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Selective decrease of dopamine content in rat carotid body during exposure to hypoxic conditions
352
Brain Research, 118 (1976) 352 355 ',i') Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Selective decrease of dopamine content in rat carotid body during exposure to hypoxic conditions
s. HELLSTROM, I. HANBAUER* and E. COSTA Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted September 9th, 1976)
The rat carotid body contains norepinephrine (NE), dopamine (DA) and serotonin (5-HT)4,5, 8. Each of these amines is stored in membrane-bound granules located in the cystoplasm of type I cells which are the most abundant cells in the rat carotid body 1. Although the role of catechotamines in chemoreceptor function remains to be elucidated, some reports have shown that in the cat, NE and DA inhibit chemoreceptor dischargOl,lL In rat carotid body, exposure to hypoxia causes a delayed increase in tyrosine hydroxylase activity without a concomitant change in dopamine-beta-hydroxytase 3. This finding suggests that during hypoxia, DA rather than NE, functions in chemoreceptor regulation. To elucidate which catecholamine participates in chemoreceptor function during hypoxia, we measured DA and NE content of the carotid body at various times after hypoxia. We used rats with unilateral transection of the sinus nerve, which was pertormed 20 days before the experiment. A desiccator (30 cm in diameter) was connected to a gas tank containing either air or a gas mixture of 5 ~ 02 and 95 ~ N2. The gas mixture was allowed to flow for 30 min through a desiccator containing 3 male Sprague-Dawley rats, weighing about 170 g. The rats were killed by cervical dislocation either immediately after the exposure to hypoxia or after they were placed for various times in room air following the hypoxia. Both carotid bodies were rapidly dissected and the catecholamines assayed by mass fragmentography as previously reported 2,5. Deuterium labeled DA ~.DA-dT) and alpha-methylnorepinephrine ~a-MNE) were used as internal standards for DA and NE respectively. The catecholamines were acylated with pentafluoropropionic anyhdride 5 and the following m / e ratios were focused: a-MNE, 190; NE, 176; DA, 176; DA-dT, 178. The catecholamine derivatives were chromatographed on a 9 ft. glass column packed with 3 ~o OV- 17 on Gas Chrom Q with the flash heater at 250 °C, the oven at 150 °C and a helium flow of 35 ml/min. The retention times of the various amines expressed in seconds were: a-MNE, 120; NE, 160; DA-d7 and DA, 320. * Section on Biochemical Pharmacology, Hypertension Endocrine Branch, National Heart, Lung and Blood Institute, Bethesda, Md. 20014, U.S.A.
353 TABLE I
Catecholamine concentrations in the carotid body of rats after exposure to hypoxic conditions for various times or injection of cholinergic drugs Catecholamine concentrations in pmoles/pair carotid body 4- S.E.M. (N = 5). In parentheses pmoles/kg i.p.
Condition
Minutes of Intact exposure Dopamine
Norepinephrine
Dopamine
Norepinephrine
30 15 30
7.2 4- 0.25 7.8 4- 0.82 8.8 4- 1.5
30 ± 3.3 18 4- 2.2* 12 4- 1.9"
9.4 4- 0.88 8.4 4- 0.55 8.1 4- 1.1
Room air 5 ~ O3 5% O3 5%O2 + atropine (42) 5 ~ O3 + hexamethonium (43) Methacholine (43)
Sinus nerve cut
30 4- 1.9 20 4- 1.5" 9.3 4- 1.9'
30
19 4- 2.2**
7.5 4- 0.35
--
--
30 30
13 4- 1.9" 17 4- 2.3*
6.3 4- 0.39 7.1 4- 0.66
-16 4- 2.2*
-9.0 4- 0.81
* P < 0.01 when compared with values of rats kept in desiccators flushed with room air. ** P < 0.01 when compared with values of rats kept for 30 min, at 5 % O8 but not injected with drugs.
