Regional rat brain content of adenosine 3′,5′-cyclic monophosphate and guanosine 3′,5′-cyclic monophosphate after acute and subacute treatment with ethanol

TOXICOLOGY

AND

Regional

APPLIED

Rat

PHARMACOLOGY

Brain

and Guanosine

ANDREW Department

Content

64, 447-455

(1982)

of Adenosine

3’,5’-Cyclic

Monophosphate

3’,5’-Cyclic Monophosphate after Subacute Treatment with Ethanol’

P. FERKO,* EMIL BOBYOCK,AND

of Pharmacology,

Hahnemann

Received

Regional Rat Brain Cyclic Monophosphate

December

Medical

College

12, 1981:

WARREN

and Hospital,

accepted

March

Acute

and

S.CHERNICK

Philadelphia,

Pennsylvania

19 IO2

29. 1982

Content After

of Adenosine 3’,5’-Cyclic Monophosphate and Guanosine 3’.5’Acute and Subacute Treatment with Ethanol. FERKO, A. P.. BOBYOCK, E., AND CHERNICK, W. S. (1982). Toxicol. Appl. Pharmacol. 64,447-455. Acute administration of ethanol (1.0, 2.0, and 5.0 g/kg, ip) to naive male rats (Sprague-Dawley) caused a dose-dependent depression of cerebellar guanosine 3’,5’-cyclic monophosphate (&IMP), and a reduction in cortical cGMP at the highest dose of ethanol. The cGMP content was not altered in the anterior hypothalamus, posterior hypothalamus, or striatum. On examining adenosine 3’,5’-cyclic monophosphate (CAMP) levels, only the area of the striatum was reduced (5.0 g/kg, ethanol). In these acute experiments there was a negative correlation between blood ethanol concentrations and body temperatures. An elevated environmental temperature (3 1 + 1 “C) to prevent hypothermia from ethanol administration indicated that hypothermia was not a contributory factor. In the subacute experiments animals showed at the end of 24 hr of ethanol inhalation less hypothermia than naive animals with similar blood ethanol concentrations, a reduction in cGMP in the cerebellum and cortex, but no alteration in regional brain CAMP. When the animals were injected with ethanol (2.0 g/kg, ip) 48 hr after removal from the chamber (ethanol vapor, 24 hr), ethanol produced no significant reduction in body temperature (tolerance), but a decrease in cerebellar cGMP. The CAMP content of the tissues was similar to control animals. Ethanol administration (2.0 g/kg, ip) 48 hr later to animals which were previously exposed to air only in the chamber (24 hr) demonstrated a reduction in body temperature as compared with tolerant animals, a decrease in cerebellar cGMP, and no depletion of CAMP in regional sections of the brain. From this in vivo study the data seem to suggest that brain cyclic nucleotides, particularly CAMP, may have a limited role in ethanol-induced intoxication and tolerance to the hypothermic effect of ethanol.

Earlier reports indicated that acute ethanol administration altered brain cyclic nucleotides. Volicer and Hurter ( 1977) showed that acute oral ethanol administration (1.0 to 6.0 g/kg) produced a dose-dependent decrease of adenosine 3’,5’-cyclic monophosphate (CAMP) in the cortex, cerebellum, pons, and medulla oblongata. Cyclic AMP

levels in the cortex, cerebellum, and pons-medulla were decreased by 59, 49, and 37% of controls, respectively. Guanosine 3’,5’cyclic monophosphate (cGMP) was also reduced in the same regions of brain as well as in the subcortex. However, Redos et al. (1976) examined the effect of acute ethanol (6.0 g/kg, po) on brain CAMP and indicated no changes in regional brain CAMP. In addition they studied ethanol and cGMP in the brain and showed that acute ethanol administration (6.0 g/kg, po) decreased cGMP

’ This work was supported in part by Biomedical Research Support Grant S07-RR05413. * To whom reprint requests should be addressed. 447

0041-008X/82/090447-09$02.00/0 Copyrtght 0 1982 by Academic Press. Inc All rights of reproduction in any form reserved

