Circulating endogenous opioids and ventilatory response to CO2 and hypoxia

Respiration Physiology (1985) 61, 85-93

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CIRCULATING E N D O G E N O U S OPIOIDS A N D VENTILATORY R E S P O N S E TO CO z A N D H Y P O X I A

T. R O C H A T 1, A.F. J U N O D 1 and R.C. G A I L L A R D 2 1Division de Pneumologie et 2Clinique Mddicale, Ddpartement de M~decine, H6pital cantonal universitaire, Gendve, Switzerland

Abstract. The role of endogenous opioids in the control of breathing is not yet well defined. Radioimmunoassays that measure fl-endorphin (BE) and met-enkephalin (MET) having recently become available, we decided to evaluate the possible relation between the blood levels of these two opioids and different hypercapnic and hypoxic ventilatory responses observed in a group of normal subjects. Ventilatory response to hypercapnia (n = 9) and to hypoxia (n = 7) were determined by classical rebreathing methods. A voluntary isocapnic normoxic hyperventilation test was used as a control. Basal levels of BE and MET did not correlate with the magnitude of the ventilatory response to either hypercapnia or hypoxia. Moreover, BE and MET levels measured repeatedly up to 30 min alter each test did not change significantly. We conclude that circulating endogenous opioids do not play a role in the control of breathing in normal humans. These results do not rule out a possible role for these substances as locally released mediators. Carbon dioxide Control of breathing

Human Opioid

Oxygen Ventilation

The role of endogenous opioids in the control of breathing is not yet well defined, probably, at least in part, because of their heterogeneity. It is now established that there are at least three families of endogenous opioid peptides: the endorphins, enkephalins and dynorphins, each family possessing its own precursor or pro-hormone. The endorphin and enkephalin families have received special attention in relation to breathing regulation (McQueen, 1983). fl-endorphin (BE) is the most active opioid in the endorphin family whose precursor is pro-opiomelanocortin. This compound also contains the structure of ACTH, fl-lipotropin (fl-LPH) and ?-MSH. Met-enkephalin (MET) is an important opioid from the enkephalin family. Like BE, it is found in the central nervous system and in peripheral tissues. Enkephalin-like immunoreactive material has been found in the glomus cells of the carotid body (Hansen et al., 1982). Experimental studies on animals have shown that opioids have a depressant effect on respiration when directly applied in the cerebrospinal fluid (Moss and Friedman, Accepted for publication 6 April 1985 0034-5687/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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1978; Florez et al., 1980) or in the carotid body (McQueen et al., 1980 and 1981). They could therefore play a role either as neural messengers or as circulating hormones in the control of breathing. In most investigations an indirect approach has been used, namely the study of the effects ofnaloxone, an opioid antagonist, on the ventilatory response to various stimuli. The results have not been uniform and have not allowed definitive conclusions. The determination of endogenous opioid levels in blood and the analysis of their relationship with the ventilatory responses to CO2 and hypoxia could represent a more direct approach. Radioimmunoassays are now available for the determination of BE (Cterminal of fl-lipotropin hormone) and MET (Jeffcoate etaL, 1978; Clement-Jones et al., 1980). However, the effects of well established physiological respiratory stimuli (hypercapnia, hypoxia) on blood levels of endogenous opioids have not yet been reported. We studied a group of normal adult subjects and tried to answer the following questions: (1) Are the ventilatory responses to hypercapnia and hypoxia related to the basal levels of BE and MET in the peripheral venous blood? (2) Do the hypercapnic and hypoxie stimuli change the basal levels of BE and/or MET?

Material and methods

Nine subjects (3 women and 6 men, age 26-44 years) were submitted on two different days to a CO2 rebreathing test and to a control test consisting of an isocapnic normoxic hyperventilation. On a third day, seven among these subjects had a hypoxic rebreathing test. All these healthy subjects were non smokers or ex-smokers. None had a history of respiratory disease and all had a normal spirometry (FEV~, FVC). The different tests were administered one to five weeks apart. They took place between 8 and 10 a.m. The subjects fasted from the preceding evening. Thirty to 45 minutes before the tests, a short flexible indwelling catheter was introduced into an antecubital vein. Blood samples of 20 ml each were then drawn immediately prior to each test (the patient being equipped with mouthpiece and noseclip, but breathing ambient room air), at the end of the test and 5, 10, 15, 20 and 30 min later. For the CO2 rebreathing test, two additional blood samples were obtained, one before the patient was equipped and installed in front of the apparatus and the other during the CO2 rebreathjng, i.e. between the 2nd and the 3rd minute of the test. Blood samples for BE and cortisol were collected into chilled heparinized tubes and immediately centrifuged for 15 min at 4 ° C. The plasma was then flash frozen and stored at - 20 °C until assayed. BE immunoreactivity (C-terminal fl-LPH) was detected after plasma extraction by a radioimmunoassay using an antiserum showing equimolar crossreactivity with fl-LPH (Jeffcoate et al., 1978). Cortisol was determined by a competitive binding method (Leclercq et al., 1969). Samples for MET assay were prepared in the same way except that the heparinized tubes contained Trasylol (aprotinin

