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Chronic cocaine administration decreases norepinephrine-induced phosphoinositide hydrolysis in rat aorta
Life Sciences, Vol. P r i n t e d in the USA
51, pp. 1675-1681
Pergamon P r e s s
CHRONIC COCAINE ADMINISTRATION DECREASES NOREPINEPHRINE. INDUCED PHOSPHOINOSITIDE HYDROLYSIS IN RAT AORTA James H. Zavecz and William MeD. Anderson Department of Pharmacology, Louisiana State University Medical Center, and the Medical Service, Overton Brooks Veterans Affairs Medical Center, Shreveport, LA. 71130 (Received in final form September 18, 1992)
Summary The effect of chronic cocaine administration on norepinephrine stimulated hydrolysis of inositol 1,4,5-trisphosphate from the membrane phosphatidylinositol phosphate pool in isolated rat aorta was investigated. Rats received saline (controls), or 10 or 20 mg/kg cocaine once a day for 15 days. This treatment resulted in a dose-dependent reduction in norepinephrine (0.36 /zM) stimulated phosphoinositide hydrolysis. The effect of acute cocaine was determined by adding 30/~M cocaine to the in vitro incubation solution. When aortas were exposed to cocaine and norepinephrine simultaneously, in vitro, inositol phosphate formation doubled. By itself, cocaine did not affect phosphoinositide hydrolysis. Contraction of aortic helical strips by norepinephrine decreased in tissues from rats chronically treated with 20 mg/kg cocaine. In vitro cocaine shifted the norepinephrine concentration/response curve to the left and increased the maximum response. Neither acute nor chronic cocaine treatment affected prazosin's apparent dissociation constant, suggesting that cocaine did not affect receptor affinity. These data suggest that chronic, but not acute cocaine administration may interfere with pharmacomechanical coupling in rat aorta.
In vascular smooth muscle, contraction occurs as the result of norepinephrine binding to a-adrenoceptors with subsequent formation of the putative second messenger, inositol 1,4,5trisphosphate (InsP3) (1-3). InsP 3 releases Ca 2÷ from the sarcoplasmic reticulum in vascular smooth muscle at a rate that is consistent with a role as a physiological second messenger, coupling chemical (neurotransmitter) stimulation with mechanical activation (4,5). Binding of InsP 3 to its receptors results in the opening of the Ca 2+ release channels of the sarcoplasmic reticulum (6), with subsequent activation of the myofilaments. Engelking and co-workers (7) have shown that cocaine interferes with the non-cyclic AMP pathway for epiCorresponding author: J.H. Zavecz, Ph.D., Department of Pharmacology, LSU Medical Center, P.O. Box 33932, Shreveport, LA 71130-3932.
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1676
Cocaine and Phosphoinositide
Hydrolysis
Vol. 51, No. 21, 1992
nephrine-induced intracellular Ca 2÷ mobilization in hepatocytes. This effect of cocaine was not associated with a change in a-receptor density or affinity, suggesting a possible effect on InsP3-induced Ca 2÷ mobilization. We have investigated whether chronic treatment with cocaine interferes with norepinephrine-induced phosphoinositide hydrolysis in rat aorta, and whether the changes mirror alterations in the contractile response of the aorta to norepinephrine. Methods Male Fischer strain 344 rats (= 250 g) were given intraperitoneal injections of either cocaine (10 or 20 mg/kg) or saline (0.3 ml) for 15 days. The animal was sacrificed 1 hr after the fifteenth injection, and the thoracic aorta was excised and cut into 4 pieces of equal dimensions after the removal of extraneous tissue. The phosphoinositide pool was labeled by incubating each tissue for 3 hr in 1 ml of Krebs-Henseleit buffer containing 8 ~Ci [3H]inositol. The buffer (pH 7.4) was continuously aerated with 95% O~-5% CO 2, and was maintained at 37°C. Following the 3 hr incorporation period, each tissue was washed in Krebs-Henseleit, and placed into 1 ml of buffer containing 10 mM LiCk After 15 min, norepinephrine was added at a concentration which produced a maximum contractile effect in controls (0.36 ~M). Tissues were exposed to norepinephrine for 1 hr with continuous aeration of the incubation mixture. In the experiments that examined the effect of acute, in vitro cocaine, the procedure was identical with the exception that 30 ~M cocaine was added to each tube 15 min prior to adding norepinephrine. In experiments examining background phosphoinositide