The identification of NE was obtained by verifying whether a ratio of about 8.33 was obtained by focusing m/e 176 and 577 at retention time of 160 sec; that of DA was obtained by verifying whether a ratio of about 2.5 was obtained at a retention time of 320 sec by focusing m/e 428 and 1762. The transection of the carotid sinus nerve (denervated carotid body) failed to change the concentration of NE or DA (Table I). Exposure of rats to hypoxia for 15 or 30 min caused a significant decrease in the DA content of intact and denervated carotid bodies fTable I). In contrast, the NE content remained unchanged in intact and denervated carotid bodies. The per cent of DA depletion during 30 min of hypoxia and the recovery time are reported in Fig. 1. The extent of DA depletion was quite similar in denervated and intact carotid body, but longer lasting in intact than in denervated carotid body. Intact
Sinus nerve cut
100
100,
80
60
7=
40
5% O= Room air
20 0
r 30
60 MINUTES
120 150
N
40
~
20
o c~
0
-
5% 02 #
Room air 30
60
150
MINUTES
Fig. 1. Concentration of D A at various times after hypoxia in intact (left panel) and denervated (right panel) carotid body. Each point represents the average of at least 5 experiments. The S.E.M. (not reported) never exceeded 15 % of the mean value. The D A values at room air (100%) in intact and sinus nerve cut carotid body arc reported in the table. * P < 0.05 when the values are compared to the D A content of rats breathing room air.
354 Previous studies had indicated that in rat carotid body, the turnover rates of N E and DA were similarL Thus, the selective decrease of the DA content caused by hypoxia could not be due to a reduced rate of its synthesis because the concentration of NE failed to change. In the absence of synthesis inhibition, the decrease of the DA content suggests that the rate of DA release might have increased during hypoxia. Since DA and NE appear to be stored in separate granules 6 and the amines are released by exocytosis, it is not probable that DA is converted into NE before being released. Thus, we provisionally suggest that hypoxia releases DA. Since this release does not require the sinus nerve, we must also suggest that mechanisms other than the firing of the sinus nerve are operative in triggering the release of DA during hypoxia. While the participation of the sinus nerve was not essential for the release of DA (Fig. 1), the duration of the DA depletion was longer if the nerve was present. Recent work by McDonald and Mitchell 6 suggests that this nerve may form reciprocal synapses with type 1 cells. The DA depletion caused by hypoxia may persist longer in intact than in denervated carotid bodies because the firing of the sinus nerve by activating the reciprocal synapses may prolong the release of DA which is initiated and maintained by a yet unknown mechanism. We could not extend to the rat a report on cat carotid body showing that epinephrine and NE content decreases wben the cats are ventilated with 8-10% 027. Our results showing that DA content decreases during hypoxia are at variance with a recent theory stating that the release of DA actually decreases during hypoxia I 0. Unfortunately, the proposal of this theory was made indirectly without actual measurements of D A content, whereas we measured the D A and N E content of carotid bodies at various times following hypoxia. Others 9 have postulated that acetylcholine participates in the response of carotid body to hypoxia. We found that methacholine (43 /~moles/kg i.p.), like hypoxia, preferentially released DA in intact and denervated carotid bodies (Table I). Moreover, atropine but not hexamethonium antagonized the decrease of DA content elicited by hypoxia (Table I). Our data support the possibility that the depletion of D A is evoked and maintained by a mechanism which involves the release of acetylcholine. This transmitter could be stored in either nerve terminals of intrinsic cholinergic neurons or glomus cellsll,lL It is possible that acetylcholine, by acting on muscarinic receptors located on the membrane of glomus cells which store DA, triggers the release of this amine. Since our results show that the presence of the sinus nerve prolongs the duration of D A depletion, a role of the reciprocal synapses in the release of DA during hypoxia cannot be excluded.
I Biscoe, T. J., Carotid body: structure and function, Physiol. Rev., 51 (1971) 437-454. 2 Costa, E., Green, A. R., Koslow, S.=H., LeFevre, H. F., Revuelta, A. V. and Wang, C., Dopamine and norepinephrine in noradrenergic axons: a study in vivo of their precursor product relationship by mass fragmentography and radiochemistry, PharmacoL Rev;, 24 (t972) 167-190. 3 Hanbauer, I., Long-term regulatory mechanisms for tyrosine hydroxylase in sympathetic ganglion and carotid body. In E. Costa, E. Giacobini and R. Paoletti (Eds.), Advances in Btochem. Psychopharmacol., Vol. 15, Raven Press, New York, 1976. 4 Hellstr6m, S., Putative neurotransmitters in carotid body: mass fragmentographic studies. In E. Costa and G. L. Gessa (Eds.), Advanc. Biochem. Psychopharmacol., Vot. 17, 1977, in press.
