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Adenosine triphosphate content in the cat carotid body under different arterial O2 and CO2 conditions
Neuroscience Letters, 50 (1984) 175-179
175
Elsevier Scientific Publishers Ireland Ltd. NSL 02921
A D E N O S I N E T R I P H O S P H A T E C O N T E N T IN T H E CAT C A R O T I D B O D Y U N D E R D I F F E R E N T A R T E R I A L Oz A N D COz C O N D I T I O N S
H. ACKER and H. STARLINGER
Max-Planck-lnstitut fiir Systemphysiologie,
Rheinlanddamm 201, D-4600 Dortmund 1 (F. R. G.)
(Received May 23rd, 1984; Revised version received June 27th, 1984; Accepted June 29th, 1984)
Key words: carotid body - adenosine triphosphate - chemoreceptive process - cat
The adenosine triphosphate (ATP) level in the carotid body has often been discussed as the crucial step in the chemoreceptive process. Therefore, the ATP level of the cat carotid body was investigated with the aid of the bioluminescence method under different stimulation conditions. Under normoxic conditions an ATP level of about 0.087 nmol/glomus was measured, which is very low in comparison to other organs. The level did not change significantly, neither under hypoxic nor hypercapnic conditions. From these results we conclude that the primary effect of the chemoreceptive process in the carotid body cannot be explained by changes of the ATP level under different stimulation conditions.
Several researchers suspected that a decrease in the intracellular adenosine triphosphate (ATP) level might be the crucial step in the chemoreceptive process of the carotid body. The evidence for this assumption is largely circumstantial, however (see ref. 4 for review). Anichkov and Belinkii [2] as well as J6els and Neil [9] suppose that hypoxia and hypercapnia cause a reduction of the A T P level which is most pronounced in the type I cells and is accompanied by a transmitter release from the type I cells which then excites synaptically connected nerve fibers in a complex way [1]. Direct measurements on the A T P level have not been carried out, apart from the qualitative histochemical measurements by B6ck [7]. Here we report the results of such measurements, which do not show significant changes of the intracellular A T P level under either hypoxic or hypercapnic conditions. Experiments were performed in cats anesthetized with pentobarbital (40-60 mg/kg). The animals were artificially ventilated, paralyzed with pancuronium bromide (5-6 m g / k g ) and thermostabilized at 37°C. The right and left carotid bodies were prepared according to the method described by Biscoe and Purves [5] leaving the sinus nerve intact. Arterial blood pressure was continuously measured through the cannulated left femoral artery with a Statham P 23 Ob transducer. The blood pressure was recorded with a two-channel ink recorder (Servogor/Berlin). Blood samples for measuring the arterial pO2 (paOz), p C O 2 ( p a C O 2 ) , and pH were obtained by means of the cannula and analyzed in a gas blood analyzer (AVL 940). 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
176
For varying the arterial pO2 or pCO2/pH, the animals were ventilated with a gas mixture of 5% 02 in N2 o r 6 % C O 2 - 2 0 ° 7 0 0 2 in N2, respectively. The carotid body and, for comparison, pieces from the neck musculature were cut out within 1 and 2 s and dropped into liquid nitrogen. After homogenizing the frozen tissue in 0.1 N HC1Oa, the probes were centrifuged for 10 min at 34,000 g, and the supernatant was used then for ATP determination. The bioluminescence method according to Strehler [12] was used for the ATP determination. In brief, the method follows the scheme: mg2 ~
ATP + luciferin + 02
---+ oxyluciferin + PPi + A M P + CO2 + light. luciferase