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1000 KIU/ml) and that the plasma was decanted into tubes containing a glycine-HC1 solution. MET was assayed by oxidizing the samples with HzO z after extraction and using an antiserum with high specificity for MET sulfoxide (Clement-Jones et al., 1980). With these methods, the intra-assay and interassay coefficients of variation were 8 and 15Yo for BE. They were 9 and 12~ for MET. A control test was performed to test for a possible effect of hyperventilation p e r se. It consisted of a voluntary hyperventilation, corresponding roughly to 50~ of the maximal voluntary ventilation, during 4 min. A rebreathing circuit similar to that of the two other tests was used but a continuous inflow of 02 (200 ml/min) and a CO 2 absorber connected in parallel to the rebreathing circuit maintained the normoxia and isocapnia. The subject had to control his breathing frequency, which was regulated by a metronome, and his tidal volume displayed on an oscilloscope. The COz test was performed according to the rebreathing method described by Read (1966). It lasted 4 min except for one subject who interrupted it after 3 min and 30 sec. The end-tidal P¢o2 (PeTco2) was monitored by a Gould Capnograph and rose by 4-6 mm Hg. min - 1. The ventilatory response to COz (CO2 sensitivity) was defined as the slope of the regression line A~/E/APETco2 and expressed as L. min- 1 .mm Hg- 1 The hypoxia test was performed according to the rebreathing method described by Rebuck and Campbell (1974), but the rebreathing bag was filled with ambient room air, instead of the 7~o CO2 mixture proposed in the original method, to avoid any hypercapnic stimulus. A CO 2 absorber maintained the PETco 2 at + 3 mm Hg from its initial value. An inflow of pure N 2 (200 ml/min) maintained the bag volume constant and also accelerated the decrease in the oxygen concentration of the circuit. The PETo2 was monitored by a Beckman oxygen analyser OM-11 and the test was interrupted after the PETo2 had reached 31-35 mm Hg. This happened after 5-7 rain and corresponded to Sao: of 59-67~o. The Sao~ was continuously monitored by a Hewlett-Packard ear oxymeter 47201 A. The ventilatory response to hypoxia (02 sensitivity) was defined as the slope of the regression line A~/E/A~ Sao~ in L. min- ~. ~oSao~-1. We only considered the points for which Sao: was ~ 91 ~ , i.e. a value corresponding to a Pao~ lower than 60 mm Hg, when the peripheral chemoreceptors were likely to be maximally stimulated. In two subjects, direct measurements of Sao~ could not be obtained. We therefore decided to calculate Sao2 values from the PETo2 assuming that PETo2 = PAo2 = Pao~. This assumption was considered valid because the subjects were all in good health and had normal spirometry. The alveolo-arterial gradient for 02 could also be considered negligible for PETo2 values ranging from 60 to 30 mm Hg. Finally, the correlation between the calculated values of Sao: and the measured values in the five other subjects was good (r = 0.98 for 62 simultaneous measurements and calculations).

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e t al.