448

FERKO,

BOBYOCK,

content. Based on the differences in experimental findings in the literature, it has been stated that the acute effects of ethanol on CAMP in the central nervous systems were uncertain (Hunt, 1979). The effects of chronic ethanol treatment on central CAMP appear to be more consistent in published work, as well as the data on the acute and chronic effects of ethanol on cGMP. Emphasis has been placed on withdrawal studies when prolonged or chronic effects of ethanol on CAMP and cGMP were reported (Volicer and Hurter, 1977; Redos et al. 1976; Eliasson et al., 1981). These investigations generally involved days or weeks of ethanol treatment to animals. Little or no information is available on the alteration of cyclic nucleotides and the development of functional tolerance to ethanol in animals. Functional tolerance, which is a central cellular adaptation to an effect of ethanol from prior exposure and occurs in the absence of enhanced ethanol biotransformation, has recently been produced in animals after a 24hr period (Ferko and Bobyock, 1979; Mullin and Ferko, 198 1). If such a short procedure of 24 hr was employed, the variability of nonspecific neural toxicity may be removed from the experiment (Hunt, 1979). This investigation studied the alterations in brain cyclic nucleotides in animals tolerant to ethanol. Functional tolerance to ethanol was produced by a 24-hr inhalation procedure (Ferko and Bobyock, 1979). In addition, acute effects of ethanol on regional brain CAMP and cGMP were examined. These experiments were of particular importance since the results may aid in rectification of the apparent controversy concerning the effects of acute ethanol administration on CAMP. Acute administration of ethanol can produce a concomitant hypothermic response in animals as the blood ethanol concentrations are increased (Freund, 1973; Ferko and Bobyock, 1979). Since hypothermia induced by ethanol may alter the activity of enzymes, membrane permeability, and cyclic nucleo-

AND

CHERNICK

tides as a secondary effect from ethanol dosage (Freund, 1979), the observed acute effects of ethanol on cyclic nucleotides when hypothermia was present should be compared with data obtained in the absence of hypothermia. Previous work assessed the role of ethanol-induced hypothermia on the rate of ethanol clearance from blood (Ferko and Bobyock, 1978). Freund ( 1979) stated that humans usually do not develop significant temperature changes during acute ethanol intoxication or withdrawal, and that the results of experiments with rodents in the absence of appropriate temperature controls may not be relevant to humans. METHODS Male Sprague-Dawley rats (Charles Rivers, Wilmington, Mass.) weighing 200 to 260 g (mean weight 235 g) were housed for 1 week prior to experimentation at 22 + 1“C with a light cycle from 800 to 2000 hr. The animals had free access to Purina Laboratory Chow (Ralston Purina Co., St. Louis, MO.) and water; however, they were fasted 18 hr prior to ethanol or saline administration but water was available ad Zibitum. The bedding used in the cages was ground corn cob, 1/8th in. (Andersons Cob Division, Maumee, Ohio). Acute experiments. Ethanol was administered (ip) to naive rats in doses of 1.0, 2.0, or 5.0 g/kg, with solutions of 10, 10, and 25%, respectively. Sodium chloride solution, 0.9%. (saline) was given to control animals (0.02 ml/g). Recta1 temperatures were measured with a lubricated clinical thermometer (Aloe Medical Co., St. Louis, MO.) inserted 2.5 cm into the rectum for 2 min immediately before and at 0.5 and 1.5 hr after injection. Blood samples were obtained by orbital sinus bleeding to determine ethanol concentrations 1.5 hr after injection just prior to death by focused microwave irradiation. Functional tolerance experiments. Animals in the chamber were exposed to ethanol vapor (nominal concentration: 28 mg/liter) by inhalation for a period of 24 hr according to the method of Ferko and Bobyock (1979). Ethanol vapor concentrations in the chamber were determined twice daily in duplicate using the enzymatic method of Lundquist (1959). Food (Purina Lab Chow) and water were available during the period of ethanol vapor exposure. Control animals were exposed to air only in the chamber for 24 hr and had restricted access to food during the 24-hr period to reflect the weights attained by the treated group (Ferko and Bobyock, 1979; Ferko et al., 1979).

