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The meaning of H2O2 generation in carotid body cells for PO2 chemoreception
Journal of the Autonomic Nervous System, 41 (1992) 41-52
41
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01327
The meaning of H202 generation in carotid body cells for PO 2 chemoreception H. Acker, B. B611ing, M.A. Delpiano, E. Dufau, A. G6rlach and G. H o l t e r m a n n Max-Planck-lnstitut fiir Sytemphysiologie, Dortmund, FRG
Key words: Hydroperoxides; Cytochrome b; Carotid body; Glutathione; Oxygen sensing; Spectroscopy; Heme protein Abstract The rat carotid body is able to generate H202 in type-I cells with the aid of an electron-transferring chain with cytochrome b as the major component as it can be detected by spectrophotometry as well as confocal laser-microscopy. This cytochrome b is reducible by hypoxia, but not by cyanide, indicating that it does not participate in the energy production by the respiratory chain. The carotid body possesses a glutathione peroxidase (GPO) which scavenges H202 and other organic hydroperoxides. The nervous chemoreceptor discharge can be inhibited by external application of hydroperoxides with a similar half maximal value (60-80 ~M) as used to stimulate GPO. A hypothetical signal chain is described which suggests the involvement of cytochrome b as an 02 sensor in PO 2 chemoreception of the carotid body and the degradation of H202 by glutathione to control the K+-conductivity of carotid body cells.
Introduction
The carotid body located at the carotis sinus is able to transduce changes of oxygen pressure in the arterial blood into nervous signals regulating respiration and circulation in order to avoid hypoxic situations in the body. The mechanism of the transducing process is still a matter of discussion. A generally accepted concept defines this process as a PO2-dependent transmitter release from carotid body type-I cells, which generates action potentials in post-synaptic nerve endings of the sinus nerve (for review see [1]). The membrane depolarisation necessary for transmitter re-
Correspondence to: H. Acker, Max-Planck-Institut fiir Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, FRG.
lease under hypoxia could be accomplished by the recently described outward rectifying potassium currents of type-I cells which are reduced under hypoxia [12,19,31]. Observations that the open-probability of K+-channels in type-I cells decreased under hypoxia [7,10] substantiated the importance of these findings for understanding PO 2 chemoreception in the carotid body. Membrane depolarisation would open voltage-dependent Ca 2+ channels increasing cytosolic calcium and herewith facilitating transmitter release [24,28]. However, the participation of intracellular calcium stores in regulating the cytosolic calcium-content, especially by mitochondria, has also been discussed [3]. The molecular mechanism of the inhibitory effect of low PO 2 on K+-channel conductivity and thus on the chemoreceptor properties of type-I cells is unknown, but the
42
involvement of a heme-type PO 2 sensor protein has been suggested [6]. The idea of heme proteins acting as PO 2 sensors in the carotid body has been published by several groups [16,18,21]. In their classical paper Mills and J6bsis [21] analyzed photometrically a cytochrome aa 3 with a low as well as with a high 0 2 affinity component in the carotid body. Biscoe and Duchen [3] supported the idea of a specialized cytochrome aa 3 by a model which located the 0 2 sensor in the carotid body mitochondria responding to oxygen changes due to a low 0 2 affinity far above the critical mitochondrial PO 2 with a mitochondrial membrane depolarization and a subsequent calcium release. In a recent paper Rumsey et al. [26] hypothesized an ATP decrease in the carotid body as the main trigger for PO 2 chemoreception starting at tissue PO 2 values below 15 Torr [32]. However, intensive studies of the different cytochromes in the respiratory chain have revealed affinity values for oxygen with a PO25 ° below 1 Torr [22] which questioned higher PO25 ° values. Cross et al. [6] carried out a detailed photometric analysis of the rat carotid body to gain more information about heme-protein characteristics in this tissue. They detected a measurable heme signal with absorbance maxima at 560 nm, 518 nm and 425 nm suggesting the presence of a b-type cytochrome. This was confirmed by pyridine hemochrome and CO spectra. This heme protein is capable of H 2 0 2 formation and seems to possess, therefore, similarities with the cytochrome b of the NAD(P)H oxidase in neutrophiles [13]. Therefore the question was addressed of whether H 2 0 2 is involved in PO 2 chemoreception, as proposed by Sies for the liver [29], with the model that glutathione peroxidase degrades hydroperoxides and thereby induces a transition in the G S H / G S S G redox couple, which, as the major cellular pool of mobile thiol groups, can change protein formations, e.g. ion channels. This paper will give further evidence for the occurrence and location of the H202-forming cytochrome b in the rat carotid body, the degradation of H 2 0 2 by glutathione peroxidase in the carotid body, and the significance of H~O2 for the nervous chemoreceptor activity. The paper
will conclude with a model proposing a PO-,sensor chain with cytochrome b as the oxygen sensor, H 2 0 2 as the effector or second messenger, and K + channels gated by glutathione [20] as the final effect for regulating the membrane potential of type-I cells in dependence on PO,.
Materials and Methods Carotid body preparation and superfusion
After prolonged flushing of the common carotid arteries with Macrodex 6% (Knoll, Glandorf, F R G ) to eliminate the red blood cells from the tissue, carotid bodies with the intact sinus nerve and the immediate vessels were excised from rats anaesthetized with a mixture of 2% chloralose, 25% urethane (0.5 ml/100 g body weight) and heparinized with 1300 units/rat. The carotid bodies were denuded of all other structures and placed in a small lucite superfusion chamber mounted on the stage of an upright microscope (Olympus, Hamburg, F R G ) for spectral analysis by light absorbance, for NAD(P)H fluorescence measurements, and nervous discharge recordings. The cleaned organs were superfused, as described by Delpiano and Acker [7], with modified Locke's solution, equilibrated with different 0 2 mixtures by using a gas mixing pump (W6sthoff, Bochum, FRG). and viewed with a 40 x water immersion objective lens and 10 × ocular lenses. Oxygenation of the superfusion medium was monitored close to the tissue with a needle PO 2 electrode whose tip was located near the objective lens. Temperature was routinely maintained at about 35°C and pH was kept at 7.36. Tissue culture
The procedures of the culture of gtomus cells by combined enzymatic and mechanical dissociation of adult rat carotid body were identical to those previously described by Nurse [22]. The carotid bodies were removed from anaesthetized rats (chloralose-urethane, see above), cleaned of surrounding tissue and incubated for 1 h in enzymatic solution (Hank's BBS, @1% collagenase, 0.1% trypsin, 0.01% DNAse, 1% penicillin/
43 streptomycin (Gibco, Eggenstein-Leopoldshofen, FRG)) at 37°C. Following inactivation of the enzyme they were mechanically teased apart and slowly triturated before being plated onto polylysin-coated cover slips. The cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO 2 for 8-12 days. The growth medium consisted of F-12 nutrient medium supplemented with 10% fetal calf serum (Gibco), 80 U/1 insulin (Sigma, Munich, FRG), 0.6% glucose, 2 mM glutamine, 1% penicillin/streptomycin, 1 /zg/ml nerve growth factor (Boehringer, Mannheim, FRG). Type-I cells in tissue culture have been identified by their round shape as well as by their glyoxylic acid-induced catecholamine fluorescence.
Photometry For light-absorption measurements, light from a halogen lamp (12V, 100W) transilluminated the carotid body deposited in the superfusion chamber in a hole of a bench which was made otherwise intransparent by sputtering with gold thus having the advantage of avoiding uncharacteristic light scattering. Therefore, only light which was guided by the carotid body tissue from the condensor to the objective (40x), was recorded by a photodiodearrayspectrophotometer (MCS 210, Zeiss, K61n, FRG) which was connected to the third ocular of the microscope trinocular head via a light guide. To demonstrate spectral characteristics of the carotid body, the organ under welloxygenated conditions served as a baseline from which spectra under different experimental conditions could automatically be subtracted by a menu-driven personal computer program, which is commercially available together with the MCS spectrometer. Light intensity changes as small as 10 -3 optical densities (OD) can be detected by this device. For NAD(P)H measurements light from a xenon arc lamp (ILC Technology, Sunnyvale, CA) which was filtered for fluorescence excitation by a 366 nm interference filter (Schott, Mainz, FRG) with a half-bandwith of 7 nm, transilluminated the carotid body tissue. The spectrum of the emitted light from the tissue passed through a 400 nm cut-off filter (Schott). Spectra were ana-
lyzed using a Veril-S-60 filter (Leitz, Wetzlar, FRG) and recorded with a photomultiplier (EMI 9502A, Hayes, UK) both placed on the third ocular tube of the microscope trinocular head. NAD(P)H fluorescence was measured at 459 + 2 nm (n = 16).