hydrolysis, the protocol was the same, but no drugs were added. At the end of the 1 hr experimental period, the tissues were frozen in liquid nitrogen. The 4 frozen tissues from a single aorta were pooled and ground to a fine powder in a Bessmer pulverizer. Phosphoinositides and inositol phosphates were extracted with 3 ml chloroform/methanol (1:2 v/v) with 0.1% HC1 overnight at 5°C. An additional 1 ml chloroform and 2 ml water were added, and the sample was centrifuged for 15 rain at 4°C to separate the phases. The aqueous phase containing the free intracellular inositol phosphates and inositol was neutralized with potassium hydroxide and placed on a 1.5 cm column of Dowex 1 x 8 anion exchange resin (400 mesh in the formate form). In some experiments only the inositol phosphates were extracted with 10% trichloroacetic acid. No differences in controls were observed using either extraction procedure. The inositol phosphates were eluted from the column by sequentially washing the column with ammonium formate of varying ionic strengths. Free inositol was eluted with 40 ml of water; inositol monophosphate (InsP,) with 20 ml of 0.2 M ammonium formate/0.1 M formic acid; inositol bisphosphate (InsP2) with 20 ml of 0.5 M ammonium formate/0.1 M formic acid; and InsP 3 with 20 ml of 1 M ammonium formate/formic acid. Radioactivity in each 2 ml fraction collected off the column was determined by liquid scintillation spectrometry. Total [3H]inositol phosphates (InsPl + InsP2 + InsP3) were used as the index of norepinephrine-induced phosphoinositide hydrolysis. The effect of cocaine on norepinephrine-induced contraction of the rat aorta was examined in helical strips cut from the thoracic aorta. After thoroughly removing all adhering tissue, the aorta was cut into a helical strip 2-3 m m wide and 2-3 cm long. One end of the strip was tied to a support and placed into a 35 ml organ bath containing Krebs-Henseleit solution (pH 7.4) at 37°C. The contents of the bath were continuously bubbled with 95% Oz5% CO 2. The other end of the tissue was attached to a force transducer (Grass model FI'03C) via a length of silk thread. After 30 rain, 200 mg resting tension was applied to the
Vol. 51, No. 21, 1992
Cocaine and Phosphoinositide
Hydrolysis
1677
strip, and the muscle was allowed to rest for another 90 min, readjusting the resting tension as needed. The Krebs-Henseleit was changed at least every 30 min. Concentration response curves for norepinephrine were generated in a non-cumulative manner with at least 30 min between drug challenges, washing the tissues at least every 10 min. Norepinephrine was given in random order (1 nM - 1.8 I~M). Only one concentration/response curve was obtained from each aorta. The apparent dissociation constant (KB) for prazosin was determined in aortas from control and chronically treated rats, as well as aortas exposed to 30 uM cocaine acutely in vitro. Norepinephrine concentration/response curves were constructed in the absence and presence of prazosin (0.1 - 3 nM). Tissues were equilibrated with prazosin for at least 30 minutes prior to exposing them to norepinephrine. K B values were calculated as previously described (8). Cocaine hydrochloride and (_+)norepinephrine bitartrate were purchased from Sigma Chemical Co., St. Louis, MO.
Results Figures 1 and 2 illustrate the effect of acute and chronic cocaine, respectively, on norepinephrine-induced [3H]inositol phosphate formation from the membrane phosphoinositide pool. Figure 1 shows that norepinephrine increased phosphoinositide hydrolysis above the background level, and that the presence of 30 /~M cocaine in the incubation medium augmented the effect of norepinephrine. Increasing the concentration of cocaine above 30/~M was without additional effect. In contrast, chronic administration of 10 or 20 mg/kg cocaine to rats for 15 days produced a decreased response to norepinephrine (Figure 2). 0.5
h_
o
~
--
o
**
ffl w 0.4
o o n- z >212 I
I---I Background r~\,~ NEpi, 0.36 pJ~ real NEpi, 0.36/4M Cocarne, 30/~M via Coca|ne, 30 p.M
O.3
D_
~
m
< z
0 212
0.2
__0_
0.1 i
i
b_ 0.0
FIG. 1 Effect of in vitro cocaine on norepinephrine-inducedinositol phosphate formation. Bars represent the mean -+ SE (n=5) fractional hydrolysis of the membrane [3H]phosphoinositide pool to [3H]inositol phosphates under each of the conditions indicated. *Significantly greater than background, P<0.005. **Significantly greater than norepinephrine, P<0.05.