ETHANOL

AND

CYCLIC

After the 24-hr exposure period the animals were removed from their respective chambers and blood samples (20 ~1) were obtained by orbital sinus bleeding (Ferko and Bobyock, 1979). Rectal temperatures were measured and body weights were also recorded. A portion of the animals which were exposed to ethanol vapor for 24 hr as well as appropriate controls were killed at the termination of the inhalation period for brain cyclic nucleotide determinations. Forty-eight hours after removal from the inhalation chamber, the remaining animals were assessed for tolerance to the hypothermic effect of ethanol. At this time the animals previously exposed to ethanol vapor or air only were each divided into two groups. The first group of the ethanol vapor or air exposed animals received 2.0 g/kg of ethanol ip. In the second group of ethanol vapor or air-exposed animals, saline (0.02 ml/g), ip, was administered. At I .5 hr after obtaining temperature readings and blood samples all animals in the experiment were killed. Elevated environmental temperature experiment. To prevent the hypothermic effect from ethanol administration in the animals, acute experiments were performed at an elevated environmental temperature of 3 I ? 1°C using an incubator.3 Body temperatures were recorded prior to placing animals in the incubator. After equilibrium for 1 hr, the animals received ethanol (2.0 g/kg, ip) or saline (0.02 ml/g, ip) and were returned to the incubator. At 1.5 hr, following rectal temperature measurements and after obtaining blood samples, the animals were killed for determination of cyclic nucleotides. Assayfor cyclic nucleotides. Animals were killed and tissue cyclic nucleotides were fixed by exposure to a focused microwave beam4 for 4.3 set similar to the methodology described by O’Callaghan et al. (1979). The excised brains were then dissected into the following regions: cortex, anterior hypothalamus, posterior hypothalamus, striatum, and cerebellum (Giowinski and Inverson, 1966; Zeman and Innes, 1963). The dissected brain regions were sonicated’ with a microtip in 1.0 ml of 6% trichloroacetic acid at a setting of 6 for 30 sec. The sonicated mixtures were centrifuged at 7000g for 20 min and the recovered pellet was saved for the determination of protein according to the method of Bradford (1976). The supernatant fraction was washed four times with 5-ml portions of water-saturated ether (3:l). Excess ether was evaporated in a water bath at 55°C. The remaining dry residue was dissolved in sodium acetate buffer (pH = 6.2). Aliquots of solution were used ’ Lab-Line B.O.D. Incubator, Lab-Line Instruments, Melrose, Park, Ill. ’ Model BN-K2, 1.3 KW, General Medical Engineering, Peabody, Mass. 5 Branson Sonifier-185, Branson Sonic Power, Danbury. Conn.

449

NUCLEOTIDES

for CAMP radioimmunoassay (Steiner et al.. 1972) with a New England Nuclear Kit. The determination for cGMP was performed in a similar manner (Steiner ef al., 1972) with a New England Kit (acetylation procedure). All samples were assayed in duplicate. Recoveries were determined by spiking trichloroacetic acidtreated samples with tritiated CAMP or cGMP f’or CAMP and cGMP, mean recovery values were 101 + 6% and 95 + 5%. respectively. Data were not corrected for recovery. O’Callaghan et al. ( 1979) showed that tissue samples did not require purification or concentration. In addition there was no significant interferenceof CAMP with specificity of the cGMP assayed. Our experiments with cyclic nucleotide phosphodiesterase (Sigma Chemical Company, St. Louis, MO.) showed that 7.8 and 9.9% remained for CAMP and cGMP, respectively. These values were in agreement with Steiner ( 1974). Phosphodiesterase incubation (40 ml/ 100 rl,45 min at 30°C. pH = 7.5, Tris buffer, followed by 2 min at 100°C 1 w;rs similar to Ho et al. (I 976). Rats were acclimated to the microwave holder to diminish the nonspecific effect introduced by handling (Mailman er al., 1979). In addition head movement of the rats was reduced by modifying the holder. The 3.5cm circular head area of the holder had two semicircular sections (width, 0.7 cm) removed from the top. They were located at 0.8 and 2.3 cm from the anterior position of the holder. Two strips of adhesive tape were placed through these openings to aid in immobilizing the head of the rat. Assay for ethanol in blood and air. Ethanol concentrations in the chamber vapor and blood were assayed enzymatically according to Lundquist (1959). The procedural details have been described elsewhere ( Ferko and Bobyock, 1977). Statistical analysis. Significant differences were determined by the Student’s t test and correlation coefficients along with level of significance (5%) were assessed according to Bancroft (1965). Multiple comparisons with a control were done by analysis of variance and Dunnett’s test ( 1964).

RESULTS

Regional Brain Content of Cyclic Nucleotides after Acute Ethanol In Table 1 the results indicated that ethanol produced a dose-dependent depression of cerebellar guanosine 3’-S-cyclic monophosphate (cGMP). In addition a reduction in cortical cGMP was observed (ethanol, 5.0 g/kg). Also blood ethanol concentrations