355 5 Hellstr6m, S. and Koslow, S. H., Biogenic amines in carotid body of adult and infant rats - - a gas chromatographic mass spectrometric assay, .4cta physioL scand., 93 (1975) 540-547. 6 McDonald, D. M. and Mitchell, R. A., The innervation of glomus cells, ganglion cells and blood vessels in the rat carotid body: a quantitative ultrastructural analysis, J. Neurocytol., 4 (1975) 177-230. 7 Mills, E. and Slotkin, T. A., Catecholamine content of the carotid body in cats ventilated with 8 - 4 0 ~ oxygen, Life Sci., 16 (1975) 1555-1562. 8 Mollmann, H., Niemeyer, D. H., Alfes, H. und Knoche, H., Mikrospektrofluorometrische Untersuchungen der biogenen Amine in Glomus caroticum des Kaninchens nach Reserpine und PCPAApplikation, Z. Zellforsch., 126 (1972) 104-115. 9 Nishi, K. and Eyzaguirre, C., The action of some cholinergic blockers on carotid body receptors in vivo, Brain Research, 33 (1971) 37-56. 10 Osborne, M. P. and Butler, P. J., New theory for receptor mechanisms of carotid body chemoreceptors, Nature (Lond.), 254 (1975) 701-703. 11 Sampson, S. R., Effects of mecamylamine on responses of carotid body chemoreceptors in vivo to physiological and pharmacological stimuli, J. Physiol. (Lond.), 212 (1971) 655~66. 12 Zapata, P., Effects of dopamine on carotid chemo- and baroreceptors in vitro, J. PhysioL (Lond.), 244 (1975) 235-251.
Brain Research, 118 (1976) 352 355 ',i') Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Selective decrease of dopamine content in rat carotid body during exposure to hypoxic conditions
s. HELLSTROM, I. HANBAUER* and E. COSTA Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted September 9th, 1976)
The rat carotid body contains norepinephrine (NE), dopamine (DA) and serotonin (5-HT)4,5, 8. Each of these amines is stored in membrane-bound granules located in the cystoplasm of type I cells which are the most abundant cells in the rat carotid body 1. Although the role of catechotamines in chemoreceptor function remains to be elucidated, some reports have shown that in the cat, NE and DA inhibit chemoreceptor dischargOl,lL In rat carotid body, exposure to hypoxia causes a delayed increase in tyrosine hydroxylase activity without a concomitant change in dopamine-beta-hydroxytase 3. This finding suggests that during hypoxia, DA rather than NE, functions in chemoreceptor regulation. To elucidate which catecholamine participates in chemoreceptor function during hypoxia, we measured DA and NE content of the carotid body at various times after hypoxia. We used rats with unilateral transection of the sinus nerve, which was pertormed 20 days before the experiment. A desiccator (30 cm in diameter) was connected to a gas tank containing either air or a gas mixture of 5 ~ 02 and 95 ~ N2. The gas mixture was allowed to flow for 30 min through a desiccator containing 3 male Sprague-Dawley rats, weighing about 170 g. The rats were killed by cervical dislocation either immediately after the exposure to hypoxia or after they were placed for various times in room air following the hypoxia. Both carotid bodies were rapidly dissected and the catecholamines assayed by mass fragmentography as previously reported 2,5. Deuterium labeled DA ~.DA-dT) and alpha-methylnorepinephrine ~a-MNE) were used as internal standards for DA and NE respectively. The catecholamines were acylated with pentafluoropropionic anyhdride 5 and the following m / e ratios were focused: a-MNE, 190; NE, 176; DA, 176; DA-dT, 178. The catecholamine derivatives were chromatographed on a 9 ft. glass column packed with 3 ~o OV- 17 on Gas Chrom Q with the flash heater at 250 °C, the oven at 150 °C and a helium flow of 35 ml/min. The retention times of the various amines expressed in seconds were: a-MNE, 120; NE, 160; DA-d7 and DA, 320. * Section on Biochemical Pharmacology, Hypertension Endocrine Branch, National Heart, Lung and Blood Institute, Bethesda, Md. 20014, U.S.A.
