1. RELATIONSHIP BETWEEN GLUTATHIONE REDOX STATE AND THE VASOCONSTRICTOR RESPONSE TO HYPOXIA IN RAT PULMONARY ARTERIES Jesus Prieto-Lloret, Jeremy P. Ward & Philip I. Aaronson Asthma, Allergy and Lung Biology, King's College London, London, UK

2. Hypoxic response & Redox hypothesis SCHUMACKER& WAYPA group
P (mmHg)
PULMONARY ARTERY
CONSTRICTION (HPV)
SYSTEMIC ARTERY
VASODILATATION
MITOCHONDRIA
Krebs cycle
Glycolisis
GLUCOSE
NADH
ETC I II
III IV
CYTOSOL
SOK Ca2+ channel +
Ca2+ SR
L-type Ca2+ channel
Ca2+ + +
[Ca2+]i
Pulmonary and vascular arteries responds in different ways to hypoxia. While pulmonary arteries contract, in what is called hypoxic pulmonary vasoconstriction, vascular arteries give a vasodilatation.
The redox theory tries to explain the relationship between reactive oxygen species and the constrictor response. There are opposite theories implying and increase or decrease of these species as the main stimuli for the constriction underlying the hypoxic pulmonary vasoconstriction. A hypoxic stimuli causes the inhibition of different complexes in the electron transport chain, that would be acting as the oxygen levels sensors, according to these theories, producing an increase or decrease in the concentration of ROS, that would activate ryanodine receptors in the sarcoplasmic reticulum giving an increase in the intracellular calcium concentration. This increase in calcium inactivates the calcium dependent and voltage gated Kv channels, resulting in membrane depolarization, and opening of calcium channels type L, that would also contribute to the increase in the concentration of intracellular calcium. This increase in calcium is the final responsible for the constriction of the smooth muscle cells and the hypoxic pulmonary vasoconstriction.
ROS K+
Kv channel
SCHUMACKER& WAYPA group
ARCHER group
HPV
PLASMA MEMBRANE
Hypoxia

3. Glutathione system & Redox state
ROS or
Effects on proteins,etc
Changes in redox state
HPV ? ?
Glutathione
Peroxidase
Glutathione
Reductase
Glucose-6P
dehydrogenase
H2O
GSH
NADPH
PPP
glucose 6-phosphate
glucose
Catalase
NADP+
GSSG
H2O2
GSSG + 2H+ + 2e-  2GSH
SOD
O2.-
ENERNST = -240 – (59.1/2) log ([GSH]2/[GSSG]) (mV)
[GSH]2/[GSSG] ratio reflects cell redox state

4. Redox potential in hypoxia
in Pulmonary Artery
in Aorta
Redox couples in cells are responsible for the electron flow and the reducing/oxidizing environment.
Between these couples glutathione is considered to be the major thiol-disulfide redox buffer of the cell, with cytosolic concentrations between 1-10mM, higher than other redox active compounds. Measurement of levels of GSH and GSSG would give us an indicator of the redox state, that would be more accurate employing the Nernst equation.
Using this approximation we found different changes in Nernst potential depending on the level of hypoxia applied to the tissue. These differences could explain the opposite response observed in hypoxia and could be the reason for the different results obtained between groups, that could be explained on the basis of different levels of hypoxia applied. Moreover, the different Nernst potential between pulmonary artery and aorta is indicating a more oxidised state for the aorta. This point could be the reason why pulmonary and vascular arteries are responding differently to hypoxia. As I said before hypoxia would be modifying ROS levels in hypoxia, that could be working in different ways depending on the basal redox level for each artery.
HYPOXIA CAUSES OXIDATION
MODERATE HYPOXIA CAUSES REDUCTION
BASAL REDOX STATE IS MORE OXIDISED IN AORTA THAN IN PULMONARY ARTERY

5. Does these oxidising agents mimic HPV?
Glutathione
Peroxidase
Glutathione
Reductase
Glucose-6P
dehydrogenase
H2O
GSH
NADPH
Catalase
glucose 6-phosphate
PPP
glucose
GSSG
NADP+
H2O2
blocking
DHEA
mercaptosuccinate
carmustine
activation
SOD
ebselen
FAD
O2.-
Carmustine blocks conversion of GSSG to GSH and NADPH to NADP+.
DHEA blocks the pentose phosphate pathway.
DEM depletes glutathione.
The cyclic reaction that converts reduced glutathione to oxydised glutathione is regulated by the enzymes glutathione peroxidase and glutathione reductase, enzymes that can be upregulated or blocked using different approaches. As blockers we used mercaptosuccinate for GPx and carmustine for GR. In the same direction we can mimic or upregulate these reactions, using ebselen, a compound that mimics the coupled conversion from GSH to GSSG regulated by GPX, or flavin adenine dinucleotide, a coenzyme for glutathione reductase that activates it.
Additionally the main reaction is coupled with other reactions, mainly the conversion from NADPH to NADP+, and the coupled conversion of glucose 6 phosphate to 6 phosphogluconolactone, both of them regulated by glucose 6 phosphate dehydrogenase, an enzyme that is modulated by hypoxia and regulates the important NADPH/NADP+ ratio, that when increased in hypoxia conduits to membrane depolarization and HPV. We can also block these reactions, using dehydroepiandrosterone or deoxyglucose. We checked the effects of some of these drugs on the glutathione levels in pulmonary arteries, and we found a marked change in the levels of total glutathione when using carmustine, DHEA and the glutathione depletor diethylmaleate, as has been described by other authors.
Caused IPA oxidation.
Does these oxidising agents mimic HPV?

