Abstract
The effects of chronic caffeine administration on ventilation and schedule-controlled behavior were studied in 12 adult rhesus monkeys. In seated subjects prepared with a head plethysmograph, ventilation was measured during exposure to air (normocapnia) and to elevated levels of CO2 (3%, 4% and 5%) mixed in air (hypercapnia). Acute administration of caffeine (10.0–30.0 mg/kg i.m.) produced marked, dose-dependent increases in ventilation during conditions of normocapnia and hypercapnia. However, daily administration of caffeine (10.0 mg/kg i.m.) for 8 consecutive days resulted in tolerance to its respiratory-stimulant effects that was surmountable with higher doses. Caffeine-tolerant subjects also were cross-tolerant to theophylline, an active metabolite of caffeine, and to rolipram and Ro 20–1724, selective phosphodiesterase inhibitors. When chronic administration was terminated and the acute effects of caffeine were redetermined, sensitivity returned to levels obtained before chronic administration within 9 days. Drug effects on behavior were studied in monkeys trained to respond under a fixed-interval schedule of stimulus termination. Acute administration of caffeine (1.0–30.0 mg/kg i.m.) produced significant rate-increasing effects on fixed-interval responding, but chronic administration resulted in tolerance that was insurmountable, such that no dose increased responding above control rates. Although the time course for development and loss of tolerance to the behavioral effects of caffeine corresponded closely with respiration, cross-tolerance did not extend to the behavioral effects of rolipram. Chronic caffeine administration had little effect on caffeine metabolism or clearance, which indicated that caffeine tolerance was pharmacodynamic. The results suggest that different neurochemical mechanisms mediate the effects of caffeine on respiration and behavior, and that inhibition of type IV phosphodiesterase plays a prominent role in caffeine-induced respiratory stimulation.
Because of the widespread use of caffeine and related xanthines as constituents of food and beverages and as therapeutic drugs, identification of mechanisms that mediate their pharmacological effects has considerable relevance for drug development and therapeutics. Numerous studies have demonstrated that caffeine and other xanthines can have significant effects on a variety of behavioral measures including schedule-controlled behavior (Glowa and Spealman, 1984; Katz and Goldberg, 1987; Spealman, 1988; Howell, 1993a), drug self-administration (Griffiths et al., 1979; Carrollet al., 1989), delayed matching-to-sample (Hudzik and Wenger, 1993) and repeated acquisition (Evans and Wenger, 1992; Buffaloet al., 1993). Over a range of doses, xanthines have significant behavioral-stimulant effects indicative of CNS stimulation (Spealman, 1988; Coffin and Spealman, 1989; Howell, 1993a; Howell and Byrd, 1993), whereas higher doses can suppress behavioral activity (Katz and Goldberg, 1987; Katz et al., 1988; Spealman, 1988;Howell, 1993a) and disrupt behavioral performances associated with learning and memory (Buffalo et al., 1993; Evans and Wenger, 1992; Hudzik and Wenger, 1993). Xanthines also have pronounced respiratory-stimulant effects that have been used in the treatment of respiratory disorders (Rall, 1990), and experimental evidence indicates that xanthines may act by affecting central mechanisms controlling respiration (Lundberg et al., 1981; Wessberg et al., 1985; Howell et al., 1990). Both caffeine and theophylline increase minute volume and sensitivity to elevated levels of inspired CO2 (Shannon et al., 1975;Mazzarelli et al., 1986; Olson and Schlitt, 1981; Howell, 1993a, b; Howell et al., 1990). These effects have been attributed to a lowering of the threshold of central chemoreceptors that are sensitive to CO2 (Davi et al., 1978; Gerhardt et al., 1979; Howell et al., 1990; Howell, 1993a, b) rather than to peripheral effects in the lung (Gerhardt et al., 1979).
