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Selective action of hypoxia on rat lung cyclic AMP
Respiration Physiology (1978) 35. 59 63 © Elsevier/North-Holland Biomedical Press
SELECTIVE ACTION OF HYPOXIA ON RAT LUNG CYCLIC AMP*
R.A. RHOADES ~ and E.G. WHITTLE 2 I Department q[' Ph.vsiology, Indiana UniversiO, School o! Medicine, 1100 West Michigan Street, Indianapolis, Indiana 46202, and 2 The Penn,Evlvania State UniversiO', University Park. Pennsylvania 16802, U.S.A.
Abstract. The effect of three different levels of 24-h hypobaric hypoxia (630 m m Hg, 520 m m Hg, and 340 m m I~g) on rat lung c A M P and c G M P was studied. Liver was also examined to evaluate comparatively with lung. Lung c A M P concentration averaged 33.6 _+ 2.2 (SE) p m o l / m g protein, and c G M P averaged 2.4_+0.21 (SE) pmol/mg protein. Twenty-four-hour hypoxia resulted in a significant 40",, decrease in lung c A M P at 520 and 340 m m Hg. The magnitude of change was the same for both hypoxic levels suggesting threshold dependency. In contrast, liver c A M P was not affected by the hypoxic exposure. Lung and liver c G M P were also relatively refractory to hypoxia. The decreased c A M P concentration seen in the hypoxic lung returned to normal level within 24 hours. These data show that acute hypoxia has a selective action on lung cAMP. Cyclic nucleotides Hypoxia
Lung
The lung is extremely rich in adenosine 3'5'-monophosphate (cAMP) and guanosine 3'5'-monophosphate (cGMP). In other tissues, these nucleotides serve as important regulators of tissue growth, smooth muscle contraction, as well as in intermediary metabolism (Andersson et al., 1972; Bergofsky and Holtzmon, 1967; Jost and Rickenberg, 1971). However, the role in which these nucleotides play in lung function is not well defined. In a previous study, (Rhoades et al., 1976) we showed that acute 24-h hypobaric hyl~oxic stress significantly decreased lung cAMP, and that the decrease was not attributed to an anorexic effect. The present studies were undertaken to further evaluate the effect of hypoxia on lung cAMP by determining: (1) if the changes observed in cAMP with the hypoxic lung exhibited threshold Acceptedjbr publication 20 May 1978 * This investigation was supported in part by Air Force Grant A F 2767. R.A.R. is supported by a Research Career Development Award K04 HL 00166. Reprint requests to : Dr. R . A . Rhoades. 59
60
R.A. RHOADES AND E. G. WHITTLE
dependency, and (2) if the decrease in cAMP was reversible. Cyclic nucleotides were also examined in liver tissue for comparative evaluation.
Methods
Male Long Evans Hooded rats (Blue Spruce Farms, Altamount, N.Y.) weighing 300-350 g were exposed to hypoxia for 24 hours. Animals were divided into three groups and one group was exposed to 630 mm Hg (1500 m), the second to 520 mm Hg (3000 m), and the third to 340 mm Hg (6000 m). The rate of ascent and descent was approximately 200 m - r a i n -~. Water was available at all times, but both control and hypoxic animals were food deprived for the 24-h period in order to separate anorexic effects from hypoxia. Ambient temperature was maintained at 23 C and animals were maintained on a 12-h light-dark cycle. Following the hypoxic exposure, animals were killed by decapitation and lungs quickly freezeclamped and stored at - 7 6 ' C . Forty to sixty mg of tissue was homogenized in 2 ml of 6~,i trichloroacetic acid; centrifuged at 2000 g at 4 ~'C. The supernatant was extracted three times with 5 ml ethyl ether saturated with water, and the aqueous phase was dried under nitrogen at 60-70 C on a steam bath. The dried aqueous portion was then resuspended in 2 ml of 0.05 M sodium acetate (pH 6.2) and assayed in replicates for cyclic nucleotides using radioimmunoassay as described previously (Rhoades et al., 1976). Tissue protein was determined by the method of Lowry et al. (1951). Statistical analyses were carried out using Student's 't" test (Snedecor, 1965).
