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Increased release of cyclic adenosine monophosphate into jugular vein in response to isoproterenol administration
Life Sciences, Vol. 35, pp. 963-967 Printed in the U.S.A.
Pergamon Press
INCREASED RELEASE OF CYCLIC ADENOSINE MONOPHOSPHATEINTO JUGULAR VEIN IN RESPONSE TO ISOPROTERENOLADMINISTRATION INorman Altszuler, 1,2Eitan Friedman, John C. Laschinger, Frank P. Catinella, Joseph N. Cunningham, J r . , Ira M. Nathan IDepartment of Pharmacology, 2Department of Psychiatry and Department of Surgery New York University School of Medicine New York, NY 10016 (Received in final form June 19, 1984)
Summary Cate~holamine administration elevates plasma cyclic AMP (cAMP) levels but the source of the cAMP is unknown. To determine possible sources, plasma cAMP level s were determined in blood vessels across the head, l i v e r , kidney and lung in anesthetized dogs infused with the B-adrenergicagonist, isoproterenol. Only the head showed an increased release of cAMP into the blood. The kidneys removed cAMP from the blood while l i v e r and lung showed no change. This in vivo demonstration of release of cAMP from the head represents--co~ibutions from brain and facial muscles and may be a useful approach to study brain involvement in the action of various hormones and drugs. Cyclic AMP is the putative second messenger for a number of hormones and neurotransmitters. Although cAMP is generated within the cell and is assumed to act l o c a l l y , s i g n i f i c a n t increases of cAMP levels in the plasma have been reported following administration of catecholamines 1-3. The source of the plasma cAMP has not been established despite extensive e f f o r t s 4. Since brain slices have been shown5 to increase cAMP in response to isoproterenol i t was of i n t e r e s t to determine i f such release occurred in vivo. Thus, this study was undertaken to determine i f stimulation of B - a d r ~ e r - ~ receptors would release cAMP by the brain in amounts that could be detected in vivo by sampling the jugular vein blood. Since the l a t t e r drains muscle tissues of the head as well as brain, a comparison was made with concentrations of cAMP in the blood across the lungs, l i v e r and kidneys. Only the head showed an increased release of cAMP, thus suggesting a potential usefulness for such specific regional sampling to study the responsiveness of B-adrenergic receptors. Methods Arterio-venous (A-V) concentrations of cyclic AMP were determined across the brain, lungs, l i v e r and kidney in 8 normal postabsorptive dogs. The dogs were anesthetized with chloralose, i00 mg/kg, i v , the skeletal muscles relaxed with metocurine iodide 2 mg/dog, and respiration was maintained by a respiratory pump. Catheters were inserted d i r e c t l y into the following blood vessels in close proximity to the tissues of interest: carotid artery, and jugular vein (brain); pulmonary artery and l e f t atrium (lung); renal artery 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
964
Cyclic AMp Release
Vol. 35, No. 9, 1984
and renal vein (kidney); and inferior vena cava, hepatic artery and portal vein (liver). Blood gases (pC02, P02), pH and blood pressure were monitored throughout the experiment on a Corning Blood Gas Analyzer and blood pressure, from the carotid artery, was recorded with a Statham P 23-26 pressure transducer on a D.R.-8 monitor (Electronics for Medicine, Co.). Blood from a normal dog was suppled i f needed in the surgical preparatory period to maintain blood pressure at 90-120 mmHg. At designated intervals, 3 ml samples of blood were drawn simultaneously from the various blood vessels into plastic syringes and transferred into chilled tubes containing 0.3 ml theophylline (180 mg percent) to inhibit phosphodiesterase. Following centrifugation, one ml aliqouts of plasma were deprotefnized with 2 ml absolute ethanol, centrifuged and the supernatant transferred to a vial for evaporation to dryness. The precipitates were resuspended in 1.5 ml of 0.05 M TRIS buffer, pH 7.4, and 20 ~1 aliqouts were used for cyclic AMP measurement by the protein binding assay of Brown et al. 6 One hour or longer was allowed following the cannulation proce~ur-es before 2 control samples were obtained, followed by infusion of isoproterenol (0.2 ug/kg/min) into the saphenous vein for 45 min and blood sampled at 15 min intervals. Resul ts Significant changes in plasma levels of cAMPoccurred across only two tissues, the head and kidneys. As shown in Fig. 1, the cAMP levels in the carotid artery and jugular vein in the basal state show a small contribution of cA~IP. Administration of isoproterenol causes an increase in the cAMP levels in both vessels but the V-A difference is markedly increased indicating an enhanced contribution of CAMP. By contrast the cAMP levels across the kidney (Fig. 2) show that