Xanthines exhibit a complex pharmacology and have several neurochemical actions that could mediate their behavioral and respiratory effects. However, two primary mechanisms involving the cyclic nucleotide system have been implicated as the bases for the effects of xanthines in the CNS. A variety of xanthines bind to specific adenosine recognition sites in vitro, and there is a close correspondence between their potency in stimulating locomotor activity in rodents and their ability to block adenosine receptors in vitro (Brunset al., 1980; Snyder et al., 1981; Katimset al., 1983). Moreover, metabolically stable analogs of adenosine are behavioral depressants, and their effects are attenuated by xanthines in a manner that indicates a competitive pharmacological antagonism (Logan and Carney, 1984; Goldberg et al. 1985;Spealman, 1988; Spealman and Coffin, 1988; Howell, 1993a; Howell and Byrd, 1993). Accordingly, the behavioral-stimulant effects of xanthines have been linked repeatedly to their capacity to block the actions of endogenous adenosine (Fredholm, 1980; Snyder et al., 1981;Daly, 1982; Howell, et al., 1997). In addition to their adenosine-antagonist effects, xanthines inhibit cyclic nucleotide PDEs responsible for the hydrolytic inactivation of cyclic AMP and cyclic GMP (De Gubareff and Sleator, 1965; Daly, 1977, 1982), sometimes at concentrations similar to those capable of blocking adenosine receptors (Smellie et al., 1979; Choi et al., 1988). Evidence indicates that the respiratory-stimulant effects of xanthines are linked more closely to PDE inhibition than to antagonism of adenosine receptors (Howell et al., 1990, 1997; Howell, 1993a). For example, type IV-selective PDE inhibitors have pronounced respiratory-stimulant effects similar to those of xanthines (Howellet al., 1990; Howell, 1993a), and the potencies of several xanthines as respiratory stimulants correspond with their potencies as PDE inhibitors (Howell et al., 1997). Collectively, these findings suggest that different neurochemical substrates involving the cyclic nucleotide system may mediate the effects of xanthines on respiration and behavior.
The study of pharmacological tolerance during chronic drug administration has proven to be an effective approach to characterize drug mechanism of action. Direct comparisons between the rate of development and loss of tolerance, and altered sensitivity to drugs with selective neurochemical actions, can provide a basis for comparing mechanisms that mediate the respiratory and behavioral effects of xanthines. Human and animal studies have reported the development of tolerance to the CNS effects of caffeine that include behavioral effects (Holtzman, 1983; Finn and Holtzman, 1986, 1987, 1988; Holtzmanet al., 1991; Evans and Griffiths, 1992) and humoral and cardiovascular effects (Robertson et al., 1981; Ammonet al., 1983). However, the mechanisms involved in caffeine tolerance are poorly understood, and specific neurochemical substrates have not been identified. Therefore, the present study established a nonhuman primate model of caffeine tolerance to investigate neurochemical mechanisms that mediate caffeine-induced changes in respiration and behavior. The effects of caffeine were compared with those of theophylline, a xanthine with nonselective PDE inhibitory effects and the primary metabolite of caffeine in macaque monkeys (Berthou et al., 1992), rolipram and Ro 20–1724, two type IV (cAMP-specific, cGMP-insensitive)-selective PDE inhibitors and cocaine, a nonxanthine stimulant. In addition, caffeine pharmacokinetics were assessed before and after chronic administration of caffeine.
Methods
Subjects
Five adult male (CF-67, RBL, RJF, RLM and RQN) and seven adult female (CF-6, CF-65, CF-61, RBS, RPZ, RQC and RYL) rhesus monkeys (Macaca mulatta) weighing between 6.8 and 14.8 kg were studied. Between experimental sessions, subjects lived in individual home cages and were provided daily access to food (commercially available monkey chow, fresh fruit and vegetables) and unrestricted access to water. Weight restrictions were not imposed. Subjects CF-65, CF-67, RBL, RJF, RLM and RQN had been studied previously under the general procedures described below and had received drugs (Howell, 1993a,b, 1995).
Apparatus
Respiration experiments.
