Respiration 38: 155-162 (1979)
A Possible Role of Lung cAM P and cGM P for Lung Histamine Release during Chronic Hypercapnia1 Norbert Voelkel, Angelika Voelkel, Ulrich Wiegers and Eckhart Kaukel I. Medizinische Universitätsklinik, Hamburg
Key Words. Chronic hypercapnia ■Lung histamine • Cyclic nucleotides • Adrenalectomy
1 Supported by the ‘Deutsche Forschungsgemeinschaft’.
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 10:23:08 PM
Abstract. The objective of this study is to determine whether chronic hypercapnia, unlike chronic hypoxia, causes lung tissue histamine release and to seek further evidence for our hypothesis that the cyclic nucleotides cAMP and cGMP play a regulatory role for lung histamine storage and release. Rats exposed chronically to hypercapnia (7% C 0 2) from 1 to 21 days demonstrated an initial decrease of the total lung histamine content: 0.32 ng//zg DNA after 6 days of hypercapnia compared with a control value of 0.75 ng/wg DNA. The decrease in lung histamine content was accompanied by a 3.8-fold increase of the lung tissue cAMP con centration, a 6.3-fold increase of the cGMP concentration, and a 4.1-fold increase of the cGMP/cAMP ratio. The concentration of both nucleotides remained elevated throughout the hypercapnic period, whereas the cGMP/cAMP ratio and the histamine concentration returned to control values. Since the adrenal glands respond to chronic hypercapnia with hypertrophy, we studied the effect of loss of adrenal function on lung tissue cyclic nucleotide and histamine contents. Adrenalectomy depletes lung histamine and leads to a moderate cGMP increase, whereas cAMP is unaffected and the cGMP/ cAMP ratio increases. Adrenalectomy does not potentiate lung histamine depletion in hypercapnic rats but prevents the increase of lung cAMP, and the cGMP accumulation is less than in non-adrenalectomized hypercapnic rats. The results suggest that (1) chronic hypercapnia leads to histamine release from rat lung tissue and (2) the cAMP increase in lung tissue seems to modulate hypercapnic histamine release and to depend on an intact adrenal gland function. The results support our hypothesis that lung histamine release during chronic hypercapnia is mediated by or associated with an increase of the lung tissue cGMP/cAMP ratio.
156
Introduction In analogy to the known influence of cyclic nucleotides on histamine release in immediate hypersensitivity reactions [1, 11], we proposed a similar regulatory role of the cyclic nucleotides for non-allergic his tamine release mechanisms [19]. Since we had recently shown that alterations of lung histamine content during chronic hypoxia were accompanied by alterations of lung cyclic nucleotide concentrations and of the cGMP/cAMP ratio [19], we were interested to see whether these findings were unique for chronic hypoxia or whether chronic hy percapnia, which also causes pulmonary hy pertension and right ventricular hypertro phy in rats [14], would lead to similar re sults. Furthermore, since a relationship be tween lung histamine and adrenal gland function has been suggested in earlier re ports [3, 9, 17] and since it has been shown that the adrenal glands have a regulating in fluence on lung cyclic nucleotide content [18], adrenal gland function, lung tissue cyclic nucleotides and histamine seem to be interrelated by some regulatory mechanism. To test our hypothesis that lung tissue histamine content is regulated by alterations of lung tissue cyclic nucleotides, we inter rupted the adrenal-pulmonary axis by ad renalectomy. Changes in lung tissue cyclic nucleotides then should change lung tissue histamine concentration. Methods Female Wistar rats with an average weight of 200 + 10 g were used. The animals were divided into groups of 6 rats. For the first experiment, rats were exposed to chronic hypercapnia lasting from 1 to 21 days and were studied after 1, 2, 3,