353 TABLE I
Catecholamine concentrations in the carotid body of rats after exposure to hypoxic conditions for various times or injection of cholinergic drugs Catecholamine concentrations in pmoles/pair carotid body 4- S.E.M. (N = 5). In parentheses pmoles/kg i.p.
Condition
Minutes of Intact exposure Dopamine
Norepinephrine
Dopamine
Norepinephrine
30 15 30
7.2 4- 0.25 7.8 4- 0.82 8.8 4- 1.5
30 ± 3.3 18 4- 2.2* 12 4- 1.9"
9.4 4- 0.88 8.4 4- 0.55 8.1 4- 1.1
Room air 5 ~ O3 5% O3 5%O2 + atropine (42) 5 ~ O3 + hexamethonium (43) Methacholine (43)
Sinus nerve cut
30 4- 1.9 20 4- 1.5" 9.3 4- 1.9'
30
19 4- 2.2**
7.5 4- 0.35
--
--
30 30
13 4- 1.9" 17 4- 2.3*
6.3 4- 0.39 7.1 4- 0.66
-16 4- 2.2*
-9.0 4- 0.81
* P < 0.01 when compared with values of rats kept in desiccators flushed with room air. ** P < 0.01 when compared with values of rats kept for 30 min, at 5 % O8 but not injected with drugs.
The identification of NE was obtained by verifying whether a ratio of about 8.33 was obtained by focusing m/e 176 and 577 at retention time of 160 sec; that of DA was obtained by verifying whether a ratio of about 2.5 was obtained at a retention time of 320 sec by focusing m/e 428 and 1762. The transection of the carotid sinus nerve (denervated carotid body) failed to change the concentration of NE or DA (Table I). Exposure of rats to hypoxia for 15 or 30 min caused a significant decrease in the DA content of intact and denervated carotid bodies fTable I). In contrast, the NE content remained unchanged in intact and denervated carotid bodies. The per cent of DA depletion during 30 min of hypoxia and the recovery time are reported in Fig. 1. The extent of DA depletion was quite similar in denervated and intact carotid body, but longer lasting in intact than in denervated carotid body. Intact
Sinus nerve cut
100
100,
80
60
7=
40
5% O= Room air
20 0
r 30
60 MINUTES
120 150
N
40
~
20
o c~
0
-
5% 02 #
Room air 30
60
150
MINUTES
Fig. 1. Concentration of D A at various times after hypoxia in intact (left panel) and denervated (right panel) carotid body. Each point represents the average of at least 5 experiments. The S.E.M. (not reported) never exceeded 15 % of the mean value. The D A values at room air (100%) in intact and sinus nerve cut carotid body arc reported in the table. * P < 0.05 when the values are compared to the D A content of rats breathing room air.