Measurement of 14202 production According to Cross et al. [6] dihydrorhodamine 123 (Molecular Probes, Eugene, OR) was dissolved in dimethylsulphoxide to give stock solutions of 50 mM. Dihydrorhodamine 123 was stored under N 2. The non-fluorescent dihydrorhodamine is converted to rhodamine in the reaction with H20 2. This fluorescent, positively charged dye is taken up by cells. Images of the fluorescent cells were obtained by using a Bio Rad MRC 500 confocal scanning optical microscope (Bio Rad, Mfinchen, FRG) mounted on a Zeiss IM405 inverted microscope (Zeiss, K61n, FRG). For measurements, carotid bodies and type-I cells in tissue culture stained with dihydrorhodamine were placed in the superfusion chamber which was mounted on the stage of the inverted microscope.
Measurement of glutathione peroxidase actit,ity Glutathione peroxidase activity was assayed according to Konz [14] using 0.17 mM H202, 0.884 mM t-butyl hydroperoxide and 1 mM cumene hydroperoxide in 1 ml assay solution. The assay solution contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM NaN 3, 1 mM GSH, 0,25 mM NAD(P)H, 1 U / m l glutathione reductase and 250 /zl sample solution. The glutathione peroxidase activity of histologically identified pig carotid bodies was measured since the minimal amount of tissue necessary for the assay (100 mg) was impossible to cover with rat carotid bodies (1 rat carotid body = 0.5 rag, 1 pig carotid body --- 8 mg). Thirty pig carotid bodies, which were dissected in the slaughterhouse, have been sonicated in potassium phosphate buffer for ten cycles of 10 s at 50 W with a Branson Soniprobe, with 30 s of cooling in an ice/water bath between the cycles. The homogenate was centrifuged at 50000 rpm for 30 rain and the supernatant was taken for the assay.
44
After 10 min of preincubation at 37°C the reaction was started with the addition of the different peroxides. The value for a blank reaction with the enzyme source replaced by buffer was substracted for each assay. The rate of reaction was recorded at room temperature following the decrease in absorbance of NAD(P)H at 334 nm. Specific activity was expressed as U / m g protein, each unit (U) representing the oxidation of 1 /zM of NAD(P)H per minute.
NAD(P)H fluorescence and chemoreceptor discharge were monitored with a multipen ink recorder.
Statistics Statistical differences were assessed using Student's t-test. Differences were considered significant with a level of P < 0.05.
Results and Discussion
Afferent chemoreceptor discharge recording Chemoreceptor discharge was recorded from the sinus nerve by the method of Delpiano and Acker [7]. Briefly, multifibre filaments (5-20 active fibres) lifted into a paraffin oil pool above the superfusion medium were recorded with the aid of a unipolar platinum electrode and displayed on a dual-beam storage oscilloscope (Tectronik 5113, K61n, FRG). The amplified spikes were fed into a window discriminator, counted with a ratemeter, and audiomonitored.
This paper tries to give a complete biological signal chain sequence which transduces changes in the PO 2 of the carotid body tissue into corresponding changes in the membrane potential of type-I cells. Therefore it seems to be recommendable to combine the description of the original findings with the development of a schematic hypothetical pathway in single steps to highlight the logical structure of the signal chain of PO 2 chemoreception in the carotid body.
rat carotid body hypoxia
hypoxio
0.0.
O.D. I
0.005
KCN+hypoxia I
400
450
500
550
600
500 650 wavelength ( n m )
I
I
I
I
|
550
'
'
'
'
I
SO0
,
,
•
•
I
6,50
Fig. 1. Light absorption difference-spectra of the rat carotid body induced by hypoxia and cyanide. The left part shows a wavelength range between 400 and 650 nm. The right part demonstrates the same spectra amplified with a wavelength range between 500 and 650 nm. O.D. = optical densities.
45 Figure 1 shows the light absorption characteristics of different heme proteins in the rat carotid body monitored by the recently developed (in our laboratory) microscope spectrophotometry using photodiodearrays for light detection. On the left side of Fig. 1 a wavelength range between 400 and 650 nm is shown and on the right side a wavelength range between 500 and 650 nm is amplified. From top to bottom to be seen are: the difference-spectrum between oxidized and hypoxia-reduced carotid body tissue, the differencespectrum between oxidized and cyanide-reduced carotid body tissue, and the difference spectrum between cyanide- and hypoxia-reduced carotid
body tissue. Significantly, distinguishable peaks can be detected under hypoxia (n = 5) [23] as cytochrome b at 432 _+ 3.9 nm and 559 _+ 0.74 nm, as cytochrome c at 551 _+ 0.74 nm, as cytochrome a a 3 at 602 _+ 1.88 nm and as F A D H at 464 _+ 2.6 nm. Cytochrome c and cytochrome a a 3 can be identified as components of the respiratory chain since they are getting reduced under hypoxia as well as by cyanide poisioning. In contrast to hypoxia, cyanide at 559 nm induces a small increase in the light absorption only. The major part of cytochrome b seems not to be directly integrated into the electrontransport of the respiratory chain since it responds to hypoxia in spite of the corn-
Fig. 2. After incubation with dihydrorhodaminecells, which produce H202, were stained with rhodamine 123 in different depths of the carotid body. Images were obtained each 15/xm with the aid of confocal laser microscopy.Fluorescence intensity is shown with white as highest and black as lowest intensity. Bar = 1 mm.
46 plete reduction of the respiratoty chain by cyanide at 432 nm and 559 nm. This cyanide insensitivity is a characteristic feature of a cytochrome b type [13] which is described to be located in the outer cell membrane, the plasma membrane of the endoplasmatic reticulum as well as in the Golgi apparatus, the outer mitochondrial membrane and the nucleus membrane (for review see [9]). The broad peak between 550 and 560 nm of the cyanide minus hypoxia difference spectrum is reminiscent of descriptions of a double peak in the alpha band at 553 and 560 nm of microsomal cytochrome b 5 [34]. However, one has to take into account that various heme proteins located in different carotid body cells might contribute to
the difference-spectra, as shown in Fig. 1, meaning that a single cell preparation together with low temperature spectrometry is necessary for the verification of a cytochrome double peak. These investigations would give more information about the number of heme groups and the O 2 affinity of the carotid body cytochrome b. A second feature of cytochrome b is its ability to generate oxygen radicals which are converted to hydrogen peroxide by superoxide dismutase (SOD) activity [9]. Therefore, the question arose whether the dihydrorhodamine assay in combination with the confocal laser microscope is able to detect H 2 0 2 in different depths of the superfused rat carotid body. The confocal microscope
Fig. 3. Rhodamine fluorescence of adult rat carotid body type-I cells in tissue after incubation with dihydrorhodamine. Fluorescence intensity is shown with white as highest and black as lowest intensity. Bar = 100 p.m.
47
scans and collects emitted light from within the plane of focus of the objective lens only. Therefore, the image obtained is the fluorescence from a discrete slice of about 10 ~ m thickness within the whole tissue. Non-fluorescent cells are not visible. Figure 2 depicts an optical sectioning of a rat carotid body showing the occurrence of H 202 indicated by the different intensities of rhodamine fluorescence. Using optical sectioning of the carotid body from the lower to the upper surface the tissue was scanned each 15 /.~m for this illustration. Figure 2 shows the rhodamine fluorescence with white as the strongest and black as the lowest intensity. Penetrating the carotid body tissue fluorescence increases with the abundance of cells. The total thickness of this carotid body can be estimated as about 480 p~m which is in close accordance with histological investigations [6]. Figure 3 shows the rhodamine fluorescence of seven type-I cells in tissue culture indicating a H 2 0 2 production also under these conditions. The cells have different diameters because fluorescence was imaged in one plane of focus only. The dark spot inside the cells represents the nucleus. Very characteristically, type-I cells in tissue culture show elongations of the cytoplasm. Figures 1, 2, and 3 confirm the results about the cytochrome b characteristics in the carotid body tissue as published by Cross et al. [6] as well as by Acker et al. [1]. It seems to be, therefore, justified to draw the first step of our hypothetical signal chain for PO 2 chemoreception in the carotid body cells: PO 2 Sensor: Effector: Cytochrome b - - e - - - O z - - S O D - - H 2 0 2 We consider three components of the signal chain. The PO 2 sensor is represented by the cytochrome b which might be part of an electron transfer chain with NAD(P)H and FAD as further members [6]. We have to assume that 0 2 radicals generated by this chain are dismutased to hydrogen peroxide which we name effector. The effector plays the role of a second messenger because U 2 0 2 production is thought to be dependent on tissue PO z. This P O 2 dependence is supported by 0 2 affinity values as reported in the literature for cytochrome b ranging between PO 2 values of 7
NADP+~.~
S 2GSH~
i~,~ ,o,~,. . . . . 0oo.o-~ ~o(~)~J
f ROOl-i
L~0'o'~'ooo . .... '~o~" I
~ - ~s~ j
\
Ro.+.~o
Fig. 4. Glutathione represents the major cellular pool of mobile sulfhydryl groups. The redox couple 2GSH/GSSG is linked to the NADP + system by glutathione reductase and to the hydroperoxide system by glutathione peroxidase.