The quantity of emitted light was measured with the bioluminescence analyzer SKAN XP 2000 (Skan AG, Basel) and recorded on an ink recorder (Servogor/ Berlin). We used either the enzyme-substrate complex, luciferin-luciferase, from firefly lantern extract (Sigma) or luciferin and luciferase as pure substances (Boehringer). The accuracy of the bioluminescence method was about 4%. In order to show that ATP levels do not change during the excision a n d / o r homogenization procedures, tissue cells of the neck musculature were excised from the living animal at the same time and were dropped into liquid nitrogen. Table I gives the ATP values for the two organs under normoxic conditions. The ATP values of the musculature are in very good agreement with data taken from the literature [12]. We therefore assume that our method yields representative ATP values for the carotid body also. Other methods for rapid carotid body or other tissue sampling like superfusion of the carotis sinus region with liquid nitrogen or excising the tissue with a forceps cooled by liquid nitrogen were not practicable in our hands, since it is impossible to identify and excise the carotid body from a deep frozen block of the carotis sinus precisely. The ATP values for the carotid body are given per organ. This is done due to the uncertainty of the carotid body weight and the heterogeneity of the carotid body cell compartments. The dry weight of the carotid body was determined by Leitner and Liaubet [10] to a mean value of 40 #g and a wet weight of about 0.5 mg. Small changes of the water content, therefore, lead to great errors of the normalized A T P values. Furthermore, the fraction of the type 1 cells in the carotid body which are known to contain the largest amount of ATP [7], varies around a mean of 20% [11]. TABLE 1 COMPARATIVE ATP VALUES Ot" CAROTID BODY AND MUSCULATURE Values expressed as mean + standard deviation; n - number of experiments. Note different dimensions. gfw, gram fresh weight. Carotid body ATP (nmot/glomus) Muscle ATP 0mlol/gfw) Normoxia
0.087 +_ 0.056 (n -: 31)
3.56 _+. 1.01 (n
16)
177
Taking both mean values into account for an approximate estimation, we calculate an ATP level of the type I cells in the carotid body of about 0.8 t~mol/g fresh weight. This is far below the A T P level of the muscle. Since the ATP values of the carotid body show a large scattering under normoxic conditions, we selected the following procedure to study the effect of hypoxia and hypercapnia. After putting the animal on the respiratory pump, a stabilizing period of 5 min was observed under normoxic conditions. After taking a blood sample for blood gas analysis, the first carotid body was excised and dropped into liquid nitrogen as a control. After ventilating 8 animals with a hypoxic mixture (paO2 25 torr, paCO2 28 torr, pH 7.28) and 9 animals with a hypercapnic gas mixture (paO2 90 torr, paCO2 49 tort, pH 7.14) for 11 min the second carotid body was excised for ATP determination. Additionally, the ATP level of 7 carotid bodies was investigated after ventilating animals with air for the same time (paO2 98 tort, paCO2 27 tort, pH 7.34). Fig. 1 shows the behavior of the blood pressure during this experimental procedure. In the first period of stabilization, the mean blood pressure ranges from 110-120 mmHg. The resection of the first carotid body always induces a slight blood pressure increase, presumably due to the loss of baroreceptor control. During the second period of the experiments, the blood pressure under normoxic and hypercapnic conditions has a tendency to higher values without any drastic change, whereas the blood pressure in the hypoxic experiments decreases to about 80 mmHg in the first minute with a following recovery to about 90 mmHg. BP/[mmHg]
150
100'
- - - Normoxie n=7 - . - - Hypercapnie n=9 Hypoxie n=8
50-
t
t
blood gas sample I. carotid body 0
o
;.
6
blood gas sample 2. carotid body
"}
lbl'lt io
Fig. 1. Behavior of the blood pressure during different experimental procedures.