During experimental sessions, the subjects were seated in a primate chair (Primate Products, Redwood City, CA) and enclosed within a ventilated, sound-attenuating chamber. Ventilation was monitored continuously with a pressure-displacement head plethysmograph, as described previously (Howell et al., 1988). A sealed, rectangular helmet (9.5 × 15 × 11 cm) made of 1/16-inch Lexan with a hole for the neck was placed over the subject’s head and served as a pressure-displacement plethysmograph. A continuous flow (10 l/min) of gas, monitored by a flowmeter (model PRO34-FMO34, Cole-Parmer Instrument Co., Chicago, IL), entered the helmet through a port in front of the subject’s face and was extracted by a vacuum pump through a port behind the subject’s head. A second flowmeter monitored the flow (10 l/min) of gas extracted by the pump. Changes in flow within the plethysmograph resulting from changes in ventilation were measured by a pressure transducer (model MP-15, Micron Instruments, Los Angeles, CA) connected to a polygraph (model 7D, Grass Instruments Co., Quincy, MA). A polygraph integrator (model 7P10F, Grass Instruments Co.) converted the flow signal to a volume measure. A microcomputer interfaced to the polygraph and to the polygraph integrator analyzed and stored data. Respiratory frequency (f) was determined directly by counting changes from positive flow to negative flow. Minute ventilation (VE) was determined by integration of the pressure-compensated plethysmograph signal after calibrated offset for bias flow, and tidal volume (VT) was calculated as the quotient of VE and f, i.e.VT = VE/f. Continuous white noise and an exhaust fan masked extraneous sounds during all sessions.
Behavioral experiments.
During experimental sessions, subjects were seated in a primate chair and enclosed within a ventilated, sound-attenuating chamber. A response panel mounted on the front of the chair was equipped with a response lever (BRS/LVE, Inc., Laurel, MD, model PRL-001) and a pair of 5-W a.c. red and white lights. The subject’s tail was held motionless in a small cuff, and two brass plates rested on a shaved portion near the end. Electrode paste (EGK Sol) minimized changes in impedance between the tail and the brass plates when a 10-mA electric stimulus of 200-msec duration was delivered. A microcomputer controlled experimental events and recorded and stored data on diskettes. Continuous white noise and an exhaust fan masked extraneous sounds during all sessions.
Pharmacokinetic experiments.
Drug plasma levels were analyzed by a reverse-phase HPLC analytical system (single pump ternary HPLC system 3004, Isco, Inc., Lincoln, NE) with a variable wavelength ultraviolet absorbance detector (model V4) and a C-18, Spherisorb ODS-2 (5.0 μm, 4.6 × 250 mm) analytical column (Isco, Inc.). A microcomputer and Chemresearch control system software package (Isco, Inc.) commanded the HPLC system, monitored the eluate for absorbance and determined peak areas.
Procedure
Respiration experiments.
Subjects CF-65, CF-67 and RQN were adapted to the experimental conditions and stable patterns of ventilation were established before the present studies. Ventilation was recorded in isolated, undisturbed subjects breathing air for an initial 25 to 30 min. Subsequently, subjects were exposed to hypercapnic conditions during which saline and drug injections were given i.m. into the thigh or calf muscle 30 min apart with the following exposure sequence: 10 min of air; 5 min of 3% CO2 in air; 5 min of 4% CO2 in air; 5 min of 5% CO2 in air; and 5 min of air. Injections were given at the start of the 10-min exposure to air to allow for absorption and distribution of the drug before subjects were exposed to elevated levels of CO2 mixed in air. Generally, two doses of a drug were studied during daily sessions lasting 2 to 3 hr, and drug experiments were conducted no more frequently than twice per week in each subject.
Behavioral experiments.
Subjects RBL, RJF and RLM were trained to press a response lever under an FI 300-sec schedule of stimulus termination with a 10-sec limited hold. A red light illuminated the experimental chamber during the interval and the limited hold. If the subject responded during the limited hold after the 300-sec interval elapsed, a white light was illuminated for 2 sec, followed by a 60-sec time-out period during which the chamber was darkened and responses had no scheduled consequences. In the absence of a response during the limited hold, an electric stimulus was delivered, followed by a 60-sec time-out. Daily experimental sessions comprised five sequential components of the FI schedule, and each component included an extended (10-min) time-out period, followed by three consecutive FIs. The total session time was approximately 140 min, and sessions were conducted 5 days per week. The effects of drugs on behavior were determined by a cumulative-dosing procedure similar to that described by Kelleher and Goldberg (1979) and Wenger (1980). A complete dose-effect curve was established during a single session for each drug by injecting (i.m.) sequentially higher doses in the thigh or calf muscle. Saline and drug injections were administered at the start of the extended (10-min) time-out periods. Typically, drug experiments were conducted on Tuesdays and Fridays, and saline (control) was administered on Thursdays. The effects of a full range of doses of each drug were determined at least twice in each subject. To preclude the presentation of excessive stimulus presentations due to drug-induced impairment of responding, the 10-mA stimulus source was disconnected on days when cumulative-dosing procedures were used. The subjects seldom received a stimulus presentation when the stimulus unit was connected, and responding was well maintained when the stimulus unit was disconnected.