Results
No differences in lung dry: wet weight ratio were observed, indicating that little edema was present. Table 1 compares cAMP and c G M P concentration of rat lung following 24-h hypoxia. The values are expressed on both a wet weight and protein basis. Lung c A M P level was not significantly affected at 630 mm Hg but showed a 40% decrease at 520 mm Hg. No further decrease in cAMP was observed at the lower hypoxic level (340 mm Hg). Lung c G M P level was not markedly affected by hypoxia with the exception of an increase at 630 mm Hg. This increase accounted for the decrease in c A M P / c G M P ratio at 630 mm Hg, whereas the decrease in the ratio observed at the other hypoxic levels was due primarily to the decrease in cAMP level. In contrast to lung, liver contained lower endogenous levels of cAMP and c G M P (table 2). Both nucleotides in liver were relatively refractory to hypoxia. Figure 1 shows cAMP concentration during a recovery from hypoxia. Animals were exposed to 340 mm Hg for 24 hours and then allowed to recover for 24, 48, and 72 hours. As seen in Figure 1, c A M P recovered and returned to control levels within 24 hours.
61
A C T I O N OF H Y P O X I A ON L U N G CYCLIC AMP TABLE 1 Lung cyclic nucleotides following 24-h hypobaric hypoxia* N ucleotide
Ambient pressure
cAMP pmol/mgprotein pmol/mgwetwt cGMP pmol/mgprotein pmol/mg wet wt cAMP/rag protein cG M P/mg pro tei n
735 mm Hg
630 mm Hg
520 mm Hg
340 mm Hg
33.6 4.7
28.8 4.1
20.1 ± 2 . 7 " 2.7 ±0.31"
19. l 2,8
±2.2 ±0.28
±3.89 ±0.55
2.4 ±0.21 0.33 ±0.03
3.9 ± 0 . 4 1 ' 0.51 ±0.06*
2.4 _+0.08 0.33 ±0.01
2.3 ±0.17 0.30 ±0.02
14
7
8
8
* All values are Mean ± SEM. * Indicates significant difference from control values (P < 0.05). n = 6 for all groups.
I
I
CONTROL 24hr HYPOBARIC HYPOXIA (349mm Hg) 24hr HYPOXIA + 24hr RECOVERY 24hr HYPOXIA + 48hr RECOVERY
50--
±2.4" ±0.29"
24hr HYPOXIA + 72hr RECOVERY Significantly different from control at p < 0.05 N/group = 6
40--
30--
20--
I0--
Fig. 1. Lung cAMP levels following recovery from 24-h hypoxic stress.
62
R.A. R H O A D E S A N D E.G. WHITTLE "FABLE 2 Liver cyclic nucleotidcs following 24-h hypocaric hypoxia +
Nucleotide
Ambient pressure 735 mm Hg
630 mm Hg
520 mm Hg
340 mm Fig
3.2 _+0.10 0.73 _+ 0.02
4.1 _+0.34 0.91 ± 0.08
3.7 ±0.29 0.83 _+0.07
3.15 ±0.367 0.70 ± 0.07
0.13 _+ 0.02 0.03 ± 0.003
0.15 _+ 0.03 0.02 _+0.005
0.14 _+0.03 0.03 _+0.004
0.12 ± 0.02 (I.03 _+0.004
25
27
26
cAMP pmol/mgprotein pmol/mg wet wt cGMP pmol/mg protein pmol/mg wet wt cAMP/mg protein cGMP/mg protein
26
* All values are Mean + SEM. * Indicates significant difference from control values (P < 0.05): n = 6 for all groups.