and 20 Torr [13]. Lowering the tissue PO 2 would lead, therefore, to a decreased H 2 0 2 content of the carotid body. One has to ask now how hydrogen peroxide could act as a second messenger. Sies [29] proposed, as already mentioned, and based on an idea as published by Kosower and Werman [15] that the degradation of H 2 0 2 by the glutathione peroxidase (GPO) would lead to a change in the equilibrium of the 2 G S H / G S S G redox couple as shown in Fig. 4. The POz-dependent H 2 0 2 production would, therefore, result in a higher GSSG content when the PO 2 is increased and in a higher GSH content when the PO 2 is lowered. Since this PO2-dependent change in oxidized or reduced glutathione might influence protein conformation (e.g. conductivity of ion channels) by regulating the availability of free SH groups it was our next step to prove this metabolic step in the carotid body. Table I gives the values for the G P O activity in the pig carotid body using different hydroperoxides as substrates. Although a direct transition of these pig carotid bodies values to the rat carotid body tissue is not possible, the data in Table I indicate that the carotid body seems to possess an active GPO. The difference-value of the GPO activities using H 2 0 2 and cumene hydroperoxide as substrate is indicative for the activity of selen-free GPO [17]. Table I clearly shows that the use of all three substrates resulted in similar values indicating that the carotid body possesses the selen-containing GPO only. The TABLE I Glutathione peroxidase acticities in the pig carotid body
H202 t-butyl hydroperoxide Cumene hydroperoxide
5.86 U / g protein 6.18 U / g protein 4.65 U / g protein
48
P02=310 Torr lOmin 100E~ E O t(.3 ~0 (J E t/] IlJ t_ O D
50.
I
"-r
E C3 Z
35 0
142 71
....~,jJM
213
cumene hydroperoxide Fig. 5. Dose-dependent oxidation of NAD(P)H in the carotid body tissue in response to a gradual increase of the cumene hydroperoxide concentration in the superfusion bath at a constant PO 2 of 310 Torr. The absolute NAD(P)H change amounts to 10% of the total fluorescence.
values in the carotid body are nearly a factor hundred lower than GPO values in the rat liver [171. We then went on to complete the list of different members of the hypothetical signal chain by superfusing the rat carotid body with the different hydroperoxides and the recording of the NAD(P)H fluorescence. According to Fig. 4, application of hydroperoxides should lead to an increased GPO activity with an increase of the GSSG content which subsequently activates the glutathione reductase (GPR) which reduces, depending on the equilibrium ratio some of the
newly formed GSSG back t o GSH with simultaneous oxidation of NAD(P)H [30]. Figure 5 shows that application of hydroperoxides, in this example cumene hydroperoxides, results in a dose-dependent fashion in a reversible oxidation of NAD(P)H confirming the presence of the 2GSH/GSSG couple also in the rat carotid body. The oxidation amounts to about 10% of the total NAD(P)H fluorescence. Table II summarizes the dose-dependent NAD(P)H oxidation in the rat carotid body induced by application of the different hydroperoxides. Taking the total NAD(P)H reaction as shown in Fig. 5 as 100%, gradual increase of the hydroperoxide dose leads to gradual decrease of the NAD(P)H fluorescence reaching a saturation between 300 and 400/xM. Half maximal values are 88 + 24.8 IzM (n = 6) for H 2 0 2, 39 + 10.7 (n = 5) /xM for cumene hydroperoxide and 42 ÷ 12,6/xM (n = 5) for t-butyl hydroperoxide. These values are statistically not significant, arguing tot the exclusive role of the selen-containing GPO as hydrogen peroxide scavenger in the rat carotid body comparable to the pig carotid body. After these indications for the importance and reactivity of the glutathione metabolism in the carotid body, one might continue with the hypothetical signal chain pathway as follows: PO 2 Sensor: Effector: Cytochrome b - - e - - - O ~ - - S O D - H20 2 2GSH/GSSG The 2GSH/GSSG couple has an equilibrium ratio of 104 meaning that on base of a high homoeostatic GSH level (10 mM), GSSG (1 /xM) is able to float depending on the H 2 0 2 supply and to serve as a signal [25]. The question was, therefore, addressed for evidences of glutathione in-
TABLE II
Reaction of NAD(P)H fluorescence in the rat carotid body to various doses o f the different hydroperoxides Hydroperoxide/zM
0
35.5
71
142
284
426
NAD(P)H fluorescence change in percentage of control H20 2 n = 6 100 60 ± 11.5
43 + 9.3
24 ± 10
6± 0
0
t-butyl hydrogenperoxide n = 5
100
25 +_ 9
14 ± 7.4
6± 4
0
0
Cumene hydroperoxide n = 5
100
28 + 12.4
3 _-2- 2.4
0
0
9± 6
49
duced ion channel conductivity changes. Glutathione-gated K + channels have been postulated by potassium flux measurements in Escherichia coil [20] which close under reduced conditions like hypoxia. More direct evidence was given by Ruppersberg et al. [27] showing that fast inactivating K + currents mediated by cloned K + channel subunits derived from rat brain expressed in Xenopus oocytes are faster inactivated by reduced glutathione. We can now complete our hypothetical signal chain pathway as follows: PO2 Sensor: Effector: Cytochrome b - - e - - - O ~ - - - S O D - - H 2 0 z Effect: 2 G S H / G S S G - - glutathione-gated K + channel Since both glutathione-gated K + channels and POz-dependent K + channels of type-I cells close under reduced and open under oxidized conditions it seems to be justified in attempting to prove whether application of hydrogen peroxide mimics hyperoxia leading to membrane hyperpolarization, cytosolic calcium decrease, reduced transmitter release and hence, depressed nervous chemoreceptor activity. Therefore we carried out experiments measuring, as shown in Fig. 6, the reaction of the nervous chemoreceptor activity to
3
30#V
Immmm
I/s
120 i
10 rain
I=
80
40
PrnO. (Torrl 338
t 19 I
t e r - butyt-hydroperoxide {~M}
338 ,
30
,
,
60
,
go
,
19 , 338 D
120
Fig. 6. Dose-dependent decrease in the nervous chemoreceptor discharge in response to a gradual increase in the t-butyl hydroperoxide concentration in the superfusion bath. The PO 2 in the bath solution amounted to 338 Torr with short hypoxic periods of 19 Torr for chemoreceptor stimulation. The figure shows original recordings of the sinus nerve action potentials photographed at point 1, 2 and 3 during the experiments. I / s = impulses per second, PmO2 = oxygen pressure in the superfusion bath,/xV = microvolt, s = 1 second, time of 10 minutes is indicated by a bar.
TABLE III
Reaction of the nercous chemoreceptor discharge of the rat carotid body to carious doses of the different hydroperoxides Hydroper- 0 oxide/zM
30
60
90
120
Chemoreceptor discharge in percentage of control H202 n=3 100 90_+ 8.7 66_+13.2 39_+11 27 _+ 4.3 t-butyl hydroperoxide n=3 100 91-+10.8 45-+ 6.7
34_+ 5.7 27_+15
cumene hydroperoxide n=2 100 106_+25.4 62_+ 4.33 44_+11.2 29+_18.9
the application of the various hydroperoxides. Fibers of the sinus nerve have been first tested for their sensitivity by a decrease of PO: in the superfusion bath. Typically, an increase in the nervous discharge can be observed (see Fig. 6). Application of hydroperoxides in different concentrations, in this case t-butyl hydroperoxide, leads to a decrease in chemoreceptor discharge. A hypoxic stimulation during this period has no response in the chemoreceptor discharge. Table III summarizes the results obtained by expressing the decrease in the nervous activity under hydroperoxide application as percentage of control. For this purpose the difference between chemoreceptor activity under a PO 2 of about 300 Torr in the superfusion bath and electrical zero of the ratemeter was taken as 100%. It is to be seen that each of the hydroperoxides leads to a decrease in the chemoreceptor activity to about 30% of the control value. Cumene hydroperoxide stimulated transiently the chemoreceptor discharge at a dose of 30/xM. Half maximal values are 77 + 13.7 p,M (n = 3) for H 2 0 2, 59 + 5.8/zM (n = 3) for t-butyl hydroperoxide and 69_+ 3.3 /zM (n = 2) for cumene hydroperoxide. The half maximal values are in the same range as the half maximal values of NAD(P)H oxidation as shown in Table II underlining the close relationship between the activity of the scavenger effect of GPO and the chemoreceptor activity under exposure of the carotid body tissue to hydroperoxides. The participation of catalase can be neglected since the organic hydroperoxides are scavenged by GPO only [30].