178 ~[ABI_E 11 ATP VALUES IN THE C A R O T I D BODY UNDER NORMO X IC , H Y P O X I C AND H Y P E R C A P N I C ('ONDITIONS Values expressed as I11eall
ff
standard devialion, gfw, gram fresh weight. Carotid body ATP (nmol/gfw)
Normoxia
0.083 ~ 0.057 (n :
7)
Normoxia
0.142 ~ 0.147 (n :
7)
Normoxia Hypoxia
0.085 ~ 0.050 (n 0.089 ~ 0.074 (n
8) 8)
Norlno:,:ia Hypercapnia
0.082 f 0.044 (n = 9) 0.133 ~ 0.069 (n : 9)
Table II gives the ATP levels for the comparative measurements of the first and second carotid body under normoxic, hypoxic and hypercapnic conditions. It is clearly to be seen that all comparative values scatter around 0.1 nmol/carotid body. The difference between values found under normoxic, hypoxic or hypercapnic conditions, are not significant, when Student's t-test is applied. When comparing the ATP levels in the carotid bodies with those in other organs, it should be noted that these levels are usually tightly regulated and, thus, do not provide a measure for ATP turnover a n d / o r metabolic rates related to this A T P turnover. Thus, we also expect a more or less constant ATP level under variable respiratory conditions. Only under ischaemic conditions, as for instance in the brain, ATP levels drop drastically [8]. Our experiments on the liver (data not shown) indicate a reduction of the ATP level of about 30% after an ischaemia of 20 s. No such drastic events are observed in our experiments. Both hypoxia and hypercapnia, which induce a pronounced nervous chemoreceptive response [1], leave the A T P level of the carotid body largely unaffected, in spite of a reduction of local flow in the carotid body under hypoxia [1] and various nervous efferent influences under hypercapnia and hypoxia [4]. Most probably this finding is a reflection of the regulation of ATP levels and does not allow us to draw easy conclusions oil the rates of either respiration or glycolysis. The lack of effect of hypoxia on the ATP levels in the carotid body is in agreement with findings on the cortex [3,6] where hypoxia also did not change the ATP level. The reason for this was proposed by tile authors to be a higher rate of glycolytic ATP production a n d / o r decrease of energy consuming processes like the acetylcholine production. Therefore, according to our measurements it seems to be doubtful that the ATP level in the carotid body is the crucial signal for 02 sensing. Ackcr, H., Delpiano, M. and Degner, F., The meaning of the PO2-field in the carotid body for tile chemoreceptive process. In H. Acker and R.G. O'Regan (Eds.), Physiology of the Peripheral Arterial Chemoreceptors, Elsevier/Norlh-Holland, Amsterdam, 1983. 2 Anichkov, S.V. and Belenkii, M.I,., Pharmacology of tile Carotid Body Chemoreceptors, Pergamon, Oxford, 1963. I
179 3 Bachelard, H.S., Lewis, L.D., Pontdn, U. and Siesj6, B.K., Mechanisms activating glycolysis in the brain in arterial hypoxia, J. Neurochem., 22 (1974) 395-401. 4 Belmonte, C. and Gonzalez, C., Mechanisms of chemoreception in the carotid body: possible models. In H. Acker and R.G. O'Regan (Eds.), Physiology of the Peripheral Arterial Chemoreceptors, Elsevier/North-Holland, Amsterdam, 1983. 5 Biscoe, T.J. and Purves, M.J., Observations on the rhythmic variation in the cat carotid body chemoreceptor activity which has the same time period as respiration, J. Physiol. (Lond.), 190 (1967) 389-412. 6 Blass, J.P. and Gibson, G.E., Consequences of mild, graded hypoxia, Advanc. Neurol., 26 (1979) 229-253. 7 B6ck, P., Adenine nucleotides in the carotid body, Cell Tiss. Res., 206 (1980) 279-290. 8 Goldberg, N.D., Passonneau, J.V. and Lowry, O.H., Effects of changes in brain metabolism on the levels of citric acid cycle intermediates, J. biol. Chem., 241 (1966) 3997-4003. 9 Joels, N. and Neil, E., The excitation mechanism of the carotid body, Brit. Med. Bull., 19 (1963) 21-24. 10 Leitner, L.M. and Liaubet, M.J., Carotid body oxygen consumption of the cat in vitro, PflOgers Arch., 323 (1971) 315-322. I 1 Seidl, E., Sch~ifer, D., Zierold, K., Acker, H. and Ltibbers, D.W., Light-microscopic and electronmicroscopic studies on the morphology of cat carotid body. In H. Acker, S. Fidone, D. Pallot, C. Eyzaguirre, D.W. LLibbers and R.W. Torrance (Eds.), Chemoreception in the Carotid Body, Springer, Berlin, 1977, pp. 1-9. 12 Strehler, P.L., Adenosin-5'-triphosphat und Creatinphosphat-Bestimmung mit Luciferase. In I-.'.U. Bergmeyer (Ed.), Methoden der Enzymatischen Analyse, Bd. 11, Verlag Chemie, Weinheim, 1974, S. 2163-2177.
