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
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
