Introduction

Around 1990, I was able to find the anti-herpetic activity in the methanol extract of digitalis leaves. Observation with an optical microscope revealed that the virus-infected human amnion cells were destroyed to pieces, whereas the virus-infected cells to which the digitalis extract was added had a brighter surface and appeared to be more alive than the non-virus-infected cells. I still remember those vividly. This impression became the driving force for my subsequent research. In this way, research to elucidate the mechanism of antiviral action of cardiac glycosides began. Around 2005, I was able to get good results by using the flow cytometer as below.

Importance of membrane potential and ATP in life activities

First, as background knowledge, I would like to confirm the most important membrane potential and ATP in biology. Here, the membrane potential is the potential generated in the entire cell including the cell membrane and the intracellular membrane, and the cell membrane potential is defined as the potential generated in the cell membrane. It is self-evident that animal cells need ATP to survive, but in humans, about 30% of all ATP is consumed by Na⁺,K⁺-ATPase present in the cell membrane. It consumes ATP, excretes intracellular Na⁺ to the outside, and takes in extracellular K⁺ as shown in Fig.1A.

As a result, the K⁺ concentration inside the cell is about 30 times higher than outside.Utilizing this intracellular and extracellular K⁺ concentration difference, K⁺ flows from the inside to the outside of the cell through the K⁺ leak channel. This flow primarily generates the cell membrane potential. This cell membrane potential plays an important role in various cellular activities such as motor control, muscle contraction, nerve transmission, sensory reception, fertilization, immunity, and cell differentiation. By the way, ATP, which is necessary for generating this cell membrane potential, is mainly produced in mitochondria using the flow of protons passing through the inner membrane, that is, the membrane potential. Thus, membrane potential and ATP are essential for all cellular activities.

High school students must learn that the flow of K⁺ from the inside to the outside of the cell through the K⁺ leak channel generates the cell membrane potential, and that the cell membrane potential is fundamental and essential for cells to survive. I would like the detailed explanation as the basis of cell activity because it is not often taken up in biology textbooks.

Although the cell membrane potential of plants and microorganisms that do not have Na⁺,K⁺-ATPase cannot be discussed in the same way as that of animals, the importance of the membrane potential itself is common to all cells.

Anti-stressor effects and changes in intracellular K⁺ concentration by ouabain

During the screening of natural substances for anti-herpetic substances, we found a strong anti-herpetic activity in the methanol extract of digitalis leaves. Subsequently, when various commercially available cardiac glycosides were tested, those activities were observed at low concentrations. Therefore, we hypothesized that inhibition of Na⁺,K⁺-ATPase, which is a common activity of cardiac glycosides, is responsible for the anti-herpetic activity. If so, cardiac glycosides may also exhibit antiviral activity against other viruses. Further expanding, considering that viruses are biological stressors for cells, to investigate the mechanism by which ouabain as a representative cardiac glycoside exhibits anti-stressor effects when various stressors other than viruses are applied to cells, the following experiments were conducted.

First, we applied anticancer drugs to human leukemia cells as chemical stressors, and investigated how the intracellular K⁺ concentration and cytotoxicity changed in the presence or absence of 43 nM ouabain using a flow cytometer. Specifically, cells were treated with an anticancer drug in the presence or absence of ouabain for 24 hours, and then stained with PBFI and PI. Here, PBFI is a reagent that binds to intracellular K⁺ and emits fluorescence, and PI is a reagent that binds to DNA of dead cells and emits fluorescence, but the fluorescence wavelengths of the two are different from each other. Then, the fluorescence intensity of PBFI was measured for about 10,000 viable cells, that is, cells not stained with PI using a flow cytometer, and the average intracellular K⁺ concentration was calculated.

In Figs.2A, 2B, and 2C, ◯ indicates viable cells, ◯ with × indicates dead cells, and red in viable cells represents K⁺, and the higher the concentration, the darker the color.

In the absence of a stressor in Fig.2A, all cells are alive and the K⁺ concentration inside the cells is higher than outside. When the cells were stressed and 50% of the total number of cells became dead cells as shown in Fig.2B, the K⁺ concentration in the surviving cells was higher than that in Fig.2A without a stressor. Next, when the same stressor was applied in the presence of ouabain as shown in Fig. 2C, 75% of the total number of cells survived, but the intracellular K⁺ concentration was lower than in the absence of ouabain in Fig.2B.

Furthermore, similar results were obtained when the number of drug types was increased to 10 or the number of cell types was increased to 5. Similar results were also obtained when the cells were oligotrophic or after exposure to ultraviolet light as a physical stressor.
From this, it was clarified that the intracellular K⁺ concentration increases due to stressors, and that ouabain suppresses the increase as well as the stressor-induced cytotoxicity.

For detailed experimental methods, please refer to our paper (J Pharm Pharmacol. 2015 Jan;67(1):126-132).

How does the intracellular K⁺ concentration increase when a stressor is applied?

In general, it is thought that when a cell weakens due to a stressor, the membrane potential decreases, that is, it approaches zero. The cause of the decrease in cell membrane potential would be that the amount of intracellular ATP decreases due to a stressor, the function of Na⁺, K⁺-ATPase decreases, the intracellular K⁺ concentration decreases, and the K⁺ concentration gradient inside and outside the cell becomes smaller, so the flow through the K⁺ leak channel weakens. However, in the above experiment, as the intracellular K⁺ concentration increased in cells weakened by a stressor, it was supposed that K⁺ leak channel was suppressed by a stressor as shown in Fig.1B.