6. Are oxidising agents able to evoke a response similar to HPV?
CARMUSTINE 100µM
From the results presented before, and as hypoxia is able to oxidise the cells, the agents able to oxidise tissue would have to evoke a response in the pulmonary artery similar to those observed in response to hypoxia, like the one that I have represented here.
When we applied carmustine we didn´t observe any response, similar result to the observed with DHEA, and DEM.
Whatever pathway is activated by hypoxia that increases the redox potential in the cells is not linked in the same way that we could obtain employing substances that modify the redox potential.
DHEA 100µM
DEM 1mM

7. Effects on HPV with pretoneMS
CARMUSTINE MS
carmustine
GSH
H2O
NADPH
PPP
H2O2
GSSG
NADP+
FAD
The next step was the study of the effects of some of these substances on the hypoxic pulmonary vasoconstriction using prostaglandin as pretone. The use of pretone will allow us to have better responses and study these effects with more precision.
In these first traces we studied again the effects of mercaptosuccinate, that, as I said before is blocking GPx, and is increasing the second phase without affecting the first, effect that is not reversed after washing. Ebselen, that mimics GPx converting the oxydised form of glutathione to the reduced is blocking both phases. I show here the effects of two different concentrations.
Going to the other side of the cyclic reaction, carmustine, glutathione reductase blocker, absolutely blocked the second phase without affecting the first one, while FAD, a known activator of the enzyme seems to increase the vasoconstrictor response.
The employment of DEM and DHEA that were increasing considerably the Nernst potential by decreasing glutathione levels in tissue are blocking HPV, both, transient and sustained phases.
ebselen
FAD
EBSELEN

8. Effects on HPV and redox potential
As a summary of the effects of all these substances on the HPV we show a table with the effects on the first and second phase are expressed as a percentage of the increase or decrease over the control response. We have represented again the effects of mercaptosuccinate, ebselen, carmustine and FAD, and other substances we tried, like the glutathione depletor diethylmaleate, dehydroepiandrosterone and deoxyglucose… These last three affected glutathione levels in the tissue, and in this way were able to block the vasoconstrictor response affecting both, first and second phase. We also tried the adition of both forms of glutathione, without clear effects over the control, but it is not sure that they could enter the cells because its big size. Finally we tried a permeable form of glutathione, ethyl esther glutathione, that was not affecting the second phase, and maybe giving a increase in the size of the first phase.
The previous results have shown that hypoxia caused a shift in the redox state in intact pulmonary. The substances that reduced total glutathione levels in tissue, causing cell oxidation consistently inhibited sustained HPV. Moreover, it looks that the cyclic reaction involving reduced and oxidised glutathione and NADPH/NADP+ are related and acts as key factors in the regulation of the vasoconstrictor response observed in intrapulmonary arteries, and the vasodilatation observed in systemic arteries in response to hypoxia.
Potential reducing agents: without redox potential data yet, but different effects on HPV

9. Summary & Conclusions
Hypoxia decreases the [GSH]2/[GSSG] ratio in IPA but not in aorta, suggesting that the cell redox state is becoming more oxidised, consistent with the mitochondrial ROS model of Schumacker and colleagues (Waypa & Schumacker 2006).
2. Carmustine, DEM and DHEA cause an oxidising shift in the [GSH]2/[GSSG] ratio, but do not mimic HPV, indicating that oxidation of the cytoplasmic is itself not sufficient to cause contraction.
Both phases of HPV were sensitive to a number of drugs which influenced cell redox state. However, the effects of these drugs on HPV did not show any obvious correlation with their effects on cytoplasmic redox potential.
4. Although preliminary, these results suggest that cytoplasmic redox state per se is not a primary determinant of contraction during HPV.

11. 1st 2nd n Eredox MS 100µM Ebs 30µM CARM 100µM (*) FAD 100µM
+1±7 ns
+47±13 ** 8 6
Ebs 30µM
-56±10
-59±22 * 5
no data
CARM 100µM
-12±1
-151±32
*** 4
(*)
FAD 100µM
-24±5
+21±7
DHEA 100µM
-65±12
-67±8
DEM 1mM
-57±8
-44±9
(***) 3
Ethyl-GSH 1mM
+24±10
+1±19

12. DEM: GST depletor
DHEA: G6Pd blocker