1678
Cocaine and Phosphoinositide
Hydrolysis
Vol. 51, No. 21, 1992
The results presented in figures 1 and 2 reflect differences in the effect of norepinephrine, not different amounts of [3H]inositol incorporated into phosphoinositides during the labeling period, since there were no statistically significant differences in total [3H]phosphoinositides among the groups. The effect of cocaine on norepinephrine-induced
dpm/mg 1750 1500 1250 1000 750
i"
500 250 0
CONTROL
10
20
COCAINE ( m g / k g ) FIG. 2 Effect of chronic cocaine treatment on norepinephrine-induced [3H]inositol phosphate formation. Bars represent the mean -+ SE (n = 3-5) [3H]inositol phosphates measured after 1 hr exposure to 0.36 I.tM norepinephrine. Controls were injected with 0.3 ml saline on the same dosing regimen as the cocaine-treated rats. *Significantly less than control, P<0.05. **Significantly less than control, P<0.0005, and significantly less than 10 mg/kg cocaine, P<0.005.
phosphoinositide hydrolysis was paralleled by the action of cocaine on the contractile response of aortic strips to norepinephrine. Figure 3 compares the effect of 30/~M cocaine in the tissue bath during the norepinephrine concentration/response experiments with the effect of 20 mg/kg cocaine administered once-a-day for 15 days. Chronic cocaine administration resulted in a reduced response to norepinephrine relative to control, whereas the presence of 30 ~M cocaine shifted the concentration/response curve to the left and increased the maximum response observed. In order to determine if our results with chronic cocaine administration were the result of changes in cq-adrenoceptor affinity, the apparent dissociation constant (K~) for the selective al-adrenoceptor antagonist, prazosin, was calculated for control tissues with and without 30 /~M cocaine present and in aortas from animals given 20 mg/kg cocaine for 15 days. Table 1 shows that there were no differences among the groups. The values for prazosin's K B are similar to those reported by others in ligand-receptor binding experiments (9).
Vol. 51, No. 21, 1992
Table 1.
Cocaine and Phosphoinositide
Hydrolysis
Prazosin affinity constants (KB) in control, acute and chronic cocaine tissues. Condition
K s (nM)
Control
0.14 _+ 0.04
Acute Cocaine (30 pM)
0.16 _+ 0.02
Chronic Cocaine (20 mg/kg)
0.17 + 0.02
f. -C ron,c eo,
~ 1751- • [ •
125
1679
• Control
• Cocaine (30 ~M)
•
T
1oo f 7~ 75 n
• "
25
0
*
o001
o01
.1
1
10
NOREPINEPHRINE (p,M) FIG. 3 Effect of in vitro cocaine (30 pM) and chronic cocaine administration (20 mg/kg) on norepinephrine-induced contraction of the rat thoracic aorta. Points are the mean +_ SE of 4-8 experiments, and represent the percentage of the maximum response to norepinephrine observed in controls. *Significantly different from control, P<0.05.
Discussion Chronic cocaine administration diminished norepinephrine-induced phosphoinositide hydrolysis in the rat thoracic aorta. Norepinephrine-stimulated inositol phosphate accumulation was reduced up to 55% by chronic treatment with 10 or 20 mg/kg cocaine injected once per day (Figure 2). On the other hand, when cocaine was present along with norepinephrine in the buffer solution bathing aortas from control rats, there was an increase in inositol phosphates (Figure 1). When contractile studies were performed under similar conditions, chronic cocaine administration resulted in reduced responses to norepinephrine. When cocaine was present in the bath (acute cocaine), norepinephrine induced greater contractions at each point on the norepinephrine concentration-response curve (Figure 3). The augmentation of the response to norepinephrine on both phosphoinositide hydrolysis and the parallel increase in developed tension can be explained by the known ability of cocaine
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Vol. 51, No. 21, 1992
to potentiate sympathetic responses by interfering with uptake of norepinephrine. This also suggests that the decreased responsiveness to norepinephrine following chronic cocaine administration was not due to cocaine's local anesthetic properties, since the decreased response was not an acute effect with cocaine. The mechanism for the effect of chronic cocaine administration may be explained in a number of ways, for example, desensitization of a~-receptors; a change in their affinity; or a change in pharmacomechanical coupling. Desensitization to the effect of norepinephrine may have occurred because of the blockade of reuptake. In support of this is the finding of Avakian and coworkers (10), who observed less epinephrine-induced tachycardia in isolated, perfused hearts from rats chronically treated with cocaine. Although one study has reported an increase in central ~,and B-receptor binding sites following chronic treatment with cocaine (11), others, using similar doses of cocaine, observed slight decreases (12). While a decrease in receptor density could explain the decreased contractions observed after chronic cocaine administration, altered receptor density may not be the sole mechanism for the actions of cocaine. Although a change in the affinity of norepinephrine for c~-receptors could explain the shift of the norepinephrine concentration/response curve to the right in aortas from chronically treated animals, altered affinity would not explain the decrease in the maximum response to norepinephrine. Furthermore, the data in table 1 showing a lack of effect of cocaine on the affinity constant of prazosin for cq-adrenoceptors strongly suggest that this is not a mechanism. Decreased contractile responses and decreased phosphoinositide hydrolysis might be explained by the direct local anesthetic action of cocaine. This seems unlikely because acute cocaine potentiated both the contraction and the formation of inositol phosphates (Figure 1) and had no effect on phosphoinositide hydrolysis by itself (Figure 1) at concentrations that block Na ÷ channels (13). Egashira and colleagues (14) have reported that cocaine can depress aortic smooth muscle by decreasing the sensitivity of the contractile proteins to Ca2÷. However, a concentration of cocaine _> 1 mM was required to produce this action. This level of cocaine is not achieved following 20 mg/kg cocaine (15). Another potential mechanism for the effect of chronic cocaine administration on norepinephrine-induced phosphoinositide hydrolysis is the interruption ofpharmacomechanical coupling (the coupling of neurotransmitter initiated events to the mechanical response). Nestler and colleagues (16) have demonstrated that chronic treatment of rats with twice daily 15 mg/kg doses of cocaine for 14 days produced a significant decrease in G-protein subunits in three different brain regions. Since c~-receptor activated phosphoinositide hydrolysis by phospholipase C has been shown to be G-protein dependent (17), a decrease in the amount of the signal transducing G-protein could lead to decreased phospholipase C activation and subsequent diminished phosphoinositide hydrolysis. The data presented suggest that chronic cocaine administration does not alter cq-receptor affinity but do not distinguish between any of the other potential mechanisms.
Acknowledgements This work was supported by grants from the American Heart Association, Louisiana Affiliate, and the Department of Veterans Affairs. The authors thank Barbara Roggero for technical assistance.
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Hydrolysis
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References .
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
M.D. CAMPBELL, R.C. DETH, R.A. PAYNE, and T.W. HONEYMAN, Eur. J. Pharmacol. 116, 129-136 (1985). V. PIJUAN and I. LITOSCH, Biochem. Biophys. Res. Comm. 15__66,240-245 (1988). R.M. RAPOPORT, J. Pharmacol. Exp. Ther. 242, 188-194 (1987). A.V. SOMLYO, M. BOND, A.P. SOMLYO, and A. SCARPA, Proc. Natn. Acad. Sci. (USA) 82, 5231-5235 (1985). J.W. WALKER, A.V. SOMLYO, Y.E. GOLDMAN, A.P. SOMLYO, and D.R. TRENTHAM, Nature 32__!,249-252 (1987). B.E. EHRLICH and J. WATRAS, Nature 336, 583-586 (1988). L.R. ENGELKING, M.S. ANWER, J. MCCONNELL, D. SULLIVAN, and L. SHUSTER, Pharmacology 40, 129-136 (1990). J.H. ZAVECZ and T.O. YELLIN, J. Pharmacol. Exp. Ther. 223, 177-182 (1982). S. YAMADA, H.I. YAMAMURA and W.R. ROESKE, J. Pharmacol. Exp. Ther. 215, 176-185 (1980). E.V. AVAKIAN, M. LEROY, K. ST. JOHN-ALLAN, F.W. SAYRE, and M.H. MALONE, Life Sci. 46, 1569-1574 (1990). S.P. BANERJEE, V.K. SHARMA, L.S. KUNG-CHEUNG, S.K. CHANDA, and S.J. RIGGI, Brain Res. 175, 119-130 (1979). M.M. SELLINGER-BARNETYE, J. MENDELS, and A. FRAZER, Neuropharmacol. 1__99,447-454 (1980). W.J. CRUMB, Jr. and C.W. CLARKSON, Biophys. J. 57, 589-599 (1990). K. EGASHIRA, K.G. MORGAN and J.P. MORGAN, J. Clin. Invest. 87, 1322-1328 (1991). A.L. MISRA, Cocaine: Chemical, Biological, Clinical, Social and Treatment Aspects. S.J. Mule (ed), 72-90, CRC Press, Cleveland (1976). E.J. NESTLER, R.Z. TERWILLIGER, J.R. WALKER, K.A. SEVARINO, and R.S. DUMAN, J. Neurochem. 55, 1079-1082 (1990). K.M. WILSON and K.P. MINNEMAN, Mol. Pharmacol. 38, 274-281 (1990).