6, 9, 11, 14 and 21 days of hypercapnia. The ani mals were exposed to a gas mixture containing 7%> C 0 2, 20% 0 2 and 73% N2 in an airtight wooden chamber covered with a glass pane and provided with a gas inlet and outlet. Anhydrous calcium sulfate was kept inside the chamber to absorb moisture; the temperature in the chamber was monitored and kept at 22 °C. To ensure a con stant 7% C 02 concentration of the chamber atmo sphere, the COz content was measured contin uously by an ultra-red absorption spectrometer (URAS; Hellige, Freiburg i. Br., FRG) and reg ulated by variation of gas inflow. The exposure was interrupted for 15 min daily for feeding and cleaning of the cages. Two control groups were kept under similar conditions as the experimental animals at room air. To correct for age and growth, the two control groups were studied 21 days apart at the beginning and end of the ex periment. To study the influence of adrenalectomy on lung metabolism in a parallel study, rats were bi laterally adrenalectomized and divided into two groups; one group was kept at room air and stud ied 1, 2, 3 and 9 days after adrenalectomy, the other group was exposed to 7% C 0 2 as described above and studied at the same intervals after ad renalectomy as the room air group. In addition, a sham adrenalectomy was performed in another group which was studied 1 and 2 days after the sham operation. All animals received laboratory chow and tap water ad libitum except for adrena lectomized rats which received normal saline [3, 9]. Experimental animals were sacrificed after ter mination of the hypercapnic exposure period. Within 15 min after removal from the hypercap nia chamber, the animals were anesthetized with 30mg/kg b. w. pentobarbital (Nembutal®). Fol lowing a median sternotomy the lungs were ex cised (leaving trachea and main bronchi in situ), blotted dry, weighed and quickly frozen between two aluminum plates that had been cooled in liq uid nitrogen. Both adrenal glands were excised, dissected free of connective tissue and weighed. Both lung and adrenal gland weights were correct ed for body weight. For the histamine assay, lung tissue was homo genized (Ultraturax; Jahnke & Kunkel, Staufen, FRG) in 9 vol of sodium phosphate buffer (pH 7.9), boiled for 10 min at 95 °C to inactivate tis
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 10:23:08 PM
Voelkel/Voelkel/Wiegers/Kaukel
A Possible Rôle of Lung cAMP and cGMP for Lung Histamine
hypoxia [18], had to be considered for chronic hy percapnia. Also, the possibility of lung edema for mation with concomitant protein leakage makes tissue protein content an unreliable parameter [6J. DNA was measured by the method of Burton [5] (DNA test kit, Boehringer, Mannheim). Results were tested for significance using twoway analysis of variance and the Student-Newman-Keuls test for multiple comparisons.
Results Absolute values, as well as values cor rected for body weight, of lung and adrenal gland weights increase significantly during hypercapnia. As shown in figure 1, the weight gain of the lungs occurs during the first days of exposure to hypercapnia; the relative lung weight increase is 15.5%> after 3 days of hypercapnia. Similarly and rapidly occurs the increase in relative adrenal gland weight, which is 36% after 3 days of hyper capnia (fig. 2). The time course for lung tissue contents of histamine, cAMP, cGMP and the cGMP/cAMP ratio is demonstrated in fig ure 3. Total lung histamine content decreas es during an initial phase of chronic hyper capnia and reaches its lowest value of 0.32 ng/,ug DNA after 6 days of chronic hy-
Fig. 1. Time course of the Iung/body weight ratio during 21 days of hypercapnia (FIco 2 = 7%>). Each data point represents the mean value and SEM of 6 animals. Two control groups (C) were studied 21 days apart. The lung/body weight ratios during hypercapnia are significantly greater than in controls (p < 0.05).