Pharmacokinetic experiments.
Subjects CF-6, CF-61, RBS, RPZ, RQC and RYL were trained to extend a leg through the front of the home cage for repeated blood withdrawals. After administration (i.m.) of 10.0 mg/kg caffeine, 1.0 ml of blood was withdrawn from the saphenous vein at 0.5, 1.0, 2.0, 6.0 and 24.0 hr postinjection. Caffeine and two primary metabolites, theophylline and paraxanthine, were extracted and assayed by a procedure described previously (Howell, 1995). Whole blood was collected in vacutainer tubes and centrifuged at 3,000 rpm for 10 min. Serum was then aspirated into collection tubes, and 40 μl of distilled water and 10 μl of 60% perchloric acid were added to the sample. After the sample was vortexed and placed in the freezer for 10 min, an additional 350 μl of distilled water was added, and the sample was centrifuged again. The supernatant was transferred to microfiltration tubes (PN MF-5500, Bioanalytical Systems, Inc., W. Lafayette, IN) containing 0.45 μm membranes (PN MF-5655) and centrifuged a third time. The filtered product was then injected into the HPLC system with a mobile phase comprising 65% 50 mM sodium phosphate buffer (pH = 2.0) and 35% methanol at a flow rate of 1.0 ml/min. The eluate was monitored for absorbance at 280 nm, and the limit of detection for caffeine and its metabolites was 200 ng/ml serum. Drug-free samples of blood were spiked with known quantities of drug (1.0–50.0 μg/ml) and carried through the extraction procedure to calibrate the process and to determine the percentage yield of recovery.
Drug administration.
On multiple occasions, caffeine (10.0 mg/kg) was administered i.m. on a chronic, daily basis with each occasion separated by at least 4 weeks of drug abstinence. During the conduct of experiments, the daily dose of caffeine was administered 10 min presession. To assess caffeine tolerance and drug cross-tolerance, the acute effects of caffeine and several other drugs were determined before and during chronic caffeine administration. When the acute effects of a drug were determined in caffeine-tolerant subjects, the daily dose of caffeine was not administered on that day.
In respiration studies, the first series of experiments characterized the development and time course of caffeine tolerance. The second and third series of experiments determined whether caffeine tolerance was surmountable and investigated cross-tolerance to rolipram, respectively. The fourth series of experiments investigated cross-tolerance to theophylline and Ro 20–1724 during the second and third weeks of chronic caffeine administration, respectively. The last series of experiments investigated potential changes in base-line sensitivity to CO2 during chronic administration of caffeine. The daily dose of caffeine was administered postsession in the latter experiments to preclude acute drug effects during CO2 determinations. Hence, subjects received chronic administration of caffeine on five separate occasions ranging from 8 to 21 days each.
In behavioral studies, the first series of experiments characterized the development and time course of caffeine tolerance, and the second series determined whether caffeine tolerance was surmountable. The third series of experiments investigated cross-tolerance to theophylline during the second week of chronic caffeine administration. The fourth series of experiments investigated cross-tolerance to rolipram and cocaine during the second and third weeks of chronic caffeine administration, respectively. In all cross-tolerance studies, the acute effects of each drug were determined immediately before chronic administration of caffeine to control for the influence of drug history. Hence, subjects received chronic administration of caffeine on four separate occasions ranging from 14 to 21 days each.