51, pp. 1675-1681
Pergamon P r e s s
CHRONIC COCAINE ADMINISTRATION DECREASES NOREPINEPHRINE. INDUCED PHOSPHOINOSITIDE HYDROLYSIS IN RAT AORTA James H. Zavecz and William MeD. Anderson Department of Pharmacology, Louisiana State University Medical Center, and the Medical Service, Overton Brooks Veterans Affairs Medical Center, Shreveport, LA. 71130 (Received in final form September 18, 1992)
Summary The effect of chronic cocaine administration on norepinephrine stimulated hydrolysis of inositol 1,4,5-trisphosphate from the membrane phosphatidylinositol phosphate pool in isolated rat aorta was investigated. Rats received saline (controls), or 10 or 20 mg/kg cocaine once a day for 15 days. This treatment resulted in a dose-dependent reduction in norepinephrine (0.36 /zM) stimulated phosphoinositide hydrolysis. The effect of acute cocaine was determined by adding 30/~M cocaine to the in vitro incubation solution. When aortas were exposed to cocaine and norepinephrine simultaneously, in vitro, inositol phosphate formation doubled. By itself, cocaine did not affect phosphoinositide hydrolysis. Contraction of aortic helical strips by norepinephrine decreased in tissues from rats chronically treated with 20 mg/kg cocaine. In vitro cocaine shifted the norepinephrine concentration/response curve to the left and increased the maximum response. Neither acute nor chronic cocaine treatment affected prazosin's apparent dissociation constant, suggesting that cocaine did not affect receptor affinity. These data suggest that chronic, but not acute cocaine administration may interfere with pharmacomechanical coupling in rat aorta.
In vascular smooth muscle, contraction occurs as the result of norepinephrine binding to a-adrenoceptors with subsequent formation of the putative second messenger, inositol 1,4,5trisphosphate (InsP3) (1-3). InsP 3 releases Ca 2÷ from the sarcoplasmic reticulum in vascular smooth muscle at a rate that is consistent with a role as a physiological second messenger, coupling chemical (neurotransmitter) stimulation with mechanical activation (4,5). Binding of InsP 3 to its receptors results in the opening of the Ca 2+ release channels of the sarcoplasmic reticulum (6), with subsequent activation of the myofilaments. Engelking and co-workers (7) have shown that cocaine interferes with the non-cyclic AMP pathway for epiCorresponding author: J.H. Zavecz, Ph.D., Department of Pharmacology, LSU Medical Center, P.O. Box 33932, Shreveport, LA 71130-3932.
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1676
Cocaine and Phosphoinositide
Hydrolysis
Vol. 51, No. 21, 1992
nephrine-induced intracellular Ca 2÷ mobilization in hepatocytes. This effect of cocaine was not associated with a change in a-receptor density or affinity, suggesting a possible effect on InsP3-induced Ca 2÷ mobilization. We have investigated whether chronic treatment with cocaine interferes with norepinephrine-induced phosphoinositide hydrolysis in rat aorta, and whether the changes mirror alterations in the contractile response of the aorta to norepinephrine. Methods Male Fischer strain 344 rats (= 250 g) were given intraperitoneal injections of either cocaine (10 or 20 mg/kg) or saline (0.3 ml) for 15 days. The animal was sacrificed 1 hr after the fifteenth injection, and the thoracic aorta was excised and cut into 4 pieces of equal dimensions after the removal of extraneous tissue. The phosphoinositide pool was labeled by incubating each tissue for 3 hr in 1 ml of Krebs-Henseleit buffer containing 8 ~Ci [3H]inositol. The buffer (pH 7.4) was continuously aerated with 95% O~-5% CO 2, and was maintained at 37°C. Following the 3 hr incorporation period, each tissue was washed in Krebs-Henseleit, and placed into 1 ml of buffer containing 10 mM LiCk After 15 min, norepinephrine was added at a concentration which produced a maximum contractile effect in controls (0.36 ~M). Tissues were exposed to norepinephrine for 1 hr with continuous aeration of the incubation mixture. In the experiments that examined the effect of acute, in vitro cocaine, the procedure was identical with the exception that 30 ~M cocaine was added to each tube 15 min prior to adding norepinephrine. In experiments examining background phosphoinositide hydrolysis, the protocol was the same, but no drugs were added. At the end of the 1 hr experimental