Results Ventilatory response to CO e and to hypoxia. Values for AVE/APETco2 (CO2 sensitivity) ranged from 0.90 to 6.03 L ' m i n - ~ ' m m H g - ~ with a mean of 2.70. Values for AVE/A~o Sao 2 (02 sensitivity) ranged from - 0.24 to - 1.88 L. m i n - ~. ~oSao - 1 with a mean of -0.98. The correlation coefficients were superior to 0.86 for the CO2 regression lines except for one subject who has r = 0.76 but also the flattest slope (0.90). For the hypoxia tests, r were always superior to 0.77 except for the same subject who has r = 0.55 but also with the flattest slope ( - 0.24). Basal levels of B E and MET. The mean levels ( + SEM) of BE, just before the tests on the 3 days were 74 + 11, 85 + 18 and 88 + 17 pg/ml. The coefficients of variation between the 3 days for each subject ranged from 0.13 to 0.61 (mean -- 0.37). On the day of the CO= test, the first basal level (before the subjects were installed in front of the apparatus) was 77 + 10 pg/ml compared with 74 + 11 pg/ml about 10 rain later, just before the test. For MET, mean levels were 107 + 13, 74 + 11 and 94 + 17 pg/ml for the 3 days, respectively. The individual coefficients of variation ranged from 0.06 to 0.76 (mean -- 0.46) and were not si~ificantly different from that for BE levels. On the day of the CO 2 test, the first basal level was 123 + 15pg/ml in comparison with 107 + 13 pg/ml about 10 min later. Basal levels of B E and M E T versus ventilatory response to hy_percapnia and hypoxia. The relationship between the basal levels of the two endogenous opioids and CO 2 or 0 2 sensitivity are shown on fig. 1. The relationship was assessed by the least-squares method and no significant correlation was found (BE vs CO= and 02 sensitivity, MET vs CO2 and 02 sensitivity: the slope of the regression lines always close to zero and coefficients of correlation equal or inferior to 0.07). BETA- ENDORPHIN / BETA-LPH

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Fig. 1. Basal levels of the two endogenous opioids beta-endorphin and met-enkephalin vs ventilatory response to CO 2 (CO 2 sens) and to hypoxia (02 sens) (see text). No significant correlation was found (least-squares method).

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Evolution of BE, MET and cortisol blood levels after the ventilatory tests. BE and MET blood levels during and for up to 30 rain after each test are detailed on figs. 2 and 3. There was no significant change in the levels of BE, MET and cortisol (data not shown) during and after the tests (Friedman two-way analysis of variance). The apparent increase of BE and MET levels after the CO 2 test was almost test

~* SEM

150-~ ,oo~ T~

Controltest ~n~9,

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10 minutes

20

30

Fig. 2. Beta-endorphin blood levels before and at~er the control test (isocapnic normoxic hyperventilation), CO 2 rebreathing test and hypoxia rebreathing test. No significant change was observed (Friedman two-way analysis of variance). test

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exclusively due to one subject whose BE level at T = 5 (5 rain after completion of the test) and MET level at T = 0 (just at the end of the test) increased about threefold together with an increase of cortisol from 1.16 #g/ml before the test to 1.86 #g/ml at T = 20. No other subject did show a clear-cut rise of one of the endogenous opioids after any of the tests.

Discussion

This study indicates that, in a group of normal subjects, the basal blood levels of two endogenous opioids, BE and MET, were not related to CO2 and hypoxic sensitivity. Furthermore, opioid blood levels did not change after a hypercapnic or hypoxic stimulus. The data obtained from our subjects were a representative sample of the normal and wide distribution of ventilatory responses t o C O 2 and hypoxia. Our values for CO2 and hypoxic sensitivity covered the range of normal values reported in the literature (Rebuck and Campbell, "1974; Rebuck and Woodley, 1975). Genetic and perhaps acquired factors play a role in determining the ventilatory response to CO2 and hypoxia (Arkinstall etal., 1974; Leitch etal., 1975). Our hypothesis was that variations in the ventilatory responses could result from different rates of production of endogenous opioids and, hence, from differences in basal blood levels. Our data clearly show that this is not the case. Several explanations can be offered. We found a day-by-day variability for BE and MET basal blood levels of about 40 Yo (coefficients of variation of 0.37 and 0.40, respectively). Read (1966) reported a mean coefficient of variation of 0.2 for the CO 2 rebreathing test. Rebuck and Campbell (1974) obtained a coefficient of variation of 0.76 for the hypoxia rebreathing test. It is therefore possible that the important variability of these biological and physiological parameters explains why no significant correlation could be obtained. Apart from the fact that BE levels are high in adrenalectomized patients and MET levels are also high in cases of renal failure (Smith et al., 1981), the significance of the basal blood levels of endogenous opioids remains unclear. Furthermore, there is no correlation between the blood levels of BE and its concentration in the cerebrospinal fluid (Jeffcoate et aL, 1978). The role of BE levels in this compartment would obviously be interesting to measure in view of the major involvement of the chemoreceptors of the medulla oblongata in the control of the response to CO2. Sequential sampling of large amounts of cerebrospinal fluid in normal subjects would be desirable, but is not feasible for ethical reasons. In relation to the sensitivity to hypoxia, the lack of correlation obtained with BE aad MET levels in peripheral bloods seems to exclude the hypothesis that the function of the peripheral chemoreceptors is influenced by these circulating agents. That does not, however, preclude their possible involvement as local mediators. Endogenous opioids could play a role in the control of breathing as locally produced neurotransmitters in