175
Elsevier Scientific Publishers Ireland Ltd. NSL 02921
A D E N O S I N E T R I P H O S P H A T E C O N T E N T IN T H E CAT C A R O T I D B O D Y U N D E R D I F F E R E N T A R T E R I A L Oz A N D COz C O N D I T I O N S
H. ACKER and H. STARLINGER
Max-Planck-lnstitut fiir Systemphysiologie,
Rheinlanddamm 201, D-4600 Dortmund 1 (F. R. G.)
(Received May 23rd, 1984; Revised version received June 27th, 1984; Accepted June 29th, 1984)
Key words: carotid body - adenosine triphosphate - chemoreceptive process - cat
The adenosine triphosphate (ATP) level in the carotid body has often been discussed as the crucial step in the chemoreceptive process. Therefore, the ATP level of the cat carotid body was investigated with the aid of the bioluminescence method under different stimulation conditions. Under normoxic conditions an ATP level of about 0.087 nmol/glomus was measured, which is very low in comparison to other organs. The level did not change significantly, neither under hypoxic nor hypercapnic conditions. From these results we conclude that the primary effect of the chemoreceptive process in the carotid body cannot be explained by changes of the ATP level under different stimulation conditions.
Several researchers suspected that a decrease in the intracellular adenosine triphosphate (ATP) level might be the crucial step in the chemoreceptive process of the carotid body. The evidence for this assumption is largely circumstantial, however (see ref. 4 for review). Anichkov and Belinkii [2] as well as J6els and Neil [9] suppose that hypoxia and hypercapnia cause a reduction of the A T P level which is most pronounced in the type I cells and is accompanied by a transmitter release from the type I cells which then excites synaptically connected nerve fibers in a complex way [1]. Direct measurements on the A T P level have not been carried out, apart from the qualitative histochemical measurements by B6ck [7]. Here we report the results of such measurements, which do not show significant changes of the intracellular A T P level under either hypoxic or hypercapnic conditions. Experiments were performed in cats anesthetized with pentobarbital (40-60 mg/kg). The animals were artificially ventilated, paralyzed with pancuronium bromide (5-6 m g / k g ) and thermostabilized at 37°C. The right and left carotid bodies were prepared according to the method described by Biscoe and Purves [5] leaving the sinus nerve intact. Arterial blood pressure was continuously measured through the cannulated left femoral artery with a Statham P 23 Ob transducer. The blood pressure was recorded with a two-channel ink recorder (Servogor/Berlin). Blood samples for measuring the arterial pO2 (paOz), p C O 2 ( p a C O 2 ) , and pH were obtained by means of the cannula and analyzed in a gas blood analyzer (AVL 940). 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
176
For varying the arterial pO2 or pCO2/pH, the animals were ventilated with a gas mixture of 5% 02 in N2 o r 6 % C O 2 - 2 0 ° 7 0 0 2 in N2, respectively. The carotid body and, for comparison, pieces from the neck musculature were cut out within 1 and 2 s and dropped into liquid nitrogen. After homogenizing the frozen tissue in 0.1 N HC1Oa, the probes were centrifuged for 10 min at 34,000 g, and the supernatant was used then for ATP determination. The bioluminescence method according to Strehler [12] was used for the ATP determination. In brief, the method follows the scheme: mg2 ~
ATP + luciferin + 02
---+ oxyluciferin + PPi + A M P + CO2 + light. luciferase