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 10:23:08 PM
sue enzyme activity and centrifuged for 10 min at 6,000 rpm (Christ, Osterode, FRG). The superna tant was used for histamine and DNA measure ment. Histamine was assayed according to the isoto pe-enzyme method of Bcaven et al. [2]. 20 /tl of the supernatant was incubated with 360 ¿
A Possible Role of Lung cAM P and cGM P for Lung Histamine Release during Chronic Hypercapnia1 Norbert Voelkel, Angelika Voelkel, Ulrich Wiegers and Eckhart Kaukel I. Medizinische Universitätsklinik, Hamburg
Key Words. Chronic hypercapnia ■Lung histamine • Cyclic nucleotides • Adrenalectomy
1 Supported by the ‘Deutsche Forschungsgemeinschaft’.
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 10:23:08 PM
Abstract. The objective of this study is to determine whether chronic hypercapnia, unlike chronic hypoxia, causes lung tissue histamine release and to seek further evidence for our hypothesis that the cyclic nucleotides cAMP and cGMP play a regulatory role for lung histamine storage and release. Rats exposed chronically to hypercapnia (7% C 0 2) from 1 to 21 days demonstrated an initial decrease of the total lung histamine content: 0.32 ng//zg DNA after 6 days of hypercapnia compared with a control value of 0.75 ng/wg DNA. The decrease in lung histamine content was accompanied by a 3.8-fold increase of the lung tissue cAMP con centration, a 6.3-fold increase of the cGMP concentration, and a 4.1-fold increase of the cGMP/cAMP ratio. The concentration of both nucleotides remained elevated throughout the hypercapnic period, whereas the cGMP/cAMP ratio and the histamine concentration returned to control values. Since the adrenal glands respond to chronic hypercapnia with hypertrophy, we studied the effect of loss of adrenal function on lung tissue cyclic nucleotide and histamine contents. Adrenalectomy depletes lung histamine and leads to a moderate cGMP increase, whereas cAMP is unaffected and the cGMP/ cAMP ratio increases. Adrenalectomy does not potentiate lung histamine depletion in hypercapnic rats but prevents the increase of lung cAMP, and the cGMP accumulation is less than in non-adrenalectomized hypercapnic rats. The results suggest that (1) chronic hypercapnia leads to histamine release from rat lung tissue and (2) the cAMP increase in lung tissue seems to modulate hypercapnic histamine release and to depend on an intact adrenal gland function. The results support our hypothesis that lung histamine release during chronic hypercapnia is mediated by or associated with an increase of the lung tissue cGMP/cAMP ratio.
156
Introduction In analogy to the known influence of cyclic nucleotides on histamine release in immediate hypersensitivity reactions [1, 11], we proposed a similar regulatory role of the cyclic nucleotides for non-allergic his tamine release mechanisms [19]. Since we had recently shown that alterations of lung histamine content during chronic hypoxia were accompanied by alterations of lung cyclic nucleotide concentrations and of the cGMP/cAMP ratio [19], we were interested to see whether these findings were unique for chronic hypoxia or whether chronic hy percapnia, which also causes pulmonary hy pertension and right ventricular hypertro phy in rats [14], would lead to similar re sults. Furthermore, since a relationship be tween lung histamine and adrenal gland function has been suggested in earlier re ports [3, 9, 17] and since it has been shown that the adrenal glands have a regulating in fluence on lung cyclic nucleotide content [18], adrenal gland function, lung tissue cyclic nucleotides and histamine seem to be interrelated by some regulatory mechanism. To test our hypothesis that lung tissue histamine content is regulated by alterations of lung tissue cyclic nucleotides, we inter rupted the adrenal-pulmonary axis by ad renalectomy. Changes in lung tissue cyclic nucleotides then should change lung tissue histamine concentration. Methods Female Wistar rats with an average weight of 200 + 10 g were used. The animals were divided into groups of 6 rats. For the first experiment, rats were exposed to chronic hypercapnia lasting from 1 to 21 days and were studied after 1, 2, 3,