50
At the moment it is not possible for us to give more evidence for our model about the importance of H202 for PO e chemoreception in the carotid body. This has to be done on the cellular level showing by the patch clamp technique, the responsivness of the PO e sensitive K + channels to glutathione with the subsequent changes of the intracellular calcium level monitored by microfluorometry. However, it might be interesting to mention that the participation of H202 in PO 2 chemoreception is discussed also in two other systems, i.e. the hypoxic vasoconstriction of the lung vessels [5] and the erythropoietin production in kidney ceils [32]. In lung vessels, H202 induces a catalase-dependent guanylate cyclase activation by interaction of the heme groups of the two enzymes leading to the cGMP-mediated relaxation of pulmonary arteries probably due to opening of K ÷ channels. This process is reversed under hypoxia when H202 production arrests, leading to a vasoconstriction [5]. The erythropoietin production in a renal carcinoma cell line can be enhanced by external application of H202 or inhibited by external application of catalase due to an enhanced scavenging of H202 [32]. The H202-producing system has not been identified in these publications. However, G6rlach et al. [11] recently identified spectrophotometrically in erythropoietin-producing hepatoma cells, a cytochrome b which is similar to the cytochrome b in the carotid body reducible by hypoxia but not by cyanide and therefore an appropriate source for H202. Future experiments have to show whether the described signal chain for PO 2 chemoreception is a general biological principle in cells acting as O 2 sensors in the body.
References 1 Acker, H., PO 2 chemoreception in arterial chemoreceptors. Ann. Rev. Physiol., 51 (1989) 835-844. 2 Acker, H., Dufau, E. Huber, J. and Sylvester, D., Indications to an NAD(P)H oxidase as a possible PO 2 sensor in the rat carotid body, FEBS Lett., 256 (1989) 75-78. 3 Biscoe, T.J. and Duchen, M.R., Monitoring PO 2 by the carotid chemoreceptor, NIPS, 5 (1990) 229-233. 4 Biscoe, T.J., Duchen, M.R., Eisner, D.A., O'Neill, S.C.
and Valdeolmillos, M., Measurements of intraceltular Ca 2+ in dissociated type-I cells of the rabbit carotid body, J. Physiol., 416 (1989) 421-434. 5 Cherry, P.D., Omar, H.A., Farrell, K.A. Stuart, J.S, and Wolin, M.S., Superoxide anion inhibits cGMP associated b o v i n e pulmonary relaxation, Am. J. Physiol., 259 (19901 H1056-HI062. 6 Cross, A.R., Henderson, L., Jones, O.T.G., Delpiano, M.A., Hentschel, J. and Acker, H., Involvement of an NAD(P)H oxidase as a POz sensor protein in the rat carotid body, Biochem. J., 272 (1990) 743-747. 7 Delpiano, M.A. and Acker, H., Hypoxic and hypercapnic responses of [Ca 2+ ]0 and [K + ]0 in the cat carotid body in vitro, Brain Res., 482 (1989) 235-246. 8 Delpiano, M A . and Hescheler, J., Evidence for a PO 2 sensitive K + channel in the type-I cell of the rabbit carotid body. FEBS Letts., 249 (1989) 195-198. 9 Galeotti, T., Borello, S. and Masotti, L.. Oxyradical sources, scavenger systems and membrane damage in cancer cells. In D.K. Das and W.B. Essman, (Eds.), Oxygen Radicals: Systemic Events and Disease Processes, Karger, Basle, 1990, pp. 129-148. 10 Ganfornina, M.D. and Lopez-Barneo, J., Single K ÷ channels in membrane patches of arterial chemoreceptor cells are modulated by 0 2 tension, Proc. Natl. Acad. Sci~ USA, 88 (1991) 2927-2930. 11 G~Srlach, A., Jelkmann, W., B611ing, B., Holtermann, G. and Acker, H., Spectrophotometric characterisation of heme proteins in erythropoietin producing hepatoma cells, Pfliigers Arch., 419 (1991) Rl16. 12 Hescheler, J., Delpiano, M.A., Acker, H. and Pietruschka, F., Ionic currents on type-I cells of the rabbit carotid body measured by voltage clamp experiments and the effect of hypoxia, Brain Res., 486 (1989) 79-88. 13 Jones, O.T.G., Cross, A.R., Hancock, J.T., Henderson, L.M. and O'Donnel, V.B., Inhibitors of NAD(P)H oxidase as guides to its mechanism, Biochem. Soc. Trans,, 19 (1991) 70-72. 14 Konz, K.H., Die Aktivit~it des Peroxid-metabolisierenden Systems in Human Leber, J. Clin. Chem. Clin. Bioehem:. 17 (1979) 353-357. 15 Kosower, E.M. and Werman, R., New step in transmitter release at the myoneural junction, Nature New Biok, 233 (1971) 121-123. 16 Lahiri, S. and DeLaney, R.G., Stimulus interaction in the response of carotid body chemoreceptor single afferent fibres. Resp. Physiol., 4 (19751 229-235. 17 Lawrence, R.A. and Burk, R.F., Glutathione peroxidase activity in selenium deficient rat liver, Biochem. Biophys. Res. Comm., 71 (1976) 951-958. 18 Lloyd, B.B., Cunningham, D.J.C. and Goode, R~C., Depression of hypoxic hyperventitation in man by sudden inspiration of carbon monoxide, In R.W. Torrance (Ed.), Arterial Chemoreceptors Blaekwell Oxford, 1968, pp. 145-148. 19 Lopez-Lopez, J., Gonzalez, C., Urena, J. and LopezBarneo, J., Low PO 2 selectively inhibits K channel avtivity
51 in chemoreceptor cells of the mammalian carotid body, J. Gen. Physiol., 93 (1989) 1001-1015. 20 Meury, J. and Robin, A., Glutathione-gated K + channels of Escherichia coli carry out K + efflux controlled by the redox state of the cell, Arch. Microbiol., 154 (1990) 475482. 21 Mills, E. and J~Sbsis, F.F., Mitochondrial respiratory chain of carotid body and chemoreceptor response to changes in oxygen tension, J. Neurophysiol., 35 (1972) 405-428. 22 Nurse, C.A., Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat, Cell Tissue Res., 261 (1990) 65-71. 23 Oshino, N., Sugano, T., Oshino, R. and Chance, B., Mitochondrial function under hypoxic conditions: the steady states of cytochrome aa 3 and their relation to mitochondrial energy states, Biochim. Biophys. Acta, 368 (1974) 298-310. 24 Pietruschka, F., Calcium influx in cultured carotid body cells is stimulated by acetylcholin and hypoxia, Brain Res., 347 (1985) 140-143. 25 Reich, J.G. and Selkov, E.E., Pools and pathways of metabolism. In J.G. Reich and E.E. Selkov, (Eds.), Energy Metabolism of the Cell, Academic Press, 1981, pp 108-190. 26 Rumsey, W.L., Iturriaga, R., Spargel, D., Lahiri, S. and Wilson, D.F., Optical measurements of the dependence of chemoreception on oxygen pressure in the cat carotid body, Am. J. Physiol., (1991) C614-C622. 27 Ruppersberg, J.P., Stocker, M., Pongs, O., Heinemann, St. H., Frank, R. and Koenen, M., Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation. Nature 352 (1991) 711-714.
28 Sato, M., Ikeda, K., Yoshizaki, K. and Koyano, H., Response of cytosolic calcium to anoxia and cyanide in cultured glomus cells of newborn rabbit carotid body, Brain Res., 551 (1991) 327-330. 29 Sies, H., Peroxisomal enzymes and oxygen metabolism in liver. In M. Ruvich, R. Coburn, S. Lahiri and B. Chance (Eds.), Tissue Hypoxia and Ischemia. Plenum Publishing Corp., New York, 1988, pp. 51-66. 30 Sies, H., Gerstenecker, Ch., Menzel, H. and Floh6, L., Oxidation in the NADP system and release of GSSG from hemoglobin free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides, FEBS Lett., 27 (1972) 171-175. 31 Stea, A. and Nurse, C.A., Whole cell and perforated patch recordings from 0 2 sensitive rat carotid body cells grown in short and long term culture, Pfliigers Arch., 418 (1991) 93-101. 32 Ueno, M., Brookins, J., Beckmann, B.S. and Fischer, J.W., Effects of reactive oxygen metabolites on erythropoietin production in renal carcinoma cells, Biochem. Biophys. Res. Commun., 154 (1988) 773-780. 33 Wilson, D.F., Rumsey, W.L., Green, Th.J. and Vanderkooi, J.M., The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration, J. Biol. Chem., 236 (1988) 2712-2718. 34 Woffendin, C., Edwards, S.W., Griffiths, A.J., The cytochromes of Dictostelium discoideum, Comp. Biochem. Physiol., 75B (1983) 53-59.