The quantity of emitted light was measured with the bioluminescence analyzer SKAN XP 2000 (Skan AG, Basel) and recorded on an ink recorder (Servogor/ Berlin). We used either the enzyme-substrate complex, luciferin-luciferase, from firefly lantern extract (Sigma) or luciferin and luciferase as pure substances (Boehringer). The accuracy of the bioluminescence method was about 4%. In order to show that ATP levels do not change during the excision a n d / o r homogenization procedures, tissue cells of the neck musculature were excised from the living animal at the same time and were dropped into liquid nitrogen. Table I gives the ATP values for the two organs under normoxic conditions. The ATP values of the musculature are in very good agreement with data taken from the literature [12]. We therefore assume that our method yields representative ATP values for the carotid body also. Other methods for rapid carotid body or other tissue sampling like superfusion of the carotis sinus region with liquid nitrogen or excising the tissue with a forceps cooled by liquid nitrogen were not practicable in our hands, since it is impossible to identify and excise the carotid body from a deep frozen block of the carotis sinus precisely. The ATP values for the carotid body are given per organ. This is done due to the uncertainty of the carotid body weight and the heterogeneity of the carotid body cell compartments. The dry weight of the carotid body was determined by Leitner and Liaubet [10] to a mean value of 40 #g and a wet weight of about 0.5 mg. Small changes of the water content, therefore, lead to great errors of the normalized A T P values. Furthermore, the fraction of the type 1 cells in the carotid body which are known to contain the largest amount of ATP [7], varies around a mean of 20% [11]. TABLE 1 COMPARATIVE ATP VALUES Ot" CAROTID BODY AND MUSCULATURE Values expressed as mean + standard deviation; n - number of experiments. Note different dimensions. gfw, gram fresh weight. Carotid body ATP (nmot/glomus) Muscle ATP 0mlol/gfw) Normoxia
0.087 +_ 0.056 (n -: 31)
3.56 _+. 1.01 (n
16)
177
Taking both mean values into account for an approximate estimation, we calculate an ATP level of the type I cells in the carotid body of about 0.8 t~mol/g fresh weight. This is far below the A T P level of the muscle. Since the ATP values of the carotid body show a large scattering under normoxic conditions, we selected the following procedure to study the effect of hypoxia and hypercapnia. After putting the animal on the respiratory pump, a stabilizing period of 5 min was observed under normoxic conditions. After taking a blood sample for blood gas analysis, the first carotid body was excised and dropped into liquid nitrogen as a control. After ventilating 8 animals with a hypoxic mixture (paO2 25 torr, paCO2 28 torr, pH 7.28) and 9 animals with a hypercapnic gas mixture (paO2 90 torr, paCO2 49 tort, pH 7.14) for 11 min the second carotid body was excised for ATP determination. Additionally, the ATP level of 7 carotid bodies was investigated after ventilating animals with air for the same time (paO2 98 tort, paCO2 27 tort, pH 7.34). Fig. 1 shows the behavior of the blood pressure during this experimental procedure. In the first period of stabilization, the mean blood pressure ranges from 110-120 mmHg. The resection of the first carotid body always induces a slight blood pressure increase, presumably due to the loss of baroreceptor control. During the second period of the experiments, the blood pressure under normoxic and hypercapnic conditions has a tendency to higher values without any drastic change, whereas the blood pressure in the hypoxic experiments decreases to about 80 mmHg in the first minute with a following recovery to about 90 mmHg. BP/[mmHg]
150
100'
- - - Normoxie n=7 - . - - Hypercapnie n=9 Hypoxie n=8
50-
t
t
blood gas sample I. carotid body 0
o
;.
6
blood gas sample 2. carotid body
"}
lbl'lt io
Fig. 1. Behavior of the blood pressure during different experimental procedures.