period, the tissues were frozen in liquid nitrogen. The 4 frozen tissues from a single aorta were pooled and ground to a fine powder in a Bessmer pulverizer. Phosphoinositides and inositol phosphates were extracted with 3 ml chloroform/methanol (1:2 v/v) with 0.1% HC1 overnight at 5°C. An additional 1 ml chloroform and 2 ml water were added, and the sample was centrifuged for 15 rain at 4°C to separate the phases. The aqueous phase containing the free intracellular inositol phosphates and inositol was neutralized with potassium hydroxide and placed on a 1.5 cm column of Dowex 1 x 8 anion exchange resin (400 mesh in the formate form). In some experiments only the inositol phosphates were extracted with 10% trichloroacetic acid. No differences in controls were observed using either extraction procedure. The inositol phosphates were eluted from the column by sequentially washing the column with ammonium formate of varying ionic strengths. Free inositol was eluted with 40 ml of water; inositol monophosphate (InsP,) with 20 ml of 0.2 M ammonium formate/0.1 M formic acid; inositol bisphosphate (InsP2) with 20 ml of 0.5 M ammonium formate/0.1 M formic acid; and InsP 3 with 20 ml of 1 M ammonium formate/formic acid. Radioactivity in each 2 ml fraction collected off the column was determined by liquid scintillation spectrometry. Total [3H]inositol phosphates (InsPl + InsP2 + InsP3) were used as the index of norepinephrine-induced phosphoinositide hydrolysis. The effect of cocaine on norepinephrine-induced contraction of the rat aorta was examined in helical strips cut from the thoracic aorta. After thoroughly removing all adhering tissue, the aorta was cut into a helical strip 2-3 m m wide and 2-3 cm long. One end of the strip was tied to a support and placed into a 35 ml organ bath containing Krebs-Henseleit solution (pH 7.4) at 37°C. The contents of the bath were continuously bubbled with 95% Oz5% CO 2. The other end of the tissue was attached to a force transducer (Grass model FI'03C) via a length of silk thread. After 30 rain, 200 mg resting tension was applied to the
Vol. 51, No. 21, 1992
Cocaine and Phosphoinositide
Hydrolysis
1677
strip, and the muscle was allowed to rest for another 90 min, readjusting the resting tension as needed. The Krebs-Henseleit was changed at least every 30 min. Concentration response curves for norepinephrine were generated in a non-cumulative manner with at least 30 min between drug challenges, washing the tissues at least every 10 min. Norepinephrine was given in random order (1 nM - 1.8 I~M). Only one concentration/response curve was obtained from each aorta. The apparent dissociation constant (KB) for prazosin was determined in aortas from control and chronically treated rats, as well as aortas exposed to 30 uM cocaine acutely in vitro. Norepinephrine concentration/response curves were constructed in the absence and presence of prazosin (0.1 - 3 nM). Tissues were equilibrated with prazosin for at least 30 minutes prior to exposing them to norepinephrine. K B values were calculated as previously described (8). Cocaine hydrochloride and (_+)norepinephrine bitartrate were purchased from Sigma Chemical Co., St. Louis, MO.
Results Figures 1 and 2 illustrate the effect of acute and chronic cocaine, respectively, on norepinephrine-induced [3H]inositol phosphate formation from the membrane phosphoinositide pool. Figure 1 shows that norepinephrine increased phosphoinositide hydrolysis above the background level, and that the presence of 30 /~M cocaine in the incubation medium augmented the effect of norepinephrine. Increasing the concentration of cocaine above 30/~M was without additional effect. In contrast, chronic administration of 10 or 20 mg/kg cocaine to rats for 15 days produced a decreased response to norepinephrine (Figure 2). 0.5
h_
o
~
--
o
**
ffl w 0.4
o o n- z >212 I
I---I Background r~\,~ NEpi, 0.36 pJ~ real NEpi, 0.36/4M Cocarne, 30/~M via Coca|ne, 30 p.M
O.3
D_
~
m
< z
0 212
0.2
__0_
0.1 i
i
b_ 0.0
FIG. 1 Effect of in vitro cocaine on norepinephrine-inducedinositol phosphate formation. Bars represent the mean -+ SE (n=5) fractional hydrolysis of the membrane [3H]phosphoinositide pool to [3H]inositol phosphates under each of the conditions indicated. *Significantly greater than background, P<0.005. **Significantly greater than norepinephrine, P<0.05.