41
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01327
The meaning of H202 generation in carotid body cells for PO 2 chemoreception H. Acker, B. B611ing, M.A. Delpiano, E. Dufau, A. G6rlach and G. H o l t e r m a n n Max-Planck-lnstitut fiir Sytemphysiologie, Dortmund, FRG
Key words: Hydroperoxides; Cytochrome b; Carotid body; Glutathione; Oxygen sensing; Spectroscopy; Heme protein Abstract The rat carotid body is able to generate H202 in type-I cells with the aid of an electron-transferring chain with cytochrome b as the major component as it can be detected by spectrophotometry as well as confocal laser-microscopy. This cytochrome b is reducible by hypoxia, but not by cyanide, indicating that it does not participate in the energy production by the respiratory chain. The carotid body possesses a glutathione peroxidase (GPO) which scavenges H202 and other organic hydroperoxides. The nervous chemoreceptor discharge can be inhibited by external application of hydroperoxides with a similar half maximal value (60-80 ~M) as used to stimulate GPO. A hypothetical signal chain is described which suggests the involvement of cytochrome b as an 02 sensor in PO 2 chemoreception of the carotid body and the degradation of H202 by glutathione to control the K+-conductivity of carotid body cells.
Introduction
The carotid body located at the carotis sinus is able to transduce changes of oxygen pressure in the arterial blood into nervous signals regulating respiration and circulation in order to avoid hypoxic situations in the body. The mechanism of the transducing process is still a matter of discussion. A generally accepted concept defines this process as a PO2-dependent transmitter release from carotid body type-I cells, which generates action potentials in post-synaptic nerve endings of the sinus nerve (for review see [1]). The membrane depolarisation necessary for transmitter re-
Correspondence to: H. Acker, Max-Planck-Institut fiir Systemphysiologie, Rheinlanddamm 201, 4600 Dortmund 1, FRG.
lease under hypoxia could be accomplished by the recently described outward rectifying potassium currents of type-I cells which are reduced under hypoxia [12,19,31]. Observations that the open-probability of K+-channels in type-I cells decreased under hypoxia [7,10] substantiated the importance of these findings for understanding PO 2 chemoreception in the carotid body. Membrane depolarisation would open voltage-dependent Ca 2+ channels increasing cytosolic calcium and herewith facilitating transmitter release [24,28]. However, the participation of intracellular calcium stores in regulating the cytosolic calcium-content, especially by mitochondria, has also been discussed [3]. The molecular mechanism of the inhibitory effect of low PO 2 on K+-channel conductivity and thus on the chemoreceptor properties of type-I cells is unknown, but the
42
involvement of a heme-type PO 2 sensor protein has been suggested [6]. The idea of heme proteins acting as PO 2 sensors in the carotid body has been published by several groups [16,18,21]. In their classical paper Mills and J6bsis [21] analyzed photometrically a cytochrome aa 3 with a low as well as with a high 0 2 affinity component in the carotid body. Biscoe and Duchen [3] supported the idea of a specialized cytochrome aa 3 by a model which located the 0 2 sensor in the carotid body mitochondria responding to oxygen changes due to a low 0 2 affinity far above the critical mitochondrial PO 2 with a mitochondrial membrane depolarization and a subsequent calcium release. In a recent paper Rumsey et al. [26] hypothesized an ATP decrease in the carotid body as the main trigger for PO 2 chemoreception starting at tissue PO 2 values below 15 Torr [32]. However, intensive studies of the different cytochromes in the respiratory chain have revealed affinity values for oxygen with a PO25 ° below 1 Torr [22] which questioned higher PO25 ° values. Cross et al. [6] carried out a detailed photometric analysis of the rat carotid body to gain more information about heme-protein characteristics in this tissue. They detected a measurable heme signal with absorbance maxima at 560 nm, 518 nm and 425 nm suggesting the presence of a b-type cytochrome. This was confirmed by pyridine hemochrome and CO spectra. This heme protein is capable of H 2 0 2 formation and seems to possess, therefore, similarities with the cytochrome b of the NAD(P)H oxidase in neutrophiles [13]. Therefore the question was addressed of whether H 2 0 2 is involved in PO 2 chemoreception, as proposed by Sies for the liver [29], with the model that glutathione peroxidase degrades hydroperoxides and thereby induces a transition in the G S H / G S S G redox couple, which, as the major cellular pool of mobile thiol groups, can change protein formations, e.g. ion channels. This paper will give further evidence for the occurrence and location of the H202-forming cytochrome b in the rat carotid body, the degradation of H 2 0 2 by glutathione peroxidase in the carotid body, and the significance of H~O2 for the nervous chemoreceptor activity. The paper
will conclude with a model proposing a PO-,sensor chain with cytochrome b as the oxygen sensor, H 2 0 2 as the effector or second messenger, and K + channels gated by glutathione [20] as the final effect for regulating the membrane potential of type-I cells in dependence on PO,.
Materials and Methods Carotid body preparation and superfusion
After prolonged flushing of the common carotid arteries with Macrodex 6% (Knoll, Glandorf, F R G ) to eliminate the red blood cells from the tissue, carotid bodies with the intact sinus nerve and the immediate vessels were excised from rats anaesthetized with a mixture of 2% chloralose, 25% urethane (0.5 ml/100 g body weight) and heparinized with 1300 units/rat. The carotid bodies were denuded of all other structures and placed in a small lucite superfusion chamber mounted on the stage of an upright microscope (Olympus, Hamburg, F R G ) for spectral analysis by light absorbance, for NAD(P)H fluorescence measurements, and nervous discharge recordings. The cleaned organs were superfused, as described by Delpiano and Acker [7], with modified Locke's solution, equilibrated with different 0 2 mixtures by using a gas mixing pump (W6sthoff, Bochum, FRG). and viewed with a 40 x water immersion objective lens and 10 × ocular lenses. Oxygenation of the superfusion medium was monitored close to the tissue with a needle PO 2 electrode whose tip was located near the objective lens. Temperature was routinely maintained at about 35°C and pH was kept at 7.36. Tissue culture
The procedures of the culture of gtomus cells by combined enzymatic and mechanical dissociation of adult rat carotid body were identical to those previously described by Nurse [22]. The carotid bodies were removed from anaesthetized rats (chloralose-urethane, see above), cleaned of surrounding tissue and incubated for 1 h in enzymatic solution (Hank's BBS, @1% collagenase, 0.1% trypsin, 0.01% DNAse, 1% penicillin/
43 streptomycin (Gibco, Eggenstein-Leopoldshofen, FRG)) at 37°C. Following inactivation of the enzyme they were mechanically teased apart and slowly triturated before being plated onto polylysin-coated cover slips. The cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO 2 for 8-12 days. The growth medium consisted of F-12 nutrient medium supplemented with 10% fetal calf serum (Gibco), 80 U/1 insulin (Sigma, Munich, FRG), 0.6% glucose, 2 mM glutamine, 1% penicillin/streptomycin, 1 /zg/ml nerve growth factor (Boehringer, Mannheim, FRG). Type-I cells in tissue culture have been identified by their round shape as well as by their glyoxylic acid-induced catecholamine fluorescence.
Photometry For light-absorption measurements, light from a halogen lamp (12V, 100W) transilluminated the carotid body deposited in the superfusion chamber in a hole of a bench which was made otherwise intransparent by sputtering with gold thus having the advantage of avoiding uncharacteristic light scattering. Therefore, only light which was guided by the carotid body tissue from the condensor to the objective (40x), was recorded by a photodiodearrayspectrophotometer (MCS 210, Zeiss, K61n, FRG) which was connected to the third ocular of the microscope trinocular head via a light guide. To demonstrate spectral characteristics of the carotid body, the organ under welloxygenated conditions served as a baseline from which spectra under different experimental conditions could automatically be subtracted by a menu-driven personal computer program, which is commercially available together with the MCS spectrometer. Light intensity changes as small as 10 -3 optical densities (OD) can be detected by this device. For NAD(P)H measurements light from a xenon arc lamp (ILC Technology, Sunnyvale, CA) which was filtered for fluorescence excitation by a 366 nm interference filter (Schott, Mainz, FRG) with a half-bandwith of 7 nm, transilluminated the carotid body tissue. The spectrum of the emitted light from the tissue passed through a 400 nm cut-off filter (Schott). Spectra were ana-
lyzed using a Veril-S-60 filter (Leitz, Wetzlar, FRG) and recorded with a photomultiplier (EMI 9502A, Hayes, UK) both placed on the third ocular tube of the microscope trinocular head. NAD(P)H fluorescence was measured at 459 + 2 nm (n = 16).