450

FERKO,

BOBYOCK,

AND

CHERNICK

and rectal temperatures were reported in Table 1. There was a negative correlation between blood ethanol concentrations and rectal temperatures measured at 1.5 hr (r = -0.99, p < 0.01). Doses of ethanol at 1.0, 2.0, and 5.0 g/kg failed to alter adenosine 3’,5’-cyclic monophosphate (CAMP), except in the area of the striatum (24.5% decrease) at the highest dose of ethanol when these values were compared with controls. The average control values (means f SE) for CAMP in the cortex, anterior hypothalamus, posterior hypothalamus, striatum, and cerebellum were 13.00 f 1.29, 12.02 kO.87, 11.70-t 1.02, 10.16 + 0.73, and 8.45 + 0.82 pmol/mg protein, respectively. Animals which received 1.0 or 2.0 g/kg of ethanol demonstrated the righting reflex 1.5 hr after drug administration, although they appeared somewhat sedated, particularly at the 2.0 g/kg dose of ethanol. A dose of 5.0 g/kg of ethanol caused the animals to lose the righting reflex by 1.5 hr; however, a few rats were still conscious and attempted to right themselves. In our laboratory the dose of 5.0 g/kg was found to be in range of the LDso for ethanol by the ip route ( LDso = 4.5 g/kg; 95% confidence limits = 3.8, 5.3 g/W.

Functional Tolerance Cyclic Nucleotides

and Regional

Brain

After 24 hr of ethanol vapor inhalation in the chamber, the animals had elevated blood ethanol concentrations and reduced rectal temperatures (Table 2). The actual ethanol vapor concentration in the chamber at 24 hr was 25.9 f 0.7 mg/liter air. Upon removal of the treated animals from the chamber, mean body weight was 216 f 2.8 g, a reduction of 9.2%. The body weight of control animals (exposed to air only and on restricted food) was 216 + 3.1 g, a loss of 6.9%. The weight changes between the treated and control animals were not significant. These

ETHANOL

AND CYCLIC

results are in agreement with earlier work (Ferko and Bobyock, 1979; Mullin and Ferko, 198 1). The effects of ethanol inhalation for 24 hr on cGMP content in the rat brain are presented in Table 2. Depression of cGMP appears to be present in all sections examined; however, significant reduction in cGMP occurred only in the cerebellum and cortex which were 83 and 50%, respectively. When the effect of ethanol inhalation on CAMP levels in the brain were examined, it was found that regional brain CAMP was not significantly different from control values, although striatal and cortical values were reduced. The average control values (animals exposed to air only for 24 hr) for CAMP in the cortex, anterior hypothalamus, posterior hypothalamus, striatum, and cerebellum were 13.41 + 1.40, 11.14 f 0.98, 11.02 rt 0.81, 7.46 + 0.74, and 9.08 -t 0.95 pmol/ mg protein, respectively. Forty-eight hours after the ethanol inhalation (24-hr period), the remaining treated animals were divided into two groups. One group received ethanol (2.0 g/kg, ip) and the other group received saline (0.02 ml/g, ip). In addition appropriate control animals (air-treated for 24 hr) were injected in a similar manner. The data for functional tolerance to ethanol and the effect of ethanol on cGMP in the brain regions are presented in Table 3. Animals which were previously exposed to ethanol vapor for 24 hr and then injected with ethanol 48 hr after removal from the chamber did not exhibit any hypothermic response to ethanol administration (2.0 g/kg) as compared with appropriate controls which were previously exposed to air only in the chamber for 24 hr. However, cGMP values were quite similar in ethanol-injected animals whether they had prior ethanol vapor or air treatment in the chamber. The cGMP content was reduced in the brain sections after ip ethanol injection, but significant differences were assessed only in the cerebellar regions when compared with appropriate controls.

NUCLEOTIDES

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FERKO, BOBYOCK,

AND CHERNICK

The amount of CAMP in the same brain sections, which were used to assay for cGMP, did not appear to be altered by ethanol injection 48 hr after the animals were removed from the chambers. Ethanol vapor-treated animals given saline (control) and killed 1.5 hr later had CAMP concentrations in the cortex, anterior hypothalamus, posterior hypothalamus, striatum, and cerebellum of 11.99+-2.11,8.76+1.12, 11.68* 1.60,8.22 + 1.08, and 9.05 f 1.06 pmol/mg protein, respectively. The air-treated group which received saline (control) had corresponding values of CAMP in the above brain sections of 13.64 f 0.82, 12.98 f 1.34, 10.90 -t 1.80, 8.80 f 0.52, and 10.40 +_0.90 pmol/mg protein, respectively. As indicated previously, the animals which received ethanol, ip, showed no significant change in brain CAMP values from their appropriate controls.