Discussion
The results of the present studies show that endogenous cAMP content in lung is markedly decreased by low O~. These effects in the lung were very distinct from those observed in liver. Also, the action of hypoxia on lung cGMP was clearly different from those seen with cAMP. All these observations demonstrate that hypoxia has a selective action on lung cAMP, and the hypoxic effect appears to be short lived since cAMP levels returned to normal within 24 hours. The selective action of hypoxia on lung cAMP also exhibits threshold dependency on hypoxia since cAMP did not progressively decrease with low Or. The mechanism by which low O, mediates its effects on cAMP in lung is not clear. The decrease in cAMP levels which we observed could be due to an inhibition of synthesis (resulting from a decrease in adenylate cyclase activity) to an increased rate of cAMP hydrolysis, or to an increased effiux from lung tissue. Lung is one of the few organs that has been shown to exhibit exceptionally high guanylate cyclase activity and cGMP content (Kuo and Kuo, 1973 ; Stoner et al., 1973) which suggests a prominent role of this nucleotide in lung function. One particular function related to low 02 is the hypoxic-induced vasoconstriction and prostaglandins release (Said et al., 1974). Stoner and associates (1973) have shown that an increase in lung cGMP results in release of prostaglandins. Since in the present investigation cGMP was not significantly changed with hypoxia, the data suggest that a cGMP-independent mechanism for hypoxic vasoconstriction may exist in the lung. Moreover, recent evidence shows that changes in pulmonary hemodynamics associated with cAMP appear to be more related to secondary
ACTION OF HYPOXIA ON LUNG CYCLIC A M P
63
effects of cardiac output than to primary pulmonary vasoconstriction (Takahashi and Ewatsuki, 1974) and the changes associated with hypoxic-induced vasoconstriction are more related to calcium levels than to cAMP levels (Kaukel et al., 1975). Thus, the functional role of cyclic nucleotides in lung hypoxia is not well defined. Another possibility is some role in glycogen metabolism. However, the decreased cAMP level seen in the hypoxic lung does not appear to be involved in glycogen metabolism since in a previous study we have shown that lung glycogen is fairly fixed and is not mobilized with 24 hours of acute hypoxic stress (Rhoades, Shaw and Eskew, 1975). At present it is difficult to pinpoint the changes seen in lung tissue cyclic nucleotide concentration to any particular function or to any particular cell type since the lung is a heterogenous organ comprised of 38 different cells.
References Andersson, R., L. Lundholm, E. Mohme-Lundholm and K. Nilsson (1972). Role of cyclic A M P and Ca 2 + in metabolic and mechanical events in smooth muscle. Adv. Cyclic Nucleotide Res. 1 : 213 229. Bergofsky, E. and S. Holtzmon (1967). A study of the mechanisms involved in the pulmonary arterial pressor response to hypoxia. Circ. Res. 20:506 519. Jost, J. P. and H. V. Rickenberg (1971). Cyclic AMP. Ann. Rev. Biochem. 40:741 774. Kaukel, E., J. Siemssen, N. Volket and V. Sill (1975). cAMP dependent and cAMP independent alterations of lung hemodynamic in the pig, Biochem. Pharm. 24:2159 2162. Kuo, J,F. and W. Kuo (1973). Regulation by /~-adrenergic receptor and muscarinic cholinergic receptor activation of intracellular cyclic AMP and cyclic G M P levels in rat lung slices. Biochem. Biophys. Res. Commun. 55: 66(~665. Lowry, O.H.. N.J. Rosebrough, A.L. Farr and R.J. Randall (1951). Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193:265 275. Rhoades, R.A., M.E. Shaw and M,L. Eskew (1975). Influence of altered 02 tension on substrate metabolism in perfused rat lung. Am. J. Physiol. 229: 1476-1479. Rhoades, R.A., R.P. Morrow and M.L. Eskew (1976). Lung cyclic AMP: Selective decrease with hypoxia (39422). Proc. Soc. Exptl. Biol. Med. 152: 480482. Said, S.l., T. Yoshida, S. Kitamaru and C. Ureim (1974). Pulmonary alveolar hypoxia: Release of prostaglandins and other humoral mediators. Science 185:1181 1182. Snedecor, G.W. (1965). The comparison of two randomized groups. In: Statistical Methods. Ames, Iowa, State University Press, pp. 85-101. Stoner, J., V.C. Manganiello and M. Vaughan (1973). Effects of bradykinin and indomethacin on cyclic G M P and cyclic A M P in lung slices. Proc. Natl. Acad. Sei. U.S.A. 70: 383(~3833. Takahashi, K. and K. lwatsuki (1974), Effects of dibutyryl cyclic A M P on the pulmonary hemodynamics. TohokuJ. Expl. Med. 113:365 370.