Measurement of 14202 production According to Cross et al. [6] dihydrorhodamine 123 (Molecular Probes, Eugene, OR) was dissolved in dimethylsulphoxide to give stock solutions of 50 mM. Dihydrorhodamine 123 was stored under N 2. The non-fluorescent dihydrorhodamine is converted to rhodamine in the reaction with H20 2. This fluorescent, positively charged dye is taken up by cells. Images of the fluorescent cells were obtained by using a Bio Rad MRC 500 confocal scanning optical microscope (Bio Rad, Mfinchen, FRG) mounted on a Zeiss IM405 inverted microscope (Zeiss, K61n, FRG). For measurements, carotid bodies and type-I cells in tissue culture stained with dihydrorhodamine were placed in the superfusion chamber which was mounted on the stage of the inverted microscope.
Measurement of glutathione peroxidase actit,ity Glutathione peroxidase activity was assayed according to Konz [14] using 0.17 mM H202, 0.884 mM t-butyl hydroperoxide and 1 mM cumene hydroperoxide in 1 ml assay solution. The assay solution contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM NaN 3, 1 mM GSH, 0,25 mM NAD(P)H, 1 U / m l glutathione reductase and 250 /zl sample solution. The glutathione peroxidase activity of histologically identified pig carotid bodies was measured since the minimal amount of tissue necessary for the assay (100 mg) was impossible to cover with rat carotid bodies (1 rat carotid body = 0.5 rag, 1 pig carotid body --- 8 mg). Thirty pig carotid bodies, which were dissected in the slaughterhouse, have been sonicated in potassium phosphate buffer for ten cycles of 10 s at 50 W with a Branson Soniprobe, with 30 s of cooling in an ice/water bath between the cycles. The homogenate was centrifuged at 50000 rpm for 30 rain and the supernatant was taken for the assay.
44
After 10 min of preincubation at 37°C the reaction was started with the addition of the different peroxides. The value for a blank reaction with the enzyme source replaced by buffer was substracted for each assay. The rate of reaction was recorded at room temperature following the decrease in absorbance of NAD(P)H at 334 nm. Specific activity was expressed as U / m g protein, each unit (U) representing the oxidation of 1 /zM of NAD(P)H per minute.
NAD(P)H fluorescence and chemoreceptor discharge were monitored with a multipen ink recorder.
Statistics Statistical differences were assessed using Student's t-test. Differences were considered significant with a level of P < 0.05.
Results and Discussion
Afferent chemoreceptor discharge recording Chemoreceptor discharge was recorded from the sinus nerve by the method of Delpiano and Acker [7]. Briefly, multifibre filaments (5-20 active fibres) lifted into a paraffin oil pool above the superfusion medium were recorded with the aid of a unipolar platinum electrode and displayed on a dual-beam storage oscilloscope (Tectronik 5113, K61n, FRG). The amplified spikes were fed into a window discriminator, counted with a ratemeter, and audiomonitored.
This paper tries to give a complete biological signal chain sequence which transduces changes in the PO 2 of the carotid body tissue into corresponding changes in the membrane potential of type-I cells. Therefore it seems to be recommendable to combine the description of the original findings with the development of a schematic hypothetical pathway in single steps to highlight the logical structure of the signal chain of PO 2 chemoreception in the carotid body.
rat carotid body hypoxia
hypoxio
0.0.
O.D. I
0.005
KCN+hypoxia I
400
450
500
550
600
500 650 wavelength ( n m )
I
I
I
I
|
550
'
'
'
'
I
SO0
,
,
•
•
I
6,50
Fig. 1. Light absorption difference-spectra of the rat carotid body induced by hypoxia and cyanide. The left part shows a wavelength range between 400 and 650 nm. The right part demonstrates the same spectra amplified with a wavelength range between 500 and 650 nm. O.D. = optical densities.
45 Figure 1 shows the light absorption characteristics of different heme proteins in the rat carotid body monitored by the recently developed (in our laboratory) microscope spectrophotometry using photodiodearrays for light detection. On the left side of Fig. 1 a wavelength range between 400 and 650 nm is shown and on the right side a wavelength range between 500 and 650 nm is amplified. From top to bottom to be seen are: the difference-spectrum between oxidized and hypoxia-reduced carotid body tissue, the differencespectrum between oxidized and cyanide-reduced carotid body tissue, and the difference spectrum between cyanide- and hypoxia-reduced carotid
body tissue. Significantly, distinguishable peaks can be detected under hypoxia (n = 5) [23] as cytochrome b at 432 _+ 3.9 nm and 559 _+ 0.74 nm, as cytochrome c at 551 _+ 0.74 nm, as cytochrome a a 3 at 602 _+ 1.88 nm and as F A D H at 464 _+ 2.6 nm. Cytochrome c and cytochrome a a 3 can be identified as components of the respiratory chain since they are getting reduced under hypoxia as well as by cyanide poisioning. In contrast to hypoxia, cyanide at 559 nm induces a small increase in the light absorption only. The major part of cytochrome b seems not to be directly integrated into the electrontransport of the respiratory chain since it responds to hypoxia in spite of the corn-
Fig. 2. After incubation with dihydrorhodaminecells, which produce H202, were stained with rhodamine 123 in different depths of the carotid body. Images were obtained each 15/xm with the aid of confocal laser microscopy.Fluorescence intensity is shown with white as highest and black as lowest intensity. Bar = 1 mm.
46 plete reduction of the respiratoty chain by cyanide at 432 nm and 559 nm. This cyanide insensitivity is a characteristic feature of a cytochrome b type [13] which is described to be located in the outer cell membrane, the plasma membrane of the endoplasmatic reticulum as well as in the Golgi apparatus, the outer mitochondrial membrane and the nucleus membrane (for review see [9]). The broad peak between 550 and 560 nm of the cyanide minus hypoxia difference spectrum is reminiscent of descriptions of a double peak in the alpha band at 553 and 560 nm of microsomal cytochrome b 5 [34]. However, one has to take into account that various heme proteins located in different carotid body cells might contribute to
the difference-spectra, as shown in Fig. 1, meaning that a single cell preparation together with low temperature spectrometry is necessary for the verification of a cytochrome double peak. These investigations would give more information about the number of heme groups and the O 2 affinity of the carotid body cytochrome b. A second feature of cytochrome b is its ability to generate oxygen radicals which are converted to hydrogen peroxide by superoxide dismutase (SOD) activity [9]. Therefore, the question arose whether the dihydrorhodamine assay in combination with the confocal laser microscope is able to detect H 2 0 2 in different depths of the superfused rat carotid body. The confocal microscope
Fig. 3. Rhodamine fluorescence of adult rat carotid body type-I cells in tissue after incubation with dihydrorhodamine. Fluorescence intensity is shown with white as highest and black as lowest intensity. Bar = 100 p.m.
47
scans and collects emitted light from within the plane of focus of the objective lens only. Therefore, the image obtained is the fluorescence from a discrete slice of about 10 ~ m thickness within the whole tissue. Non-fluorescent cells are not visible. Figure 2 depicts an optical sectioning of a rat carotid body showing the occurrence of H 202 indicated by the different intensities of rhodamine fluorescence. Using optical sectioning of the carotid body from the lower to the upper surface the tissue was scanned each 15 /.~m for this illustration. Figure 2 shows the rhodamine fluorescence with white as the strongest and black as the lowest intensity. Penetrating the carotid body tissue fluorescence increases with the abundance of cells. The total thickness of this carotid body can be estimated as about 480 p~m which is in close accordance with histological investigations [6]. Figure 3 shows the rhodamine fluorescence of seven type-I cells in tissue culture indicating a H 2 0 2 production also under these conditions. The cells have different diameters because fluorescence was imaged in one plane of focus only. The dark spot inside the cells represents the nucleus. Very characteristically, type-I cells in tissue culture show elongations of the cytoplasm. Figures 1, 2, and 3 confirm the results about the cytochrome b characteristics in the carotid body tissue as published by Cross et al. [6] as well as by Acker et al. [1]. It seems to be, therefore, justified to draw the first step of our hypothetical signal chain for PO 2 chemoreception in the carotid body cells: PO 2 Sensor: Effector: Cytochrome b - - e - - - O z - - S O D - - H 2 0 2 We consider three components of the signal chain. The PO 2 sensor is represented by the cytochrome b which might be part of an electron transfer chain with NAD(P)H and FAD as further members [6]. We have to assume that 0 2 radicals generated by this chain are dismutased to hydrogen peroxide which we name effector. The effector plays the role of a second messenger because U 2 0 2 production is thought to be dependent on tissue PO z. This P O 2 dependence is supported by 0 2 affinity values as reported in the literature for cytochrome b ranging between PO 2 values of 7
NADP+~.~
S 2GSH~
i~,~ ,o,~,. . . . . 0oo.o-~ ~o(~)~J
f ROOl-i
L~0'o'~'ooo . .... '~o~" I
~ - ~s~ j
\
Ro.+.~o
Fig. 4. Glutathione represents the major cellular pool of mobile sulfhydryl groups. The redox couple 2GSH/GSSG is linked to the NADP + system by glutathione reductase and to the hydroperoxide system by glutathione peroxidase.