Elevated Environmental

0 7 0 +I z

d +I Co id m

? 0 +I 0 G m

P

m

t-

* 7 0 +I c-4 ;r

00

Temperature

Study

Naive animals were placed in an elevated environmental temperature of 31 -t 1 “C in this experiment. The results are shown in Table 4. Animals which received ethanol (2.0 g/kg, ip) had significant blood concentrations 1.5 hr later, but they did not manifest a reduction in body temperature. Rectal temperatures of ethanol-injected animals were not significantly different from saline controls during the entire experiment in which the animals were maintained at 31 ? 1°C. However, cGMP was decreased in the cerebellum and posterior hypothalamus of the ethanol-treated animals. When the five regions of the brain were examined for CAMP in the ethanol-dosed animals, the levels of CAMP were quite similar to those which were present in the saline controls. The mean values of CAMP for controls were 14.35 f 0.55, 11.46 f 1.33, 11.46 f 0.81, 9.16 t 0.56, and 9.87 f 0.58 pmol/ mg protein in the cortex, anterior hypothalamus, posterior hypothalamus, striatum, and cerebellum, respectively.

ETHANOL

AND CYCLIC

DISCUSSION In examining the cyclic nucleotide system, other investigators reported that ethanol had no significant influence on adenylate cyclase activity (Kuriyama and Israel, 1973; Volicer et al., 1977) whereas Rabin and Molinoff ( 198 1) showed activation of adenylate cyclase by ethanol. Also phosphodiesterase activity was shown to be unaltered after ethanol treatment (Volicer et al., 1977; Kuriyama and Israel, 1973). Although ethanol may activate adenylate cyclase in vitro, the in vivo steady-state experiments on cyclic nucleotides and acute ethanol administration in the present investigation failed to show any significant changes in CAMP in the brain. These results were in contrast to the work of Volicer and Hurter ( 1977) but confirmed the findings of others (Redos et al., 1976; Frye et al., 1981). Brain cGMP content was reduced in certain areas after acute ethanol administration (Table 1). This effect of ethanol on brain cGMP was somewhat similar to other works (Hunt et al., 1977; Volicer and Hurter, 1977; Frye et al., 198 1). The exact mechanism for the effect of ethanol on cGMP appears to be unknown. It had been reported that ethanol did not modify the activity of either guanylate cyclase or phosphodiesterase under in vitro conditions (Hunt et al., 1977). In addition, Hunt et al. (1977) suggested that in vivo calcium ions may regulate guanylate cyclase activity and, therefore, cGMP content in the brain, since ethanol blocked inward calcium current (Bergmann et al. 1974) and reduced brain calcium content (Ross et al. 1974). Recent studies, however, showed that ethanol failed to reduce regional brain calcium content following acute and chronic administration of ethanol (Ferko and Bobyock, 1980; Hood and Harris, 1979). Although it appears that the exact mechanism for the reduction of cerebellar cGMP has not been delineated, several investigators suggested that a portion of the drug induced decrease in cGMP content in

NUCLEOTIDES

454

FERKO, BOBYOCK,

vivo may be secondary to altered motor activity in animals following drug administration (Lundberg et al. 1979). In this work, experiments were performed at an elevated environmental temperature to prevent ethanol-induced hypothermia. Freund (1979) suggested that hypothermia may be a factor in the biochemical effects attributed to ethanol. The results (Table 4) indicated that even in the absence of hypothermia, acute ethanol administration still depressed cerebellar cGMP. Therefore, it can be stated that ethanol-induced hypothermia did not play a significant role in the reduction of cGMP. The higher control values for cerebellar cGMP in Table 4 may be related to the increased activity of the animals in the incubator (Lundberg et al., 1979). It was also shown in this investigation that animals developed tolerance to the hypothermic effect of ethanol at the end of 24 hr of ethanol inhalation (Table 2 compared with Table 1). These data provided evidence to support a suggestion which was advanced in earlier work (Mullin and Ferko, 198 1). In addition, animals manifested tolerance to the hypothermic effect of ethanol 48 hr after ethanol vapor (Table 3); however, they did not develop tolerance to the effect of ethanol on cGMP in the cerebellum. When a longer period of ethanol administration (4 days, po) was used, Hunt et al. (1977) reported significantly less depletion of cGMP in the cerebellum than was found after a single dose of equivalent blood ethanol concentrations (373 + 9.8 mg/dl). Therefore, tolerance to the effect of ethanol on a cyclic nucleotide may be time dependent. However, our experiment (24-hr exposure to ethanol vapor) was designed for a shorter time period to reduce the possibility of nonspecific neural toxicity (Hunt, 1979). The data presented from this work appear to suggest that cyclic nucleotides, particularly CAMP, may have a limited role in the phenomena of intoxication and tolerance associated with ethanol administration.

AND CHERNICK

ACKNOWLEDGMENTS The authors express their sincere appreciation to Drs. A. Gould and E. J. Barbieri and Ms. S. Goodman for their assistance and to Mrs. L. Bush and Ms. J. Addario for help in preparation of the manuscript.

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