SELECTIVE ACTION OF HYPOXIA ON RAT LUNG CYCLIC AMP*
R.A. RHOADES ~ and E.G. WHITTLE 2 I Department q[' Ph.vsiology, Indiana UniversiO, School o! Medicine, 1100 West Michigan Street, Indianapolis, Indiana 46202, and 2 The Penn,Evlvania State UniversiO', University Park. Pennsylvania 16802, U.S.A.
Abstract. The effect of three different levels of 24-h hypobaric hypoxia (630 m m Hg, 520 m m Hg, and 340 m m I~g) on rat lung c A M P and c G M P was studied. Liver was also examined to evaluate comparatively with lung. Lung c A M P concentration averaged 33.6 _+ 2.2 (SE) p m o l / m g protein, and c G M P averaged 2.4_+0.21 (SE) pmol/mg protein. Twenty-four-hour hypoxia resulted in a significant 40",, decrease in lung c A M P at 520 and 340 m m Hg. The magnitude of change was the same for both hypoxic levels suggesting threshold dependency. In contrast, liver c A M P was not affected by the hypoxic exposure. Lung and liver c G M P were also relatively refractory to hypoxia. The decreased c A M P concentration seen in the hypoxic lung returned to normal level within 24 hours. These data show that acute hypoxia has a selective action on lung cAMP. Cyclic nucleotides Hypoxia
Lung
The lung is extremely rich in adenosine 3'5'-monophosphate (cAMP) and guanosine 3'5'-monophosphate (cGMP). In other tissues, these nucleotides serve as important regulators of tissue growth, smooth muscle contraction, as well as in intermediary metabolism (Andersson et al., 1972; Bergofsky and Holtzmon, 1967; Jost and Rickenberg, 1971). However, the role in which these nucleotides play in lung function is not well defined. In a previous study, (Rhoades et al., 1976) we showed that acute 24-h hypobaric hyl~oxic stress significantly decreased lung cAMP, and that the decrease was not attributed to an anorexic effect. The present studies were undertaken to further evaluate the effect of hypoxia on lung cAMP by determining: (1) if the changes observed in cAMP with the hypoxic lung exhibited threshold Acceptedjbr publication 20 May 1978 * This investigation was supported in part by Air Force Grant A F 2767. R.A.R. is supported by a Research Career Development Award K04 HL 00166. Reprint requests to : Dr. R . A . Rhoades. 59
60
R.A. RHOADES AND E. G. WHITTLE
dependency, and (2) if the decrease in cAMP was reversible. Cyclic nucleotides were also examined in liver tissue for comparative evaluation.
Methods
Male Long Evans Hooded rats (Blue Spruce Farms, Altamount, N.Y.) weighing 300-350 g were exposed to hypoxia for 24 hours. Animals were divided into three groups and one group was exposed to 630 mm Hg (1500 m), the second to 520 mm Hg (3000 m), and the third to 340 mm Hg (6000 m). The rate of ascent and descent was approximately 200 m - r a i n -~. Water was available at all times, but both control and hypoxic animals were food deprived for the 24-h period in order to separate anorexic effects from hypoxia. Ambient temperature was maintained at 23 C and animals were maintained on a 12-h light-dark cycle. Following the hypoxic exposure, animals were killed by decapitation and lungs quickly freezeclamped and stored at - 7 6 ' C . Forty to sixty mg of tissue was homogenized in 2 ml of 6~,i trichloroacetic acid; centrifuged at 2000 g at 4 ~'C. The supernatant was extracted three times with 5 ml ethyl ether saturated with water, and the aqueous phase was dried under nitrogen at 60-70 C on a steam bath. The dried aqueous portion was then resuspended in 2 ml of 0.05 M sodium acetate (pH 6.2) and assayed in replicates for cyclic nucleotides using radioimmunoassay as described previously (Rhoades et al., 1976). Tissue protein was determined by the method of Lowry et al. (1951). Statistical analyses were carried out using Student's 't" test (Snedecor, 1965).
