF and their enhancement by aldosterone, both locally in the PVN or in the general CSF. MRs, benzamil-blockable Na(+) channels or transporters, and "ouabain" can be functionally active in the PVN, but in Wistar rats appear not to contribute to the pressor responses to short-term increases in CSF [Na(+)].

Differential expression of Na+/K+-ATPase alpha-subunits in mouse hippocampal interneurones and pyramidal cells.

The sodium pump (Na+/K+-ATPase), maintains intracellular and extracellular concentrations of sodium and potassium by catalysing ATP. Three sodium pump alpha subunits, ATP1A1, ATP1A2 and ATP1A3, are expressed in brain. We compared their role in pyramidal cells and a subset of interneurones in the subiculum. Interneurones were identified by their expression of GFP under the GAD-65 promoter. We used the sensitivity to the cardiac glycoside, ouabain, to discriminate between different alpha subunit isoforms. GFP-positive interneurones were depolarized by nanomolar doses of ouabain, but higher concentrations were needed to depolarize pyramidal cells. Comparison of pump currents in these cells revealed a current sensitive to low doses of ouabain in interneurones, while micromolar doses of ouabain were needed to suppress the pump current in subicular pyramidal cells. As predicted, nanomolar doses of ouabain increased the frequency but not the amplitudes of IPSPs in pyramidal cells. Immunostaining confirmed a differential distribution of alpha-subunits of the Na+/K+-ATPase in subicular interneurones and pyramidal cells. In conclusion, these data suggest that while ATP1A3-isoforms regulate sodium and potassium homeostasis in subicular interneurones, ATP1A1-isoforms assume this function in pyramidal cells. This differential expression of sodium pump isoforms may contribute to differences in resting membrane potential of subicular interneurones and pyramidal cells.

Antiarrhythmic Properties of Acetaminophen in the Dog

Mongrel dogs bred for research and weighing 25 +/- 3 kg were used to test the hypothesis that acetaminophen has antiar-rhythmic properties. Only ventricular arrhythmias defined by the Lambeth Conventions were investigated. Dogs were exposed either to 60 mins of regional myocardial ischemia followed by 180 mins of reperfusion (n = 14) or were administered a high dose of ouabain (n = 14). Both groups of 14 dogs were further divided into vehicle and acetaminophen treatment groups (n = 7 in each). During selected 10-min intervals, we recorded the numbers of ventricular premature beats, ventricular salvos, ventricular bigeminy, ventricular tachycardia (nonsustained and sustained), and we recorded the heart rate, systemic arterial blood pressure, and left ventricular function. Neither heart rate nor the number of ventricular arrhythmias differed significantly under baseline conditions. Conversely, the combined average number of ventricular ectopic beats during ischemia and reperfusion was significantly less in the presence of acetaminophen (28 +/- 4 vs. 6 +/- 1; P < 0.05). Similarly, percent ectopy during reperfusion in vehicle- and acetaminophen-treated dogs was 1.4 +/- 0.4 and 0.4 +/- 0.2, respectively (P < 0.05). The number of all ventricular ectopic beats except ventricular salvos was also significantly reduced in the presence of acetaminophen. Similar results were obtained with ouabain. Our results reveal that systemic administration of a therapeutic dose of acetaminophen has previously unreported antiarrhythmic effects in the dog.

Intrahippocampal microinjection of an exquisitely low dose of ouabain mimics NaCl loading and stimulates a bufadienolide Na/K-ATPase inhibitor