and 20 Torr [13]. Lowering the tissue PO 2 would lead, therefore, to a decreased H 2 0 2 content of the carotid body. One has to ask now how hydrogen peroxide could act as a second messenger. Sies [29] proposed, as already mentioned, and based on an idea as published by Kosower and Werman [15] that the degradation of H 2 0 2 by the glutathione peroxidase (GPO) would lead to a change in the equilibrium of the 2 G S H / G S S G redox couple as shown in Fig. 4. The POz-dependent H 2 0 2 production would, therefore, result in a higher GSSG content when the PO 2 is increased and in a higher GSH content when the PO 2 is lowered. Since this PO2-dependent change in oxidized or reduced glutathione might influence protein conformation (e.g. conductivity of ion channels) by regulating the availability of free SH groups it was our next step to prove this metabolic step in the carotid body. Table I gives the values for the G P O activity in the pig carotid body using different hydroperoxides as substrates. Although a direct transition of these pig carotid bodies values to the rat carotid body tissue is not possible, the data in Table I indicate that the carotid body seems to possess an active GPO. The difference-value of the GPO activities using H 2 0 2 and cumene hydroperoxide as substrate is indicative for the activity of selen-free GPO [17]. Table I clearly shows that the use of all three substrates resulted in similar values indicating that the carotid body possesses the selen-containing GPO only. The TABLE I Glutathione peroxidase acticities in the pig carotid body
H202 t-butyl hydroperoxide Cumene hydroperoxide
5.86 U / g protein 6.18 U / g protein 4.65 U / g protein
48
P02=310 Torr lOmin 100E~ E O t(.3 ~0 (J E t/] IlJ t_ O D
50.
I
"-r
E C3 Z
35 0
142 71
....~,jJM
213
cumene hydroperoxide Fig. 5. Dose-dependent oxidation of NAD(P)H in the carotid body tissue in response to a gradual increase of the cumene hydroperoxide concentration in the superfusion bath at a constant PO 2 of 310 Torr. The absolute NAD(P)H change amounts to 10% of the total fluorescence.
values in the carotid body are nearly a factor hundred lower than GPO values in the rat liver [171. We then went on to complete the list of different members of the hypothetical signal chain by superfusing the rat carotid body with the different hydroperoxides and the recording of the NAD(P)H fluorescence. According to Fig. 4, application of hydroperoxides should lead to an increased GPO activity with an increase of the GSSG content which subsequently activates the glutathione reductase (GPR) which reduces, depending on the equilibrium ratio some of the
newly formed GSSG back t o GSH with simultaneous oxidation of NAD(P)H [30]. Figure 5 shows that application of hydroperoxides, in this example cumene hydroperoxides, results in a dose-dependent fashion in a reversible oxidation of NAD(P)H confirming the presence of the 2GSH/GSSG couple also in the rat carotid body. The oxidation amounts to about 10% of the total NAD(P)H fluorescence. Table II summarizes the dose-dependent NAD(P)H oxidation in the rat carotid body induced by application of the different hydroperoxides. Taking the total NAD(P)H reaction as shown in Fig. 5 as 100%, gradual increase of the hydroperoxide dose leads to gradual decrease of the NAD(P)H fluorescence reaching a saturation between 300 and 400/xM. Half maximal values are 88 + 24.8 IzM (n = 6) for H 2 0 2, 39 + 10.7 (n = 5) /xM for cumene hydroperoxide and 42 ÷ 12,6/xM (n = 5) for t-butyl hydroperoxide. These values are statistically not significant, arguing tot the exclusive role of the selen-containing GPO as hydrogen peroxide scavenger in the rat carotid body comparable to the pig carotid body. After these indications for the importance and reactivity of the glutathione metabolism in the carotid body, one might continue with the hypothetical signal chain pathway as follows: PO 2 Sensor: Effector: Cytochrome b - - e - - - O ~ - - S O D - H20 2 2GSH/GSSG The 2GSH/GSSG couple has an equilibrium ratio of 104 meaning that on base of a high homoeostatic GSH level (10 mM), GSSG (1 /xM) is able to float depending on the H 2 0 2 supply and to serve as a signal [25]. The question was, therefore, addressed for evidences of glutathione in-
TABLE II
Reaction of NAD(P)H fluorescence in the rat carotid body to various doses o f the different hydroperoxides Hydroperoxide/zM
0
35.5
71
142
284
426
NAD(P)H fluorescence change in percentage of control H20 2 n = 6 100 60 ± 11.5
43 + 9.3
24 ± 10
6± 0
0
t-butyl hydrogenperoxide n = 5
100
25 +_ 9
14 ± 7.4
6± 4
0
0
Cumene hydroperoxide n = 5
100
28 + 12.4
3 _-2- 2.4
0
0
9± 6
49
duced ion channel conductivity changes. Glutathione-gated K + channels have been postulated by potassium flux measurements in Escherichia coil [20] which close under reduced conditions like hypoxia. More direct evidence was given by Ruppersberg et al. [27] showing that fast inactivating K + currents mediated by cloned K + channel subunits derived from rat brain expressed in Xenopus oocytes are faster inactivated by reduced glutathione. We can now complete our hypothetical signal chain pathway as follows: PO2 Sensor: Effector: Cytochrome b - - e - - - O ~ - - - S O D - - H 2 0 z Effect: 2 G S H / G S S G - - glutathione-gated K + channel Since both glutathione-gated K + channels and POz-dependent K + channels of type-I cells close under reduced and open under oxidized conditions it seems to be justified in attempting to prove whether application of hydrogen peroxide mimics hyperoxia leading to membrane hyperpolarization, cytosolic calcium decrease, reduced transmitter release and hence, depressed nervous chemoreceptor activity. Therefore we carried out experiments measuring, as shown in Fig. 6, the reaction of the nervous chemoreceptor activity to
3
30#V
Immmm
I/s
120 i
10 rain
I=
80
40
PrnO. (Torrl 338
t 19 I
t e r - butyt-hydroperoxide {~M}
338 ,
30
,
,
60
,
go
,
19 , 338 D
120
Fig. 6. Dose-dependent decrease in the nervous chemoreceptor discharge in response to a gradual increase in the t-butyl hydroperoxide concentration in the superfusion bath. The PO 2 in the bath solution amounted to 338 Torr with short hypoxic periods of 19 Torr for chemoreceptor stimulation. The figure shows original recordings of the sinus nerve action potentials photographed at point 1, 2 and 3 during the experiments. I / s = impulses per second, PmO2 = oxygen pressure in the superfusion bath,/xV = microvolt, s = 1 second, time of 10 minutes is indicated by a bar.
TABLE III
Reaction of the nercous chemoreceptor discharge of the rat carotid body to carious doses of the different hydroperoxides Hydroper- 0 oxide/zM
30
60
90
120
Chemoreceptor discharge in percentage of control H202 n=3 100 90_+ 8.7 66_+13.2 39_+11 27 _+ 4.3 t-butyl hydroperoxide n=3 100 91-+10.8 45-+ 6.7
34_+ 5.7 27_+15
cumene hydroperoxide n=2 100 106_+25.4 62_+ 4.33 44_+11.2 29+_18.9
the application of the various hydroperoxides. Fibers of the sinus nerve have been first tested for their sensitivity by a decrease of PO: in the superfusion bath. Typically, an increase in the nervous discharge can be observed (see Fig. 6). Application of hydroperoxides in different concentrations, in this case t-butyl hydroperoxide, leads to a decrease in chemoreceptor discharge. A hypoxic stimulation during this period has no response in the chemoreceptor discharge. Table III summarizes the results obtained by expressing the decrease in the nervous activity under hydroperoxide application as percentage of control. For this purpose the difference between chemoreceptor activity under a PO 2 of about 300 Torr in the superfusion bath and electrical zero of the ratemeter was taken as 100%. It is to be seen that each of the hydroperoxides leads to a decrease in the chemoreceptor activity to about 30% of the control value. Cumene hydroperoxide stimulated transiently the chemoreceptor discharge at a dose of 30/xM. Half maximal values are 77 + 13.7 p,M (n = 3) for H 2 0 2, 59 + 5.8/zM (n = 3) for t-butyl hydroperoxide and 69_+ 3.3 /zM (n = 2) for cumene hydroperoxide. The half maximal values are in the same range as the half maximal values of NAD(P)H oxidation as shown in Table II underlining the close relationship between the activity of the scavenger effect of GPO and the chemoreceptor activity under exposure of the carotid body tissue to hydroperoxides. The participation of catalase can be neglected since the organic hydroperoxides are scavenged by GPO only [30].