1678
Cocaine and Phosphoinositide
Hydrolysis
Vol. 51, No. 21, 1992
The results presented in figures 1 and 2 reflect differences in the effect of norepinephrine, not different amounts of [3H]inositol incorporated into phosphoinositides during the labeling period, since there were no statistically significant differences in total [3H]phosphoinositides among the groups. The effect of cocaine on norepinephrine-induced
dpm/mg 1750 1500 1250 1000 750
i"
500 250 0
CONTROL
10
20
COCAINE ( m g / k g ) FIG. 2 Effect of chronic cocaine treatment on norepinephrine-induced [3H]inositol phosphate formation. Bars represent the mean -+ SE (n = 3-5) [3H]inositol phosphates measured after 1 hr exposure to 0.36 I.tM norepinephrine. Controls were injected with 0.3 ml saline on the same dosing regimen as the cocaine-treated rats. *Significantly less than control, P<0.05. **Significantly less than control, P<0.0005, and significantly less than 10 mg/kg cocaine, P<0.005.
phosphoinositide hydrolysis was paralleled by the action of cocaine on the contractile response of aortic strips to norepinephrine. Figure 3 compares the effect of 30/~M cocaine in the tissue bath during the norepinephrine concentration/response experiments with the effect of 20 mg/kg cocaine administered once-a-day for 15 days. Chronic cocaine administration resulted in a reduced response to norepinephrine relative to control, whereas the presence of 30 ~M cocaine shifted the concentration/response curve to the left and increased the maximum response observed. In order to determine if our results with chronic cocaine administration were the result of changes in cq-adrenoceptor affinity, the apparent dissociation constant (K~) for the selective al-adrenoceptor antagonist, prazosin, was calculated for control tissues with and without 30 /~M cocaine present and in aortas from animals given 20 mg/kg cocaine for 15 days. Table 1 shows that there were no differences among the groups. The values for prazosin's K B are similar to those reported by others in ligand-receptor binding experiments (9).
Vol. 51, No. 21, 1992
Table 1.
Cocaine and Phosphoinositide
Hydrolysis
Prazosin affinity constants (KB) in control, acute and chronic cocaine tissues. Condition
K s (nM)
Control
0.14 _+ 0.04
Acute Cocaine (30 pM)
0.16 _+ 0.02
Chronic Cocaine (20 mg/kg)
0.17 + 0.02
f. -C ron,c eo,
~ 1751- • [ •
125
1679
• Control
• Cocaine (30 ~M)
•
T
1oo f 7~ 75 n
• "
25
0
*
o001
o01
.1
1
10
NOREPINEPHRINE (p,M) FIG. 3 Effect of in vitro cocaine (30 pM) and chronic cocaine administration (20 mg/kg) on norepinephrine-induced contraction of the rat thoracic aorta. Points are the mean +_ SE of 4-8 experiments, and represent the percentage of the maximum response to norepinephrine observed in controls. *Significantly different from control, P<0.05.
Discussion Chronic cocaine administration diminished norepinephrine-induced phosphoinositide hydrolysis in the rat thoracic aorta. Norepinephrine-stimulated inositol phosphate accumulation was reduced up to 55% by chronic treatment with 10 or 20 mg/kg cocaine injected once per day (Figure 2). On the other hand, when cocaine was present along with norepinephrine in the buffer solution bathing aortas from control rats, there was an increase in inositol phosphates (Figure 1). When contractile studies were performed under similar conditions, chronic cocaine administration resulted in reduced responses to norepinephrine. When cocaine was present in the bath (acute cocaine), norepinephrine induced greater contractions at each point on the norepinephrine concentration-response curve (Figure 3). The augmentation of the response to norepinephrine on both phosphoinositide hydrolysis and the parallel increase in developed tension can be explained by the known ability of cocaine
1680
Cocaine and Phosphoinositide Hydrolysis
Vol. 51, No. 21, 1992