Brain endogenous ouabain (EOU) raises blood pressure (BP) via an angiotensin II (ATII)-sensitive pathway in NaCl-loaded Dahl salt-sensitive rats (DSS). Brain EOU activates central and adrenocortical renin-angiotensin systems, and stimulates marinobufagenin, a vasoconstrictor and natriuretic inhibitor of sodium pump. We studied effects of acute NaCl loading (17 mmol/kg NaCl, intraperitoneally) on levels of EOU and marinobufagenin in several brain areas in DSS. We then studied effects of intrahippocampal administration of very-low-dose ouabain (60 pg) on EOU, marinobufagenin, BP, sodium excretion, and sodium-pump activity in the aorta and renal medulla in the absence and presence of anti-marinobufagenin and anti-ouabain antibodies, and losartan. NaCl loading of DSS induced transient increases of EOU in the hippocampus and amygdala (15 min; 300%), supraoptical nucleus of hypothalamus (SON) (30 min, 230%) and pituitary (30 min; 85%), and ATII elevation in the SON (30 min). Intrahippocampal administration of ouabain (60 pg) stimulated ATII in the SON, produced natriuresis, 40 mmHg rise in BP, inhibition of sodium-pump in the renal medulla (19.6%) and aorta (25%), and a two-fold increase in renal marinobufagenin excretion. Pretreatment of rats with anti-marinobufagenin antibody prevented ouabain-induced pressor and natriuretic responses and sodium-pump inhibition. Pressor responses to ouabain were also prevented by losartan (intravenously) and by administration of anti-ouabain antibody into the SON. NaCl loading of DSS induces a cascade of events, triggered by brain EOU and ATII. Intrahippocampal administration of a low-dose ouabain mimics effects of NaCl loading and stimulates marinobufagenin, which produces natriuresis, and inhibits the vascular sodium-pump, inducing an increase in BP.

Effects of purified endogenous inhibitor of the Na +/Ca 2+ exchanger on ouabain-induced arrhythmias in the atria and ventricle strips of guinea pig

Previous studies demonstrated that the purified endogenous inhibitor (NCX IF) of the cardiac Na +/Ca 2+ exchanger (NCX1) has the capacity to modulate cardiac muscle contractility. Here, we tested the effects of purified NCX IF on arrhythmias induced by ouabain in the atria and ventricle strips of guinea pig. For the sake of comparison NCX IF was compared to lidocaine and KB-R7943. In the ventricle strip, NCX IF (∼ 10 U/ml) results in rapid, complete and stable inhibition of ouabain-induced arrhythmias (the inhibition of arrhythmia is not followed by revival of irregular contractions). Under similar experimental conditions the atria strips require somewhat higher doses of NCX IF (25–50 U/ml) for complete suppression of arrhythmia. In the atria strip, NCX IF (10–25 U/ml) increases the threshold dose (1 μM) of ouabain for arrhythmia onset 2.2 ± 0.5-fold ( n = 5, p < 0.05) as well as prolongs the lag-phase for arrhythmia appearance 4.0 ± 0.5-fold ( n = 5, p < 0.01). The lag period for arrhythmia onset was also lengthened (2.0 ± 0.4-fold) by NCX IF in the ventricle strips ( n = 6, p < 0.002). At low frequency of pacing (1 Hz), all three tested substances, lidocaine, KB-R7943, and NCX IF can effectively suppress the ouabain-induced arrhythmia. However, at higher frequency (2 Hz), lidocaine is ineffective in suppressing arrhythmia, whereas KB-R7943 becomes pro-arrhythmic. In contrast to reference drugs, NCX IF retains its anti-arrhythmic capacity at high frequencies, either in the atria ( n = 6, p < 0.01) or ventricle ( n = 5, p < 0.05) strips. In conclusion, NCX IF results in rapid, effective and stable suppression of arrhythmia both in the atria and ventricle preparations under conditions at which the reference drugs become ineffective.