354 Previous studies had indicated that in rat carotid body, the turnover rates of N E and DA were similarL Thus, the selective decrease of the DA content caused by hypoxia could not be due to a reduced rate of its synthesis because the concentration of NE failed to change. In the absence of synthesis inhibition, the decrease of the DA content suggests that the rate of DA release might have increased during hypoxia. Since DA and NE appear to be stored in separate granules 6 and the amines are released by exocytosis, it is not probable that DA is converted into NE before being released. Thus, we provisionally suggest that hypoxia releases DA. Since this release does not require the sinus nerve, we must also suggest that mechanisms other than the firing of the sinus nerve are operative in triggering the release of DA during hypoxia. While the participation of the sinus nerve was not essential for the release of DA (Fig. 1), the duration of the DA depletion was longer if the nerve was present. Recent work by McDonald and Mitchell 6 suggests that this nerve may form reciprocal synapses with type 1 cells. The DA depletion caused by hypoxia may persist longer in intact than in denervated carotid bodies because the firing of the sinus nerve by activating the reciprocal synapses may prolong the release of DA which is initiated and maintained by a yet unknown mechanism. We could not extend to the rat a report on cat carotid body showing that epinephrine and NE content decreases wben the cats are ventilated with 8-10% 027. Our results showing that DA content decreases during hypoxia are at variance with a recent theory stating that the release of DA actually decreases during hypoxia I 0. Unfortunately, the proposal of this theory was made indirectly without actual measurements of D A content, whereas we measured the D A and N E content of carotid bodies at various times following hypoxia. Others 9 have postulated that acetylcholine participates in the response of carotid body to hypoxia. We found that methacholine (43 /~moles/kg i.p.), like hypoxia, preferentially released DA in intact and denervated carotid bodies (Table I). Moreover, atropine but not hexamethonium antagonized the decrease of DA content elicited by hypoxia (Table I). Our data support the possibility that the depletion of D A is evoked and maintained by a mechanism which involves the release of acetylcholine. This transmitter could be stored in either nerve terminals of intrinsic cholinergic neurons or glomus cellsll,lL It is possible that acetylcholine, by acting on muscarinic receptors located on the membrane of glomus cells which store DA, triggers the release of this amine. Since our results show that the presence of the sinus nerve prolongs the duration of D A depletion, a role of the reciprocal synapses in the release of DA during hypoxia cannot be excluded.
I Biscoe, T. J., Carotid body: structure and function, Physiol. Rev., 51 (1971) 437-454. 2 Costa, E., Green, A. R., Koslow, S.=H., LeFevre, H. F., Revuelta, A. V. and Wang, C., Dopamine and norepinephrine in noradrenergic axons: a study in vivo of their precursor product relationship by mass fragmentography and radiochemistry, PharmacoL Rev;, 24 (t972) 167-190. 3 Hanbauer, I., Long-term regulatory mechanisms for tyrosine hydroxylase in sympathetic ganglion and carotid body. In E. Costa, E. Giacobini and R. Paoletti (Eds.), Advances in Btochem. Psychopharmacol., Vol. 15, Raven Press, New York, 1976. 4 Hellstr6m, S., Putative neurotransmitters in carotid body: mass fragmentographic studies. In E. Costa and G. L. Gessa (Eds.), Advanc. Biochem. Psychopharmacol., Vot. 17, 1977, in press.
355 5 Hellstr6m, S. and Koslow, S. H., Biogenic amines in carotid body of adult and infant rats - - a gas chromatographic mass spectrometric assay, .4cta physioL scand., 93 (1975) 540-547. 6 McDonald, D. M. and Mitchell, R. A., The innervation of glomus cells, ganglion cells and blood vessels in the rat carotid body: a quantitative ultrastructural analysis, J. Neurocytol., 4 (1975) 177-230. 7 Mills, E. and Slotkin, T. A., Catecholamine content of the carotid body in cats ventilated with 8 - 4 0 ~ oxygen, Life Sci., 16 (1975) 1555-1562. 8 Mollmann, H., Niemeyer, D. H., Alfes, H. und Knoche, H., Mikrospektrofluorometrische Untersuchungen der biogenen Amine in Glomus caroticum des Kaninchens nach Reserpine und PCPAApplikation, Z. Zellforsch., 126 (1972) 104-115. 9 Nishi, K. and Eyzaguirre, C., The action of some cholinergic blockers on carotid body receptors in vivo, Brain Research, 33 (1971) 37-56. 10 Osborne, M. P. and Butler, P. J., New theory for receptor mechanisms of carotid body chemoreceptors, Nature (Lond.), 254 (1975) 701-703. 11 Sampson, S. R., Effects of mecamylamine on responses of carotid body chemoreceptors in vivo to physiological and pharmacological stimuli, J. Physiol. (Lond.), 212 (1971) 655~66. 12 Zapata, P., Effects of dopamine on carotid chemo- and baroreceptors in vitro, J. PhysioL (Lond.), 244 (1975) 235-251.