to potentiate sympathetic responses by interfering with uptake of norepinephrine. This also suggests that the decreased responsiveness to norepinephrine following chronic cocaine administration was not due to cocaine's local anesthetic properties, since the decreased response was not an acute effect with cocaine. The mechanism for the effect of chronic cocaine administration may be explained in a number of ways, for example, desensitization of a~-receptors; a change in their affinity; or a change in pharmacomechanical coupling. Desensitization to the effect of norepinephrine may have occurred because of the blockade of reuptake. In support of this is the finding of Avakian and coworkers (10), who observed less epinephrine-induced tachycardia in isolated, perfused hearts from rats chronically treated with cocaine. Although one study has reported an increase in central ~,and B-receptor binding sites following chronic treatment with cocaine (11), others, using similar doses of cocaine, observed slight decreases (12). While a decrease in receptor density could explain the decreased contractions observed after chronic cocaine administration, altered receptor density may not be the sole mechanism for the actions of cocaine. Although a change in the affinity of norepinephrine for c~-receptors could explain the shift of the norepinephrine concentration/response curve to the right in aortas from chronically treated animals, altered affinity would not explain the decrease in the maximum response to norepinephrine. Furthermore, the data in table 1 showing a lack of effect of cocaine on the affinity constant of prazosin for cq-adrenoceptors strongly suggest that this is not a mechanism. Decreased contractile responses and decreased phosphoinositide hydrolysis might be explained by the direct local anesthetic action of cocaine. This seems unlikely because acute cocaine potentiated both the contraction and the formation of inositol phosphates (Figure 1) and had no effect on phosphoinositide hydrolysis by itself (Figure 1) at concentrations that block Na ÷ channels (13). Egashira and colleagues (14) have reported that cocaine can depress aortic smooth muscle by decreasing the sensitivity of the contractile proteins to Ca2÷. However, a concentration of cocaine _> 1 mM was required to produce this action. This level of cocaine is not achieved following 20 mg/kg cocaine (15). Another potential mechanism for the effect of chronic cocaine administration on norepinephrine-induced phosphoinositide hydrolysis is the interruption ofpharmacomechanical coupling (the coupling of neurotransmitter initiated events to the mechanical response). Nestler and colleagues (16) have demonstrated that chronic treatment of rats with twice daily 15 mg/kg doses of cocaine for 14 days produced a significant decrease in G-protein subunits in three different brain regions. Since c~-receptor activated phosphoinositide hydrolysis by phospholipase C has been shown to be G-protein dependent (17), a decrease in the amount of the signal transducing G-protein could lead to decreased phospholipase C activation and subsequent diminished phosphoinositide hydrolysis. The data presented suggest that chronic cocaine administration does not alter cq-receptor affinity but do not distinguish between any of the other potential mechanisms.
Acknowledgements This work was supported by grants from the American Heart Association, Louisiana Affiliate, and the Department of Veterans Affairs. The authors thank Barbara Roggero for technical assistance.
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References .
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
M.D. CAMPBELL, R.C. DETH, R.A. PAYNE, and T.W. HONEYMAN, Eur. J. Pharmacol. 116, 129-136 (1985). V. PIJUAN and I. LITOSCH, Biochem. Biophys. Res. Comm. 15__66,240-245 (1988). R.M. RAPOPORT, J. Pharmacol. Exp. Ther. 242, 188-194 (1987). A.V. SOMLYO, M. BOND, A.P. SOMLYO, and A. SCARPA, Proc. Natn. Acad. Sci. (USA) 82, 5231-5235 (1985). J.W. WALKER, A.V. SOMLYO, Y.E. GOLDMAN, A.P. SOMLYO, and D.R. TRENTHAM, Nature 32__!,249-252 (1987). B.E. EHRLICH and J. WATRAS, Nature 336, 583-586 (1988). L.R. ENGELKING, M.S. ANWER, J. MCCONNELL, D. SULLIVAN, and L. SHUSTER, Pharmacology 40, 129-136 (1990). J.H. ZAVECZ and T.O. YELLIN, J. Pharmacol. Exp. Ther. 223, 177-182 (1982). S. YAMADA, H.I. YAMAMURA and W.R. ROESKE, J. Pharmacol. Exp. Ther. 215, 176-185 (1980). E.V. AVAKIAN, M. LEROY, K. ST. JOHN-ALLAN, F.W. SAYRE, and M.H. MALONE, Life Sci. 46, 1569-1574 (1990). S.P. BANERJEE, V.K. SHARMA, L.S. KUNG-CHEUNG, S.K. CHANDA, and S.J. RIGGI, Brain Res. 175, 119-130 (1979). M.M. SELLINGER-BARNETYE, J. MENDELS, and A. FRAZER, Neuropharmacol. 1__99,447-454 (1980). W.J. CRUMB, Jr. and C.W. CLARKSON, Biophys. J. 57, 589-599 (1990). K. EGASHIRA, K.G. MORGAN and J.P. MORGAN, J. Clin. Invest. 87, 1322-1328 (1991). A.L. MISRA, Cocaine: Chemical, Biological, Clinical, Social and Treatment Aspects. S.J. Mule (ed), 72-90, CRC Press, Cleveland (1976). E.J. NESTLER, R.Z. TERWILLIGER, J.R. WALKER, K.A. SEVARINO, and R.S. DUMAN, J. Neurochem. 55, 1079-1082 (1990). K.M. WILSON and K.P. MINNEMAN, Mol. Pharmacol. 38, 274-281 (1990).