Low Doses of Ouabain Protect from Serum Deprivation–Triggered Apoptosis and Stimulate Kidney Cell Proliferation via Activation of NF-κB

It now generally is agreed that Na,K-ATPase, in addition to its role in the maintenance of Na+ and K+ gradients across the cell membrane, plays a role in communicating information from the extracellular environment to intracellular signaling pathways. It was reported recently that interaction between ouabain-bound Na,K-ATPase and the 1,4,5-trisphosphate receptor (IP3R) triggers slow calcium oscillations and activation of NF-kappaB. Here it is demonstrated that this signaling pathway can serve to prevent cell death and promote cell growth. Rat renal proximal tubular cells in primary culture first were grown in the presence of 10% serum and then exposed to 0.2% serum for 24 h to induce apoptosis. Serum starvation increased the apoptotic index from 1.21 +/- 0.26 to 14.01 +/- 1.17%. Ouabain in concentrations that did not inhibit Na,K-ATPase activity (1 to 10 nM) completely abolished the apoptotic effect of serum starvation. Ouabain protection from apoptosis was not observed when release of calcium from intracellular stores via the IP3R was prevented. It was shown that the NH2 terminal tail of the Na,K-ATPase alpha subunit plays a key role in ouabain-triggered calcium oscillations. It was found that ouabain did not protect from apoptosis in serum-deprived cells that expressed a mutant Na,K-ATPase alpha subunit with deletion of the NH2 terminal tail. Ouabain exposure (10 nM for 24 h) significantly increased translocation of NF-kappaB from cytoplasm to nucleus. Helenalin, an inhibitor of NF-kappaB, abolished the antiapoptotic effect of ouabain. Ouabain (0.1 to 10 nM) also was found to stimulate proliferation and increase the viability of kidney cells. These effects were abolished when release of calcium via the IP3R was prevented.

Resveratrol, a natural ingredient of grape skin: Antiarrhythmic efficacy and ionic mechanisms

Resveratrol has been demonstrated to produce a variety of biological actions. Accumulating line of evidence supported the view that resveratrol may exert protective effect on the cardiovascular system. The aim of the study was to assess the antiarrhythmic profile as well as electrophysiological properties of resveratrol. We observe the antiarrhythmic effect of resveratrol on aconitine induced rat arrhythmia, ouabain induced guinea pig arrhythmia, and coronary ligation induced rat arrhythmia animal models. Resveratrol significantly and dose-dependently increased the doses of aconitine and ouabain required to induce the arrhythmia indexes. In coronary ligation induced rat arrhythmia model, resveratrol shortened duration of arrhythmia, decreased incidence of ventricular tachycardia and mortality. Electrophysiological experiment revealed that resveratrol could shorten APD through inhibition of I Ca and selective enhancement of I Ks without an effect on I Kr.