Traditionally, K⁺ leak channels are thought to be fully open at all times, but they may be blocked by various stressors.
However, it is currently unclear whether a stressor directly acts on the K⁺ leak channel to inhibit it or indirectly inhibits it, and we would like to wait for future research. As supporting evidence, Liu et al. reported in 2005 that cells overexpressing K⁺ leak channels have higher resistance to stressors than the original cells. Although the K⁺ leak channel is the most important channel among K⁺ channels, research on it has been delayed the most, so I would like to expect the future progress in research, including the relationship with stressors.

How does ouabain suppress the stressor-induced cytotoxicity?

As shown in Fig.1C, when cells are stressed in the presence of ouabain, the stressor would inhibit the K⁺ leak channel and reduce the amount of K⁺ efflux from the inside of the cell, but ouabain would inhibit Na⁺, K⁺-ATPase and reduce the amount of K⁺ transported from the outside to the inside of the cell.

As described above, the cell membrane potential is determined mainly by the magnitude of the K⁺ flow from the inside to the outside of the cell, so it is thought that ouabain restored the cell membrane potential that had been reduced by a stressor.

By the way, it is thought that when cells are stressed, the production of ATP in the cells decreases, and the amount of ATP in the cells decreases. However, if ouabain reduces the ATP consumption of Na⁺,K⁺-ATPase, it is thought that the decrease in intracellular ATP due to a stressor can be suppressed.

Thus, ouabain is thought to exert anti-stressor effect by suppressing the stressor-induced decrease in cell membrane potential and intracellular ATP content.

Prospects for clinical application

Although ouabain is often used in laboratory experiments as described above, it is not used clinically because it is not listed in the Japanese Pharmacopoeia. On the other hand, although cardiac glycosides such as digoxin are listed in the Japanese Pharmacopoeia, they are rarely used clinically mainly due to their accumulation and narrow safety margin. However, around 2010, as a DIG (digitalis investigation group) trial, about 7,000 heart failure patients in the United States were followed up for about 3 years with or without the administration of about half the dose of digoxin that showed cardiotonic effects. As a result, the QOL of formers was improved.

By the way, the crude drug Senso (dried toad venom) in Kampo medicine Rokushingan or Kyushin contains cardiotonic steroid bufalin that exibits the same mechanism of action as cardiac glycosides. As bufalin is excreted faster than digoxin, Senso is relatively safe. Considering the results of the DIG trial, it seems that Senso can be expected to have anti-stressor effect at about half of the amount that exhibits cardiotonic effect.

First, as a specific anti-stressor effect of Senso, carcinogenesis preventive effect can be considered, because it is thought that normal cells undergo canceration when they are repeatedly exposed to various stressors, such as ultraviolet irradiation, exposure to irritants, and viral infections. In addition, vaccines are administered to prevent viral infections such as corona virus, but drinking Senso may prevent viral infections. Furthermore, Dr. Kakitsuka’s group at Kyoto University is trying to prevent the development of Alzheimer’s disease by inhibiting ATPase, as various stressors can reduce the amount of ATP in cells and cause Alzheimer’s disease. However, taking Senso as a drug with ATPase inhibitory action may prevent dementia.

In other words, just as aspirin is used not only as an antipyretic analgesic but also in low doses to prevent thrombosis, Senso may be used not only as a cardiotonic but also as an anti-aging drug in low doses. However, Senso has the disadvantage of being expensive and difficult to obtain, so I would like to expect research on the synthesis of safe and inexpensive Na⁺,K⁺-ATPase inhibitors, or digitoxigenin whose structure is very similar to bufalin.

My message

As described above, ATP is produced by membrane potential in mitochondria, and cell membrane potential is formed by ATP on cell membrane. It is no exaggeration to say that membrane potential and ATP are the two wheels of a cell, that is, life. Cellular stressors damage both wheels, and cardiac glycosides appear to prevent their damages, at least in humans. In that sense, I believe that my research has caused a stir in the fields of cell biology and preventive medicine. As a future subject, in the field of cell biology, we must confirm whether the K⁺ leak channel, which plays a major role in the cell membrane potential, is inhibited by stressors. In the field of preventive medicine, it seems necessary to conduct development research on inhibitors of Na⁺,K⁺-ATPase, which is the target of anti-stressor action. Finally, the hypothesis presented at the Pharmaceutical Society of Japan in 2014 is revised as follows.

1. Since dead cells have zero membrane potential and ATP content, those of stressor-weakened cells close to zero.
2. Various stressors (eg, stimulants, UV irradiation, virus infection, etc.) inhibit the K⁺ leak channel in the cell membrane and decrease the cell membrane potential, while also decreasing the amount of intracellular ATP.
3. Senso shows anti-aging effects such as carcinogenesis prevention, dementia prevention and viral infection prevention by inhibiting Na⁺,K⁺-ATPase and suppressing reduction of cell membrane potential and intracellular ATP amount due to stressors.
4. Endogenous ouabain-like substances are anti-stressor substances.

Please send your comments, impressions, questions, etc. to Masayuki Takechi at mt471019@gmail.com.

Researcher information

Former Kinki University Faculty of Pharmaceutical Sciences Professor Masayuki Takechi

I posted the same content as this homepage in the October 2022 issue of Kenkojuku Tsushin. In addition, it was published as an article on October 27, 2022 by Pharmaceutical News.

I have been taking 1mg of Senso after each meal for about 3 years now, and my blood circulation has improved and I have become more energetic.