Results
No differences in lung dry: wet weight ratio were observed, indicating that little edema was present. Table 1 compares cAMP and c G M P concentration of rat lung following 24-h hypoxia. The values are expressed on both a wet weight and protein basis. Lung c A M P level was not significantly affected at 630 mm Hg but showed a 40% decrease at 520 mm Hg. No further decrease in cAMP was observed at the lower hypoxic level (340 mm Hg). Lung c G M P level was not markedly affected by hypoxia with the exception of an increase at 630 mm Hg. This increase accounted for the decrease in c A M P / c G M P ratio at 630 mm Hg, whereas the decrease in the ratio observed at the other hypoxic levels was due primarily to the decrease in cAMP level. In contrast to lung, liver contained lower endogenous levels of cAMP and c G M P (table 2). Both nucleotides in liver were relatively refractory to hypoxia. Figure 1 shows cAMP concentration during a recovery from hypoxia. Animals were exposed to 340 mm Hg for 24 hours and then allowed to recover for 24, 48, and 72 hours. As seen in Figure 1, c A M P recovered and returned to control levels within 24 hours.
61
A C T I O N OF H Y P O X I A ON L U N G CYCLIC AMP TABLE 1 Lung cyclic nucleotides following 24-h hypobaric hypoxia* N ucleotide
Ambient pressure
cAMP pmol/mgprotein pmol/mgwetwt cGMP pmol/mgprotein pmol/mg wet wt cAMP/rag protein cG M P/mg pro tei n
735 mm Hg
630 mm Hg
520 mm Hg
340 mm Hg
33.6 4.7
28.8 4.1
20.1 ± 2 . 7 " 2.7 ±0.31"
19. l 2,8
±2.2 ±0.28
±3.89 ±0.55
2.4 ±0.21 0.33 ±0.03
3.9 ± 0 . 4 1 ' 0.51 ±0.06*
2.4 _+0.08 0.33 ±0.01
2.3 ±0.17 0.30 ±0.02
14
7
8
8
* All values are Mean ± SEM. * Indicates significant difference from control values (P < 0.05). n = 6 for all groups.
I
I
CONTROL 24hr HYPOBARIC HYPOXIA (349mm Hg) 24hr HYPOXIA + 24hr RECOVERY 24hr HYPOXIA + 48hr RECOVERY
50--
±2.4" ±0.29"
24hr HYPOXIA + 72hr RECOVERY Significantly different from control at p < 0.05 N/group = 6
40--
30--
20--
I0--
Fig. 1. Lung cAMP levels following recovery from 24-h hypoxic stress.
62
R.A. R H O A D E S A N D E.G. WHITTLE "FABLE 2 Liver cyclic nucleotidcs following 24-h hypocaric hypoxia +
Nucleotide
Ambient pressure 735 mm Hg
630 mm Hg
520 mm Hg
340 mm Fig
3.2 _+0.10 0.73 _+ 0.02
4.1 _+0.34 0.91 ± 0.08
3.7 ±0.29 0.83 _+0.07
3.15 ±0.367 0.70 ± 0.07
0.13 _+ 0.02 0.03 ± 0.003
0.15 _+ 0.03 0.02 _+0.005
0.14 _+0.03 0.03 _+0.004
0.12 ± 0.02 (I.03 _+0.004
25
27
26
cAMP pmol/mgprotein pmol/mg wet wt cGMP pmol/mg protein pmol/mg wet wt cAMP/mg protein cGMP/mg protein
26
* All values are Mean + SEM. * Indicates significant difference from control values (P < 0.05): n = 6 for all groups.