FISH OSMOREGULATE WITH OUABAIN

Ouabain, a potent inhibitor of the enzyme Na+/K+-ATPase, is known to be a steroid hormone in mammals that is involved in blood pressure and volume regulation. Recently, a group from the University of Hawaii examined whether ouabain also acts as a hormone in fish and reported that ouabain is present in the plasma and various tissues of the euryhaline Mozambique tilapia, Oreochromis mossambicus. When the researchers transferred tilapia between fresh and salty water, they discovered that changes in plasma concentrations of ouabain in tilapia were well correlated with changes in plasma osmolality and cortisol, an important hormone during salinity change. Now, this same research group has followed up with an equally intriguing analysis of the functional role of ouabain in fish osmoregulation.The present study set out to establish whether ouabain interacts with growth hormone and prolactin (known osmoregulatory hormones in fish) in the same manner as cortisol. During seawater acclimation, cortisol stimulates Na+/K+-ATPase activity and the proliferation of chloride cells that are responsible for the active extrusion of Cl-. In addition, cortisol stimulates the release of growth hormone, which is also involved in seawater adaptation, and inhibits the release of prolactin, an important hormone in freshwater osmoregulation, from the pituitary gland. Prolactin release is dependent on calcium entering pituitary gland cells and the synthesis of cAMP, both of which are prevented by cortisol.To assess the role of ouabain in fish osmoregulation, Kajimura, Grau and the group examined the release of osmoregulatory hormones by tilapia pituitary glands. They isolated whole pituitary glands from tilapia and placed them individually in a 96-well plate containing 200 μl of incubation media. With this in vitro preparation, they measured prolactin and growth hormone release from tilapia pituitary glands in response to different doses of ouabain. To see if ouabain, like cortisol, inhibits prolactin release by preventing calcium uptake and cAMP synthesis, they measured prolactin release in response to calcium (using the calcium ionophore A23187) and to cAMP (using the analogue dbcAMP and the phosphodiesterase inhibitor IBMX) in the presence or absence of ouabain. The group found that at physiological concentrations,ouabain exerted a dose-dependent inhibitory effect on prolactin, whereas supra-physiological doses of ouabain stimulated prolactin release. By contrast, ouabain stimulated growth hormone release at lower concentrations but had an inhibitory effect at higher levels. Both A23187 and dbcAMP + IBMX had stimulatory effects on prolactin release, and these effects were inhibited by ouabain when it was also placed in the incubation media. This suggests that, like cortisol, ouabain regulates the mechanism of prolactin release. Furthermore, using microspectrofluorometry and a calcium-sensitive dye, the team was able to quantify `real-time' changes in calcium levels within pituitary cells. Their data indicated that at physiological concentrations,ouabain rapidly reduces intracellular calcium concentrations, just as cortisol does.The group has shown convincingly that, similar to the actions of cortisol during salinity change, ouabain plays an important role in fish osmoregulation by inhibiting the release of prolactin via the Ca2+ and cAMP signal transduction pathways. The team believes that ouabain in the hypothalamus of the brain may be involved in the regulation of pituitary prolactin release and hope to prove this hypothesis in the future by using a ouabain-specific antibody and immunocytochemistry. Until then, we will wait for more news about this exciting new fish hormone!

Cardiovascular effects of the aqueous extract of Gynostemma pentaphyllum Makino

In the present study, the cardiovascular activity of the aqueous extract of Gynostemma pentaphyllum Makino leaves was investigated in the anaestetized guinea-pigs and has been compared with two of its isolated gypenosides (III, VIII) and with verapamil, a well-known Ca-antagonistic drug. The results obtained showed that the intravenous administration of the decoction of G. pentaphyllum (2.5, 5 and 10mg/kg) produced a protective effect against pitressin-induced coronaryspasm, arrhythmias and pressor response. Extract also increased the dose of ouabain required to cause ventricular tachyarrhythmias and lethality. Further extract reversed ouabain-induced persistent ventricular tachycardia and restored sinus rhythm in a dose-dependent manner. The results obtained have also shown that gypenosides III and VIII caused similar protective effects in both experimental models used; however, the duration of the action is lower than that of the extract containing corresponding quantities of gypenosides III and VIII.

Physiological concentrations of ouabain rapidly inhibit prolactin release from the tilapia pituitary