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the central nervous system and in peripheral chemoreceptors (McQueen, 1983). Individual differences in basal tissue concentrations of opioids are not necessarily reflected by basal blood levels (Rossier et al., 1977). The second question we tried to answer was: does a hypercapnic or hypoxic stimulus affect the blood levels of BE and MET*. The hypothesis was that these opioids could function as humoral negative feed-back against the induced ventilatory response. Several arguments could sustain this hypothesis. The role of opioid peptides as a negative feed-back in the ventilatory response to profound hypoxia was postulated by Chernick et al. (1980): they observed that the duration of the primary apnea resulting from asphyxia in newborn rabbits was reduced following a pretreatment with naloxone. They concluded that endogenous opioids, released during asphyxia, were a major factor in the suppression of the medullary neuronal discharge during primary apnea. Whether this release of endogenous opioids would occur locally or could affect blood levels of these compounds is still an open question. Wardlaw et al. (1979) have shown that BE levels in the umbilical cord plasma of human newborns was elevated as a function of their degree of acidosis and hypoxia. We could not detect any rise in BE levels in our adult subjects after a hypoxic test. It is true, however, that the potential role of opioids in the control of breathing in adults and newborns might differ. Our hypoxic stimulus did not have the severity and the duration of the hypoxic episodes reported by Chernick et al. (1980) in animals and by Wardlaw et aL (1979) in human newborns. That MET could play a role in the response of the peripheral chemoreceptors to hypoxia was suggested by the experiments reported by McQueen et al. (1980). They showed that the local application of MET in the carotid arteries of cats markedly depressed the activity of the peripheral chemoreceptors, whereas BE and morphin had only a moderate depressant effect. Pokorsky and Lahiri (1981) also showed that the intravenous administration of naloxone under hypoxic conditions increased the activity of the cat peripheral chemoreceptors when the Pao2 was as low as 35 mm Hg. Although these observations suggest that enkephalins produced locally could modulate the function of the carotid body, another hypothesis could be considered invoking the release of MET from another site of production into the circulation. Our results do not support this second hypothesis. It cannot be excluded however that sudden and short-lasting changes in MET blood levels have occurred which whould have been missed because of the rate of blood sampling used. MET in blood has a very short half-life of less than one minute (R. Smith, personal communication). A possible effect of hypercapnia on blood levels of endogenous opioids could have been postulated on the basis of the effects of naloxone on CO 2 response. Tabona et al. (1982) found an increase in CO 2 sensitivity in normals and in occlusion pressure in patients with chronic obstructive pulmonary disease following treatment with 0.1 mg/kg naloxone i.v. Fleetham et al. (1980), on the other hand, found an increase in the response to CO2 in only two out of six normal subjects. It could be that the release of opioids takes place only in some subjects and not in others. The dose of naloxone can also be a critical determinant, either because this drug has a different affinity for the various

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types of opioid receptors or because naloxone may have a direct pharmacological effect on its own (Sawynok etal., 1979). Exercise is known to produce a transient rise in endogenous opioid levels in the blood of untrained subjects (Carr et aL, 1981) as well as of athletes (Farrell et al., 1982). A recent study indicates that MET may also rise under similar conditions (Howlett et al., 1984). Our data indicate that the increase in ventilation associated with hypercapnia, hypoxia or voluntary hyperventilation does not result in increased blood levels of circulating opioids. Thus, an increase in ventilation alone is unlikely to be responsible for the high levels of opioids found after exercise. The rise of BE and MET presented by one of our subjects after the CO 2 test deserves a brief comment. It may have resulted from a non-specific stress reaction as shown by the rise in cortisol level 20 min after the test. Stress situations are known to provoke a rise of BE blood levels, but not of those of MET. The parallel increase in both peptides in our subject remains an isolated finding difficult to interpret. In conclusion, circulating BE and MET do not appear to play a role in the ventilatory response to CO 2 and hypoxia in normal adults. This negative finding does not rule out a possible role for endogenous opioids in the control of breathing as locally released mediators.

Acknowledgements The authors are greatly indebted to Professor L. H. Rees and Dr. V. Clement-Jones for the gift of fl-LPH and met-enkephalin antibodies, to Dr. A.M. Riondel for measuring plasma cortisol levels, to Mrs. M.C. Jacquier, H. Steinort and D. Turnill for their technical assistance, to Mrs. M. Huesser for the preparation of the manuscript.

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