Discussion
The results of the present studies show that endogenous cAMP content in lung is markedly decreased by low O~. These effects in the lung were very distinct from those observed in liver. Also, the action of hypoxia on lung cGMP was clearly different from those seen with cAMP. All these observations demonstrate that hypoxia has a selective action on lung cAMP, and the hypoxic effect appears to be short lived since cAMP levels returned to normal within 24 hours. The selective action of hypoxia on lung cAMP also exhibits threshold dependency on hypoxia since cAMP did not progressively decrease with low Or. The mechanism by which low O, mediates its effects on cAMP in lung is not clear. The decrease in cAMP levels which we observed could be due to an inhibition of synthesis (resulting from a decrease in adenylate cyclase activity) to an increased rate of cAMP hydrolysis, or to an increased effiux from lung tissue. Lung is one of the few organs that has been shown to exhibit exceptionally high guanylate cyclase activity and cGMP content (Kuo and Kuo, 1973 ; Stoner et al., 1973) which suggests a prominent role of this nucleotide in lung function. One particular function related to low 02 is the hypoxic-induced vasoconstriction and prostaglandins release (Said et al., 1974). Stoner and associates (1973) have shown that an increase in lung cGMP results in release of prostaglandins. Since in the present investigation cGMP was not significantly changed with hypoxia, the data suggest that a cGMP-independent mechanism for hypoxic vasoconstriction may exist in the lung. Moreover, recent evidence shows that changes in pulmonary hemodynamics associated with cAMP appear to be more related to secondary
ACTION OF HYPOXIA ON LUNG CYCLIC A M P
63
effects of cardiac output than to primary pulmonary vasoconstriction (Takahashi and Ewatsuki, 1974) and the changes associated with hypoxic-induced vasoconstriction are more related to calcium levels than to cAMP levels (Kaukel et al., 1975). Thus, the functional role of cyclic nucleotides in lung hypoxia is not well defined. Another possibility is some role in glycogen metabolism. However, the decreased cAMP level seen in the hypoxic lung does not appear to be involved in glycogen metabolism since in a previous study we have shown that lung glycogen is fairly fixed and is not mobilized with 24 hours of acute hypoxic stress (Rhoades, Shaw and Eskew, 1975). At present it is difficult to pinpoint the changes seen in lung tissue cyclic nucleotide concentration to any particular function or to any particular cell type since the lung is a heterogenous organ comprised of 38 different cells.
References Andersson, R., L. Lundholm, E. Mohme-Lundholm and K. Nilsson (1972). Role of cyclic A M P and Ca 2 + in metabolic and mechanical events in smooth muscle. Adv. Cyclic Nucleotide Res. 1 : 213 229. Bergofsky, E. and S. Holtzmon (1967). A study of the mechanisms involved in the pulmonary arterial pressor response to hypoxia. Circ. Res. 20:506 519. Jost, J. P. and H. V. Rickenberg (1971). Cyclic AMP. Ann. Rev. Biochem. 40:741 774. Kaukel, E., J. Siemssen, N. Volket and V. Sill (1975). cAMP dependent and cAMP independent alterations of lung hemodynamic in the pig, Biochem. Pharm. 24:2159 2162. Kuo, J,F. and W. Kuo (1973). Regulation by /~-adrenergic receptor and muscarinic cholinergic receptor activation of intracellular cyclic AMP and cyclic G M P levels in rat lung slices. Biochem. Biophys. Res. Commun. 55: 66(~665. Lowry, O.H.. N.J. Rosebrough, A.L. Farr and R.J. Randall (1951). Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193:265 275. Rhoades, R.A., M.E. Shaw and M,L. Eskew (1975). Influence of altered 02 tension on substrate metabolism in perfused rat lung. Am. J. Physiol. 229: 1476-1479. Rhoades, R.A., R.P. Morrow and M.L. Eskew (1976). Lung cyclic AMP: Selective decrease with hypoxia (39422). Proc. Soc. Exptl. Biol. Med. 152: 480482. Said, S.l., T. Yoshida, S. Kitamaru and C. Ureim (1974). Pulmonary alveolar hypoxia: Release of prostaglandins and other humoral mediators. Science 185:1181 1182. Snedecor, G.W. (1965). The comparison of two randomized groups. In: Statistical Methods. Ames, Iowa, State University Press, pp. 85-101. Stoner, J., V.C. Manganiello and M. Vaughan (1973). Effects of bradykinin and indomethacin on cyclic G M P and cyclic A M P in lung slices. Proc. Natl. Acad. Sei. U.S.A. 70: 383(~3833. Takahashi, K. and K. lwatsuki (1974), Effects of dibutyryl cyclic A M P on the pulmonary hemodynamics. TohokuJ. Expl. Med. 113:365 370.