6, 9, 11, 14 and 21 days of hypercapnia. The ani mals were exposed to a gas mixture containing 7%> C 0 2, 20% 0 2 and 73% N2 in an airtight wooden chamber covered with a glass pane and provided with a gas inlet and outlet. Anhydrous calcium sulfate was kept inside the chamber to absorb moisture; the temperature in the chamber was monitored and kept at 22 °C. To ensure a con stant 7% C 02 concentration of the chamber atmo sphere, the COz content was measured contin uously by an ultra-red absorption spectrometer (URAS; Hellige, Freiburg i. Br., FRG) and reg ulated by variation of gas inflow. The exposure was interrupted for 15 min daily for feeding and cleaning of the cages. Two control groups were kept under similar conditions as the experimental animals at room air. To correct for age and growth, the two control groups were studied 21 days apart at the beginning and end of the ex periment. To study the influence of adrenalectomy on lung metabolism in a parallel study, rats were bi laterally adrenalectomized and divided into two groups; one group was kept at room air and stud ied 1, 2, 3 and 9 days after adrenalectomy, the other group was exposed to 7% C 0 2 as described above and studied at the same intervals after ad renalectomy as the room air group. In addition, a sham adrenalectomy was performed in another group which was studied 1 and 2 days after the sham operation. All animals received laboratory chow and tap water ad libitum except for adrena lectomized rats which received normal saline [3, 9]. Experimental animals were sacrificed after ter mination of the hypercapnic exposure period. Within 15 min after removal from the hypercap nia chamber, the animals were anesthetized with 30mg/kg b. w. pentobarbital (Nembutal®). Fol lowing a median sternotomy the lungs were ex cised (leaving trachea and main bronchi in situ), blotted dry, weighed and quickly frozen between two aluminum plates that had been cooled in liq uid nitrogen. Both adrenal glands were excised, dissected free of connective tissue and weighed. Both lung and adrenal gland weights were correct ed for body weight. For the histamine assay, lung tissue was homo genized (Ultraturax; Jahnke & Kunkel, Staufen, FRG) in 9 vol of sodium phosphate buffer (pH 7.9), boiled for 10 min at 95 °C to inactivate tis
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 10:23:08 PM
Voelkel/Voelkel/Wiegers/Kaukel
A Possible Rôle of Lung cAMP and cGMP for Lung Histamine
hypoxia [18], had to be considered for chronic hy percapnia. Also, the possibility of lung edema for mation with concomitant protein leakage makes tissue protein content an unreliable parameter [6J. DNA was measured by the method of Burton [5] (DNA test kit, Boehringer, Mannheim). Results were tested for significance using twoway analysis of variance and the Student-Newman-Keuls test for multiple comparisons.
Results Absolute values, as well as values cor rected for body weight, of lung and adrenal gland weights increase significantly during hypercapnia. As shown in figure 1, the weight gain of the lungs occurs during the first days of exposure to hypercapnia; the relative lung weight increase is 15.5%> after 3 days of hypercapnia. Similarly and rapidly occurs the increase in relative adrenal gland weight, which is 36% after 3 days of hyper capnia (fig. 2). The time course for lung tissue contents of histamine, cAMP, cGMP and the cGMP/cAMP ratio is demonstrated in fig ure 3. Total lung histamine content decreas es during an initial phase of chronic hyper capnia and reaches its lowest value of 0.32 ng/,ug DNA after 6 days of chronic hy-
Fig. 1. Time course of the Iung/body weight ratio during 21 days of hypercapnia (FIco 2 = 7%>). Each data point represents the mean value and SEM of 6 animals. Two control groups (C) were studied 21 days apart. The lung/body weight ratios during hypercapnia are significantly greater than in controls (p < 0.05).
Downloaded by: Nagoya University 133.6.82.173 - 1/12/2019 10:23:08 PM
sue enzyme activity and centrifuged for 10 min at 6,000 rpm (Christ, Osterode, FRG). The superna tant was used for histamine and DNA measure ment. Histamine was assayed according to the isoto pe-enzyme method of Bcaven et al. [2]. 20 /tl of the supernatant was incubated with 360 ¿