178 ~[ABI_E 11 ATP VALUES IN THE C A R O T I D BODY UNDER NORMO X IC , H Y P O X I C AND H Y P E R C A P N I C ('ONDITIONS Values expressed as I11eall
ff
standard devialion, gfw, gram fresh weight. Carotid body ATP (nmol/gfw)
Normoxia
0.083 ~ 0.057 (n :
7)
Normoxia
0.142 ~ 0.147 (n :
7)
Normoxia Hypoxia
0.085 ~ 0.050 (n 0.089 ~ 0.074 (n
8) 8)
Norlno:,:ia Hypercapnia
0.082 f 0.044 (n = 9) 0.133 ~ 0.069 (n : 9)
Table II gives the ATP levels for the comparative measurements of the first and second carotid body under normoxic, hypoxic and hypercapnic conditions. It is clearly to be seen that all comparative values scatter around 0.1 nmol/carotid body. The difference between values found under normoxic, hypoxic or hypercapnic conditions, are not significant, when Student's t-test is applied. When comparing the ATP levels in the carotid bodies with those in other organs, it should be noted that these levels are usually tightly regulated and, thus, do not provide a measure for ATP turnover a n d / o r metabolic rates related to this A T P turnover. Thus, we also expect a more or less constant ATP level under variable respiratory conditions. Only under ischaemic conditions, as for instance in the brain, ATP levels drop drastically [8]. Our experiments on the liver (data not shown) indicate a reduction of the ATP level of about 30% after an ischaemia of 20 s. No such drastic events are observed in our experiments. Both hypoxia and hypercapnia, which induce a pronounced nervous chemoreceptive response [1], leave the A T P level of the carotid body largely unaffected, in spite of a reduction of local flow in the carotid body under hypoxia [1] and various nervous efferent influences under hypercapnia and hypoxia [4]. Most probably this finding is a reflection of the regulation of ATP levels and does not allow us to draw easy conclusions oil the rates of either respiration or glycolysis. The lack of effect of hypoxia on the ATP levels in the carotid body is in agreement with findings on the cortex [3,6] where hypoxia also did not change the ATP level. The reason for this was proposed by tile authors to be a higher rate of glycolytic ATP production a n d / o r decrease of energy consuming processes like the acetylcholine production. Therefore, according to our measurements it seems to be doubtful that the ATP level in the carotid body is the crucial signal for 02 sensing. Ackcr, H., Delpiano, M. and Degner, F., The meaning of the PO2-field in the carotid body for tile chemoreceptive process. In H. Acker and R.G. O'Regan (Eds.), Physiology of the Peripheral Arterial Chemoreceptors, Elsevier/Norlh-Holland, Amsterdam, 1983. 2 Anichkov, S.V. and Belenkii, M.I,., Pharmacology of tile Carotid Body Chemoreceptors, Pergamon, Oxford, 1963. I
179 3 Bachelard, H.S., Lewis, L.D., Pontdn, U. and Siesj6, B.K., Mechanisms activating glycolysis in the brain in arterial hypoxia, J. Neurochem., 22 (1974) 395-401. 4 Belmonte, C. and Gonzalez, C., Mechanisms of chemoreception in the carotid body: possible models. In H. Acker and R.G. O'Regan (Eds.), Physiology of the Peripheral Arterial Chemoreceptors, Elsevier/North-Holland, Amsterdam, 1983. 5 Biscoe, T.J. and Purves, M.J., Observations on the rhythmic variation in the cat carotid body chemoreceptor activity which has the same time period as respiration, J. Physiol. (Lond.), 190 (1967) 389-412. 6 Blass, J.P. and Gibson, G.E., Consequences of mild, graded hypoxia, Advanc. Neurol., 26 (1979) 229-253. 7 B6ck, P., Adenine nucleotides in the carotid body, Cell Tiss. Res., 206 (1980) 279-290. 8 Goldberg, N.D., Passonneau, J.V. and Lowry, O.H., Effects of changes in brain metabolism on the levels of citric acid cycle intermediates, J. biol. Chem., 241 (1966) 3997-4003. 9 Joels, N. and Neil, E., The excitation mechanism of the carotid body, Brit. Med. Bull., 19 (1963) 21-24. 10 Leitner, L.M. and Liaubet, M.J., Carotid body oxygen consumption of the cat in vitro, PflOgers Arch., 323 (1971) 315-322. I 1 Seidl, E., Sch~ifer, D., Zierold, K., Acker, H. and Ltibbers, D.W., Light-microscopic and electronmicroscopic studies on the morphology of cat carotid body. In H. Acker, S. Fidone, D. Pallot, C. Eyzaguirre, D.W. LLibbers and R.W. Torrance (Eds.), Chemoreception in the Carotid Body, Springer, Berlin, 1977, pp. 1-9. 12 Strehler, P.L., Adenosin-5'-triphosphat und Creatinphosphat-Bestimmung mit Luciferase. In I-.'.U. Bergmeyer (Ed.), Methoden der Enzymatischen Analyse, Bd. 11, Verlag Chemie, Weinheim, 1974, S. 2163-2177.