the kidney extracts CAMPduring the basal state as well as during the isoproterenol infusion. The plasma levels of cAMP across the liver (Fig. 3) and across the lungs (Fig. 4) show no detectable differences indicating that these organs are not contributing to the circulating cAMP. Discussion The present study demonstrates that there are greater increases in cAMP concentration in the jugular vein than in the carotid artery following administration of the B-adrenergic agonist, isoproterenol. The possibility that this increment in cAMPlevels in the jugular vein might be due to diminished cerebral blood flow is highly unlikely because in numerous studies, using thermoclearance7, 8 or 85Kr clearance in dogs9, isoprotorenol was found to actually increase blood flow. I t is reasonable, therefore, to assume that isoproterenol did indeed cause an increased release of cAMP into the circulation. The relative contribution of the brain to the cAMPeffluent in the jugular vein cannot be ascertained at this time. Since the jugular vein drains tissues other than brain, their potential contribution to the cAMP levels in the jugular vein must be kept in mind. However, since the brain, pituitary and pineal body have been shown to have a particularly high specific activity of adenylate cyclase and CAMP, these tissues may be major contributors to the elevated levels of c#/~P in the jugular vein following isoproterenol administration. Furthermore, although the catecholamines do not penetrate the brain readily, they do reach the circumventricular areas of the brain, and the hypothalmus, as well as the pituitary and pineal body, thus reinforcing the importance of these tissues as sources of CAMPin the jugular vein. The present study shows that cAMP is released into the jugular vein following B-adrenergic stimulation. The extent of this contribution to the
Vol. 35, No. 9, 1984
Cyclic AMP Release
1
ISOPROTERENOL 0.2 I~g/kg/min
965
I
160
i JUGULAR VEIN
////
120
CAROTIDART.
/I
o
E
~
80
o.
g.
40 V-A 0 0
-15
15
30
45
MINUTES
FIG. 1. Plasma cAMP concentrations during infusion of isoprotereno] (0.2 ,g/kg/min) into normal dogs. Bar graphs, based on subtraction of arterial from venous levels of cAMP, depict the increment of cAMP across the head. ISOPROTERENOL 0.2 p.,g/kg/min
120
I
I
I RENAL ART.
¢.) ¢b
"
80
~ RENAL VEIN
0
E Q.
u
40
z v
i@"
"A-V
n -15
n 0
I
n 15
30
N
45
MINUTES
FIG. 2. Plasma cAMP concentrations in blood vessels across the kidney during infusion of i soproterenol into normal dogs. The cAMP in vein is lower than in artery and the discrepancy increases during infusion indicating increased uptake of cAMP by kidney. Bar graphs (Arterial-venous cAMP) show that discrepancy increases during infusion.
966
Cyclic AMP Release
I
ISOPROTERENOL
Vol. 35, No. 9, 1984
0.2p.9/kg/min
I
120 HEPATIC ART. P O R T A L V. INF. V E N A CAVA
80 o
E
Q. (L '(I U
40
I -15
0
I 0
1 15
I 30
I 45
MINUTES
Fig. 3. Lack of change in plasma concentration of cAMP across the l i v e r during isoproterenol infusion in nomal dogs.
I
ISOPROTERENOL
0.2 I~g/kg/min
t
120
"~
PULMONARY ART. LEFT ATRIUM
80
o
E
Q. (Z.
40
_ z
. =
.
.
.
.
. =
¢J
0
I -15
I
I
I
0
15
30
45
MINUTES
Fig. 4. Lack of change in the arterio-venous concentration gradient across the lung during isoproterenol infusion in normal dogs.
Vol. 35, No. 9, 1984
Cyclic AMp Release
967
overall rise in plasma CAMPwas not determined, but from the marked increase in arterial cAMPand the small increment in cAMP in the jugular vein, i t is probable that tissues other than the brain are the major contributors to the rise in plasma cAMP induced by isoproterenol. The present findings rule out the lung and l i v e r as sources of plasma cAMP, leaving skeletal muscle and vasculature as l i k e l y sources. The physiologic significance of elevations of plasma cAMP remains to be elucidated. Since cAMP does not penetrate cell membranes readily, i t is unlikely to be internalized, but conceivably i t could act as mediator or modify the effect of other neurohumoral agents through an action on the surface membrane. At any rate release of cAMP by a tissue presumably reflects activation of the adenylate cyclase system and therefore measurement of cAMP changes across such tissue during hormonal or drug treatment could serve as an indicator of the involvement of the tissue in the observed responses. In the case of the brain this approach may be applicable in studying the mode of action of certain antidepressant drugs, e.g., imipramine, which affect the function of the B-adrenergic receptors in the brain I0-14. Acknowledgements This study was supported in part by a grant from the Mental Health C1ini cal Research Center (MH35976) Department of Psychiatry, New York University Medical Center and in part by Research Scientist Development Award MHO0208 to E.F. Referen ces 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11 12. 13. 14.