50
At the moment it is not possible for us to give more evidence for our model about the importance of H202 for PO e chemoreception in the carotid body. This has to be done on the cellular level showing by the patch clamp technique, the responsivness of the PO e sensitive K + channels to glutathione with the subsequent changes of the intracellular calcium level monitored by microfluorometry. However, it might be interesting to mention that the participation of H202 in PO 2 chemoreception is discussed also in two other systems, i.e. the hypoxic vasoconstriction of the lung vessels [5] and the erythropoietin production in kidney ceils [32]. In lung vessels, H202 induces a catalase-dependent guanylate cyclase activation by interaction of the heme groups of the two enzymes leading to the cGMP-mediated relaxation of pulmonary arteries probably due to opening of K ÷ channels. This process is reversed under hypoxia when H202 production arrests, leading to a vasoconstriction [5]. The erythropoietin production in a renal carcinoma cell line can be enhanced by external application of H202 or inhibited by external application of catalase due to an enhanced scavenging of H202 [32]. The H202-producing system has not been identified in these publications. However, G6rlach et al. [11] recently identified spectrophotometrically in erythropoietin-producing hepatoma cells, a cytochrome b which is similar to the cytochrome b in the carotid body reducible by hypoxia but not by cyanide and therefore an appropriate source for H202. Future experiments have to show whether the described signal chain for PO 2 chemoreception is a general biological principle in cells acting as O 2 sensors in the body.
References 1 Acker, H., PO 2 chemoreception in arterial chemoreceptors. Ann. Rev. Physiol., 51 (1989) 835-844. 2 Acker, H., Dufau, E. Huber, J. and Sylvester, D., Indications to an NAD(P)H oxidase as a possible PO 2 sensor in the rat carotid body, FEBS Lett., 256 (1989) 75-78. 3 Biscoe, T.J. and Duchen, M.R., Monitoring PO 2 by the carotid chemoreceptor, NIPS, 5 (1990) 229-233. 4 Biscoe, T.J., Duchen, M.R., Eisner, D.A., O'Neill, S.C.
and Valdeolmillos, M., Measurements of intraceltular Ca 2+ in dissociated type-I cells of the rabbit carotid body, J. Physiol., 416 (1989) 421-434. 5 Cherry, P.D., Omar, H.A., Farrell, K.A. Stuart, J.S, and Wolin, M.S., Superoxide anion inhibits cGMP associated b o v i n e pulmonary relaxation, Am. J. Physiol., 259 (19901 H1056-HI062. 6 Cross, A.R., Henderson, L., Jones, O.T.G., Delpiano, M.A., Hentschel, J. and Acker, H., Involvement of an NAD(P)H oxidase as a POz sensor protein in the rat carotid body, Biochem. J., 272 (1990) 743-747. 7 Delpiano, M.A. and Acker, H., Hypoxic and hypercapnic responses of [Ca 2+ ]0 and [K + ]0 in the cat carotid body in vitro, Brain Res., 482 (1989) 235-246. 8 Delpiano, M A . and Hescheler, J., Evidence for a PO 2 sensitive K + channel in the type-I cell of the rabbit carotid body. FEBS Letts., 249 (1989) 195-198. 9 Galeotti, T., Borello, S. and Masotti, L.. Oxyradical sources, scavenger systems and membrane damage in cancer cells. In D.K. Das and W.B. Essman, (Eds.), Oxygen Radicals: Systemic Events and Disease Processes, Karger, Basle, 1990, pp. 129-148. 10 Ganfornina, M.D. and Lopez-Barneo, J., Single K ÷ channels in membrane patches of arterial chemoreceptor cells are modulated by 0 2 tension, Proc. Natl. Acad. Sci~ USA, 88 (1991) 2927-2930. 11 G~Srlach, A., Jelkmann, W., B611ing, B., Holtermann, G. and Acker, H., Spectrophotometric characterisation of heme proteins in erythropoietin producing hepatoma cells, Pfliigers Arch., 419 (1991) Rl16. 12 Hescheler, J., Delpiano, M.A., Acker, H. and Pietruschka, F., Ionic currents on type-I cells of the rabbit carotid body measured by voltage clamp experiments and the effect of hypoxia, Brain Res., 486 (1989) 79-88. 13 Jones, O.T.G., Cross, A.R., Hancock, J.T., Henderson, L.M. and O'Donnel, V.B., Inhibitors of NAD(P)H oxidase as guides to its mechanism, Biochem. Soc. Trans,, 19 (1991) 70-72. 14 Konz, K.H., Die Aktivit~it des Peroxid-metabolisierenden Systems in Human Leber, J. Clin. Chem. Clin. Bioehem:. 17 (1979) 353-357. 15 Kosower, E.M. and Werman, R., New step in transmitter release at the myoneural junction, Nature New Biok, 233 (1971) 121-123. 16 Lahiri, S. and DeLaney, R.G., Stimulus interaction in the response of carotid body chemoreceptor single afferent fibres. Resp. Physiol., 4 (19751 229-235. 17 Lawrence, R.A. and Burk, R.F., Glutathione peroxidase activity in selenium deficient rat liver, Biochem. Biophys. Res. Comm., 71 (1976) 951-958. 18 Lloyd, B.B., Cunningham, D.J.C. and Goode, R~C., Depression of hypoxic hyperventitation in man by sudden inspiration of carbon monoxide, In R.W. Torrance (Ed.), Arterial Chemoreceptors Blaekwell Oxford, 1968, pp. 145-148. 19 Lopez-Lopez, J., Gonzalez, C., Urena, J. and LopezBarneo, J., Low PO 2 selectively inhibits K channel avtivity
51 in chemoreceptor cells of the mammalian carotid body, J. Gen. Physiol., 93 (1989) 1001-1015. 20 Meury, J. and Robin, A., Glutathione-gated K + channels of Escherichia coli carry out K + efflux controlled by the redox state of the cell, Arch. Microbiol., 154 (1990) 475482. 21 Mills, E. and J~Sbsis, F.F., Mitochondrial respiratory chain of carotid body and chemoreceptor response to changes in oxygen tension, J. Neurophysiol., 35 (1972) 405-428. 22 Nurse, C.A., Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat, Cell Tissue Res., 261 (1990) 65-71. 23 Oshino, N., Sugano, T., Oshino, R. and Chance, B., Mitochondrial function under hypoxic conditions: the steady states of cytochrome aa 3 and their relation to mitochondrial energy states, Biochim. Biophys. Acta, 368 (1974) 298-310. 24 Pietruschka, F., Calcium influx in cultured carotid body cells is stimulated by acetylcholin and hypoxia, Brain Res., 347 (1985) 140-143. 25 Reich, J.G. and Selkov, E.E., Pools and pathways of metabolism. In J.G. Reich and E.E. Selkov, (Eds.), Energy Metabolism of the Cell, Academic Press, 1981, pp 108-190. 26 Rumsey, W.L., Iturriaga, R., Spargel, D., Lahiri, S. and Wilson, D.F., Optical measurements of the dependence of chemoreception on oxygen pressure in the cat carotid body, Am. J. Physiol., (1991) C614-C622. 27 Ruppersberg, J.P., Stocker, M., Pongs, O., Heinemann, St. H., Frank, R. and Koenen, M., Regulation of fast inactivation of cloned mammalian IK(A) channels by cysteine oxidation. Nature 352 (1991) 711-714.
28 Sato, M., Ikeda, K., Yoshizaki, K. and Koyano, H., Response of cytosolic calcium to anoxia and cyanide in cultured glomus cells of newborn rabbit carotid body, Brain Res., 551 (1991) 327-330. 29 Sies, H., Peroxisomal enzymes and oxygen metabolism in liver. In M. Ruvich, R. Coburn, S. Lahiri and B. Chance (Eds.), Tissue Hypoxia and Ischemia. Plenum Publishing Corp., New York, 1988, pp. 51-66. 30 Sies, H., Gerstenecker, Ch., Menzel, H. and Floh6, L., Oxidation in the NADP system and release of GSSG from hemoglobin free perfused rat liver during peroxidatic oxidation of glutathione by hydroperoxides, FEBS Lett., 27 (1972) 171-175. 31 Stea, A. and Nurse, C.A., Whole cell and perforated patch recordings from 0 2 sensitive rat carotid body cells grown in short and long term culture, Pfliigers Arch., 418 (1991) 93-101. 32 Ueno, M., Brookins, J., Beckmann, B.S. and Fischer, J.W., Effects of reactive oxygen metabolites on erythropoietin production in renal carcinoma cells, Biochem. Biophys. Res. Commun., 154 (1988) 773-780. 33 Wilson, D.F., Rumsey, W.L., Green, Th.J. and Vanderkooi, J.M., The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration, J. Biol. Chem., 236 (1988) 2712-2718. 34 Woffendin, C., Edwards, S.W., Griffiths, A.J., The cytochromes of Dictostelium discoideum, Comp. Biochem. Physiol., 75B (1983) 53-59.