Ouabain, a cardiac glycoside and inhibitor of Na +, K +-ATPase, is now believed to be a steroid hormone in mammals. We have recently identified ouabain immunoreactivity in the plasma of the tilapia, a euryhaline teleost. Changes in plasma concentrations of immunoreactive ouabain (20–40 pM) in response to salinity change were well correlated with the changes in plasma osmolality and cortisol. Our previous studies have shown that cortisol rapidly inhibits prolactin (PRL) release from the tilapia pituitary by suppressing intracellular Ca 2+ ([Ca 2+] i) and cAMP. In the present study, low doses of ouabain (10–1000 pM) inhibited PRL release dose-dependently during 2–24 h of incubation. There was no effect on growth hormone (GH) release, except for a significant increase at 1000 pM during 8–24 h of incubation. Significant dose-related increases in PRL release were observed at higher doses of ouabain (100–1000 nM), whereas significant inhibition was seen in GH release at 1000 nM during 2–24 h of incubation. Ouabain at 1–100 pM had no effect on Na +, K +-ATPase activity of the pituitary homogenate. The enzyme activity was inhibited by higher concentrations of ouabain, 10% at 1 nM, 15% at 10 nM, 28% at 100 nM, and 45% at 1000 nM. Ouabain also attenuated stimulation of PRL release by the Ca 2+ ionophore, A23187, and by a combination of dibutyryl cAMP and a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthin. Intracellular Ca 2+ concentrations were monitored in the dispersed PRL cells with the Ca 2+-sensitive dye, fura-2. Ouabain at 1 nM reversibly reduced [Ca 2+] i within seconds, whereas 1 μM ouabain increased [Ca 2+] i. A rapid reduction in [Ca 2+] i was also observed when PRL cells were exposed to 1 μM cortisol, whereas there was no consistent effect at 1 nM. These results suggest that ouabain at physiological concentrations rapidly inhibits PRL release from the tilapia pituitary by suppressing intracellular Ca 2+ and cAMP metabolism. The stimulation of PRL release by high concentrations of ouabain (100–1000 nM) may result from an increase in [Ca 2+] i, and subsequent depolarization due to the inhibition of Na +, K +-ATPase activity.

Positive inotropic effect of ouabain on isolated heart is accompanied by activation of signal pathways that link Na+/K+-ATPase to ERK1/2.

Exposure of cultured rat cardiac myocytes to ouabain is known to cause the interaction of Na+/K+-ATPase with adjacent proteins, leading to activation of multiple signal transduction pathways, regulation of growth-related genes, and hypertrophy. The aim of this work was to determine if the proximal signaling events identified in cultured myocytes also occur in isolated intact hearts of rat and guinea pig in response to positive inotropic doses of ouabain. Langendorff rat heart preparations were exposed to 50 microM ouabain to produce positive inotropy without toxicity, and assayed for Src kinase, protein kinase C, and extracellular signal-regulated kinases 1 and 2 (ERK(1/2)). These kinases were rapidly activated by ouabain as in cultured cells. In isolated guinea pig hearts, 1 microM ouabain caused ERK(1/2) activation comparable to the effect of 50 microM ouabain in rat heart and consistent with the higher ouabain sensitivity of the contractility of guinea pig heart. These data show that the proximal ouabain-induced signal pathways previously noted in cultured cells are not artifacts of dispersion/culturing of myocytes, and are not the peculiar properties of the rat heart with its relatively low ouabain sensitivity. They also suggest that treatment with positive inotropic doses of cardiac glycosides is likely to be associated with changes in the cardiac phenotype.

Na,K-ATPase as a signal transducer.

Recent studies have indicated that Na,K-ATPase may, in addition to being the key regulator of intracellular Na(+) and K(+) concentration, act as a signal transducer. Despite extensive research, the biological role for ouabain, a natural ligand of Na,K-ATPase, is not well understood. We have reported that exposure of rat proximal tubular cells (RPTC) to doses of ouabain that inhibit the Na,K-ATPase activity by less than 50% (10 nM - 500 micro M), will induce intracellular [Ca(2+)](i) oscillations and that this calcium signal leads to activation of the transcription factor NF-kappaB. The ouabain-induced calcium oscillations were blocked by an inhibitor of the IP(3) receptors but not by phospholipase C inhibitors nor by cellular depletion of IP(3), suggesting that the calcium signal is not due to phospholipase C-mediated IP(3) release. Fluorescence resonance energy transfer (FRET) studies suggested a close proximity between the Na,K-ATPase and IP(3) receptor. Our findings demonstrate a novel principle for calcium signaling via Na,K-ATPase.

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase.

Ouabain binding to Na(+)/K(+)-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na(+)/K(+)-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of alpha(1)- and alpha(2)-isoforms of Na(+)/K(+)-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and alpha-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar alpha(1)-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and alpha(2)-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na(+)/K(+)-ATPase to ERK1/2 and [Ca(2+)](i) are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and alpha(2)-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.