J.H. BALL, N.I. KAMINSKY, J.G. HARDMAN,A.E. BROADUCS,E.W. SUTHERLAND and G.W. LIDDLE, J. Clin. Invest. 51, 2124-29 (1972). S. NISTRUP MADSEN, S. ENGKJAERCHRIS-T-ENSEN, A. PRANGEHANSENand J. THODE, Clin. Endocrinol. (Oxf) 15, 431-437 (1981). P. BARNES, G. FITZGERALD, M. BROI~-and C. DOLLERY, N. Engl. J. Med. 303, 263-267 (1980). S. NISTRUP HOLMEGAARD, Acta Endocrinologia, Supplement 290, 5-47 (1982). J. SCHULTZand G. KLEEFELD, Pharmacology 18, 162-167 (11Tl!T). B.L. BROWN,J.D.M. ALBANO, R.P. EKINS, A.M__SGHERZI and W. TAMPION, Biochem. J. 121, 561-563 (1971). P. AUBINEAU,T. SEYLAZ, R. SERCOMBEand H. MAMO, Brain Res. 61, 153-161 (1973). R. SERCOMBE, P. AUBINEAU, L. EDVINSSON, H. MAMO, C.H. OWMANand J. SEYLAZ, Pflugers Archiv. 368, 241-244 (1977). C. XANALATOSand I.M. JAME~Clin. Sci. 42, 63-68 (1972). M.S. EBADI, B. WEISS and E. COSTA, J. Ne~ochem. 18, 183-192 (1971). E.W. SUTHERLAND,J.W. RALL and T. MENON, J. B i o l . ~ e m . 237, 1220-27 (1962). F. SULSERand P.L. MOBLEY, in Neuroreceptors, Basic and Clinical Aspects. Eds. E. USDIN, W.E. BUNNEYand J.M. DAVIS. J. Wiley and Sons, Inc., 1981. J. VETULANI and F. SULSER, Nature 257, 495-496 (1975). S.P. BANERJEE, L.S. KUNG, S.J. RIGG~nd S.K. CHANDA, Nature, 268, 455-456 (1977).
Pergamon Press
INCREASED RELEASE OF CYCLIC ADENOSINE MONOPHOSPHATEINTO JUGULAR VEIN IN RESPONSE TO ISOPROTERENOLADMINISTRATION INorman Altszuler, 1,2Eitan Friedman, John C. Laschinger, Frank P. Catinella, Joseph N. Cunningham, J r . , Ira M. Nathan IDepartment of Pharmacology, 2Department of Psychiatry and Department of Surgery New York University School of Medicine New York, NY 10016 (Received in final form June 19, 1984)
Summary Cate~holamine administration elevates plasma cyclic AMP (cAMP) levels but the source of the cAMP is unknown. To determine possible sources, plasma cAMP level s were determined in blood vessels across the head, l i v e r , kidney and lung in anesthetized dogs infused with the B-adrenergicagonist, isoproterenol. Only the head showed an increased release of cAMP into the blood. The kidneys removed cAMP from the blood while l i v e r and lung showed no change. This in vivo demonstration of release of cAMP from the head represents--co~ibutions from brain and facial muscles and may be a useful approach to study brain involvement in the action of various hormones and drugs. Cyclic AMP is the putative second messenger for a number of hormones and neurotransmitters. Although cAMP is generated within the cell and is assumed to act l o c a l l y , s i g n i f i c a n t increases of cAMP levels in the plasma have been reported following administration of catecholamines 1-3. The source of the plasma cAMP has not been established despite extensive e f f o r t s 4. Since brain slices have been shown5 to increase cAMP in response to isoproterenol i t was of i n t e r e s t to determine i f such release occurred in vivo. Thus, this study was undertaken to determine i f stimulation of B - a d r ~ e r - ~ receptors would release cAMP by the brain in amounts that could be detected in vivo by sampling the jugular vein blood. Since the l a t t e r drains muscle tissues of the head as well as brain, a comparison was made with concentrations of cAMP in the blood across the lungs, l i v e r and kidneys. Only the head showed an increased release of cAMP, thus suggesting a potential usefulness for such specific regional sampling to study the responsiveness of B-adrenergic receptors. Methods Arterio-venous (A-V) concentrations of cyclic AMP were determined across the brain, lungs, l i v e r and kidney in 8 normal postabsorptive dogs. The dogs were anesthetized with chloralose, i00 mg/kg, i v , the skeletal muscles relaxed with metocurine iodide 2 mg/dog, and respiration was maintained by a respiratory pump. Catheters were inserted d i r e c t l y into the following blood vessels in close proximity to the tissues of interest: carotid artery, and jugular vein (brain); pulmonary artery and l e f t atrium (lung); renal artery 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
964
Cyclic AMp Release
Vol. 35, No. 9, 1984
and renal vein (kidney); and inferior vena cava, hepatic artery and portal vein (liver). Blood gases (pC02, P02), pH and blood pressure were monitored throughout the experiment on a Corning Blood Gas Analyzer and blood pressure, from the carotid artery, was recorded with a Statham P 23-26 pressure transducer on a D.R.-8 monitor (Electronics for Medicine, Co.). Blood from a normal dog was suppled i f needed in the surgical preparatory period to maintain blood pressure at 90-120 mmHg. At designated intervals, 3 ml samples of blood were drawn simultaneously from the various blood vessels into plastic syringes and transferred into chilled tubes containing 0.3 ml theophylline (180 mg percent) to inhibit phosphodiesterase. Following centrifugation, one ml aliqouts of plasma were deprotefnized with 2 ml absolute ethanol, centrifuged and the supernatant transferred to a vial for evaporation to dryness. The precipitates were resuspended in 1.5 ml of 0.05 M TRIS buffer, pH 7.4, and 20 ~1 aliqouts were used for cyclic AMP measurement by the protein binding assay of Brown et al. 6 One hour or longer was allowed following the cannulation proce~ur-es before 2 control samples were obtained, followed by infusion of isoproterenol (0.2 ug/kg/min) into the saphenous vein for 45 min and blood sampled at 15 min intervals. Resul ts Significant changes in plasma levels of cAMPoccurred across only two tissues, the head and kidneys. As shown in Fig. 1, the cAMP levels in the carotid artery and jugular vein in the basal state show a small contribution of cA~IP. Administration of isoproterenol causes an increase in the cAMP levels in both vessels but the V-A difference is markedly increased indicating an enhanced contribution of CAMP. By contrast the cAMP levels across the kidney (Fig. 2) show that the kidney extracts CAMPduring the basal state as well as during the isoproterenol infusion. The plasma levels of cAMP across the liver (Fig. 3) and across the lungs (Fig. 4) show no detectable differences indicating that these organs are not contributing to the circulating cAMP. Discussion The present study demonstrates that there are greater increases in cAMP concentration in the jugular vein than in the carotid artery following administration of the B-adrenergic agonist, isoproterenol. The possibility that this increment in cAMPlevels in the jugular vein might be due to diminished cerebral blood flow is highly unlikely because in numerous studies, using thermoclearance7, 8 or 85Kr clearance in dogs9, isoprotorenol was found to actually increase blood flow. I t is reasonable, therefore, to assume that isoproterenol did indeed cause an increased release of cAMP into the circulation. The relative contribution of the brain to the cAMPeffluent in the jugular vein cannot be ascertained at this time. Since the jugular vein drains tissues other than brain, their potential contribution to the cAMP levels in the jugular vein must be kept in mind. However, since the brain, pituitary and pineal body have been shown to have a particularly high specific activity of adenylate cyclase and CAMP, these tissues may be major contributors to the elevated levels of c#/~P in the jugular vein following isoproterenol administration. Furthermore, although the catecholamines do not penetrate the brain readily, they do reach the circumventricular areas of the brain, and the hypothalmus, as well as the pituitary and pineal body, thus reinforcing the importance of these tissues as sources of CAMPin the jugular vein. The present study shows that cAMP is released into the jugular vein following B-adrenergic stimulation. The extent of this contribution to the
Vol. 35, No. 9, 1984
Cyclic AMP Release
1
ISOPROTERENOL 0.2 I~g/kg/min
965
I
160
i JUGULAR VEIN
////
120
CAROTIDART.
/I
o
E
~
80
o.
g.
40 V-A 0 0
-15
15
30
45
MINUTES
FIG. 1. Plasma cAMP concentrations during infusion of isoprotereno] (0.2 ,g/kg/min) into normal dogs. Bar graphs, based on subtraction of arterial from venous levels of cAMP, depict the increment of cAMP across the head. ISOPROTERENOL 0.2 p.,g/kg/min
120
I
I
I RENAL ART.
¢.) ¢b
"
80
~ RENAL VEIN
0
E Q.
u
40
z v
i@"
"A-V
n -15
n 0
I
n 15
30
N
45
MINUTES
FIG. 2. Plasma cAMP concentrations in blood vessels across the kidney during infusion of i soproterenol into normal dogs. The cAMP in vein is lower than in artery and the discrepancy increases during infusion indicating increased uptake of cAMP by kidney. Bar graphs (Arterial-venous cAMP) show that discrepancy increases during infusion.
966
Cyclic AMP Release
I
ISOPROTERENOL
Vol. 35, No. 9, 1984
0.2p.9/kg/min
I
120 HEPATIC ART. P O R T A L V. INF. V E N A CAVA
80 o
E
Q. (L '(I U
40
I -15
0
I 0
1 15
I 30
I 45
MINUTES
Fig. 3. Lack of change in plasma concentration of cAMP across the l i v e r during isoproterenol infusion in nomal dogs.
I
ISOPROTERENOL
0.2 I~g/kg/min
t
120
"~
PULMONARY ART. LEFT ATRIUM
80
o
E
Q. (Z.
40
_ z
. =
.
.
.
.
. =
¢J
0
I -15
I
I
I
0
15
30
45
MINUTES
Fig. 4. Lack of change in the arterio-venous concentration gradient across the lung during isoproterenol infusion in normal dogs.
Vol. 35, No. 9, 1984
Cyclic AMp Release
967
overall rise in plasma CAMPwas not determined, but from the marked increase in arterial cAMPand the small increment in cAMP in the jugular vein, i t is probable that tissues other than the brain are the major contributors to the rise in plasma cAMP induced by isoproterenol. The present findings rule out the lung and l i v e r as sources of plasma cAMP, leaving skeletal muscle and vasculature as l i k e l y sources. The physiologic significance of elevations of plasma cAMP remains to be elucidated. Since cAMP does not penetrate cell membranes readily, i t is unlikely to be internalized, but conceivably i t could act as mediator or modify the effect of other neurohumoral agents through an action on the surface membrane. At any rate release of cAMP by a tissue presumably reflects activation of the adenylate cyclase system and therefore measurement of cAMP changes across such tissue during hormonal or drug treatment could serve as an indicator of the involvement of the tissue in the observed responses. In the case of the brain this approach may be applicable in studying the mode of action of certain antidepressant drugs, e.g., imipramine, which affect the function of the B-adrenergic receptors in the brain I0-14. Acknowledgements This study was supported in part by a grant from the Mental Health C1ini cal Research Center (MH35976) Department of Psychiatry, New York University Medical Center and in part by Research Scientist Development Award MHO0208 to E.F. Referen ces 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11 12. 13. 14.
J.H. BALL, N.I. KAMINSKY, J.G. HARDMAN,A.E. BROADUCS,E.W. SUTHERLAND and G.W. LIDDLE, J. Clin. Invest. 51, 2124-29 (1972). S. NISTRUP MADSEN, S. ENGKJAERCHRIS-T-ENSEN, A. PRANGEHANSENand J. THODE, Clin. Endocrinol. (Oxf) 15, 431-437 (1981). P. BARNES, G. FITZGERALD, M. BROI~-and C. DOLLERY, N. Engl. J. Med. 303, 263-267 (1980). S. NISTRUP HOLMEGAARD, Acta Endocrinologia, Supplement 290, 5-47 (1982). J. SCHULTZand G. KLEEFELD, Pharmacology 18, 162-167 (11Tl!T). B.L. BROWN,J.D.M. ALBANO, R.P. EKINS, A.M__SGHERZI and W. TAMPION, Biochem. J. 121, 561-563 (1971). P. AUBINEAU,T. SEYLAZ, R. SERCOMBEand H. MAMO, Brain Res. 61, 153-161 (1973). R. SERCOMBE, P. AUBINEAU, L. EDVINSSON, H. MAMO, C.H. OWMANand J. SEYLAZ, Pflugers Archiv. 368, 241-244 (1977). C. XANALATOSand I.M. JAME~Clin. Sci. 42, 63-68 (1972). M.S. EBADI, B. WEISS and E. COSTA, J. Ne~ochem. 18, 183-192 (1971). E.W. SUTHERLAND,J.W. RALL and T. MENON, J. B i o l . ~ e m . 237, 1220-27 (1962). F. SULSERand P.L. MOBLEY, in Neuroreceptors, Basic and Clinical Aspects. Eds. E. USDIN, W.E. BUNNEYand J.M. DAVIS. J. Wiley and Sons, Inc., 1981. J. VETULANI and F. SULSER, Nature 257, 495-496 (1975). S.P. BANERJEE, L.S. KUNG, S.J. RIGG~nd S.K. CHANDA, Nature, 268, 455-456 (1977).