Data Analysis
Results were calculated for individual subjects and combined to derive the mean for the group. In respiration studies, the last 3 min of each 5-min exposure to CO2 were used for data analysis to allow time for ventilation to reach a steady state. In behavioral experiments, response rates were computed separately for each FI component by dividing the total number of responses in a component by the total time the red light was present. Mean control rates were determined for each monkey by averaging response rates for all saline control sessions. The effects of drugs on responding were expressed as a percent of control rate obtained when saline was administered. In pharmacokinetic experiments, caffeine half-life (t1/2) was calculated by least-squares linear-regression analysis of ln(Ct/Cpeak) as a function of time where Ct = concentration at time t, Cpeak = peak concentration and ln(Ct /Cpeak = −(0.693/t1/2)t. A repeated-measures analysis of variance with Tukey’s post hoc multiple comparisons was used to determine the statistical significance of drug treatment conditions, and statistical significance was accepted at the 95% level of confidence (P < .05). In some figures, the S.E.M. or 95% confidence limits were presented only for the control data to avoid figures that appeared cluttered and difficult to interpret.
Drugs
The drugs studied were caffeine and theophylline (Sigma Chemical Co., St. Louis, MO), cocaine hydrochloride (National Institute on Drug Abuse, Rockville, MD), rolipram (Berlex Laboratories, Inc., Cedar Knolls, NJ) and Ro 20–1724 (Research Biochemicals, Inc., Natick, MA). Caffeine and theophylline were dissolved in 0.9% saline containing sodium benzoate, and doses are expressed in terms of the free base of the drugs. Ro 20–1724 was dissolved in 45% (w/v) aqueous 2-hydroxypropyl-β-cyclodextrin. Cocaine and rolipram were dissolved in 0.9% saline. Injection volumes were 0.5 to 3.0 ml.
Results
Respiration Experiments
During control conditions when subjects breathed air alone, f averaged 19.0 ± 1.5 breaths/min, VTaveraged 78.9 ± 9.8 ml and VE averaged 1.5 ± .2 l/min for the group of three monkeys. Exposure to elevated levels of CO2 in air produced marked increases in ventilation above levels recorded during exposure to air alone (fig. 1). There was a concentration-dependent increase in f, VT and VE as inspired CO2increased to a maximum of 5%. During the first 2 to 3 min of subsequent exposure to air, ventilation decreased and returned to prior values. Polygraph tracings also illustrate caffeine-induced changes in ventilation (fig. 1). On the first day of chronic, daily administration of 10.0 mg/kg caffeine, there was a marked increase in ventilation during exposure both to air alone and to elevated levels of CO2 in air. However, caffeine had little effect on ventilation by day 8 of chronic administration.
In figure 2, the data are summarized for the group of three monkeys, and f and VE are shown as a function of CO2 concentration during control conditions and after caffeine administration. On the first day of chronic administration, the effects of caffeine were similar to those obtained previously in acute administration experiments. Caffeine produced significant increases in f and VE during exposure to air alone and during exposure to 3%, 4% and 5% CO2 in air (fig. 2, top). Caffeine-induced increases in VE were caused primarily by increases in f with little change in VT. However, by day 5 of chronic administration, the effects of caffeine were less pronounced (data not shown), and by day 8, neither f nor VE differed significantly from nondrug, baseline conditions. When chronic administration was terminated and the acute effects of caffeine were redetermined, sensitivity returned to levels obtained before chronic administration within 9 days, and caffeine produced significant increases in f and VE (fig.2, bottom). Moreover, control ventilation was characteristic of prechronic conditions, providing no evidence of drug withdrawal.
In subsequent experiments, 10.0 mg/kg caffeine was administered daily until complete tolerance was evident, and on day 9, the acute effects of 30.0 mg/kg caffeine were determined. The higher dose of caffeine produced significant increases in f and VE, but the effects were similar to those obtained with 10.0 mg/kg in nontolerant subjects (fig. 3, top). In another series of experiments, the effects of rolipram (0.03 mg/kg) were compared with those of caffeine in nontolerant and caffeine-tolerant subjects. The acute effects of rolipram were very similar to those of caffeine in nontolerant subjects. Rolipram produced significant increases in f and VE during exposure to air alone and during exposure to 3%, 4% and 5% CO2 in air (fig. 3, bottom). However, compared with nontolerant subjects, the acute effects of rolipram were less pronounced during chronic administration of caffeine.
Additional experiments investigated the respiratory effects of theophylline and Ro 20–1724 administered as cumulative doses in nontolerant and caffeine-tolerant subjects. Like rolipram, the acute effects of theophylline (10.0 and 30.0 mg/kg) and Ro 20–1724 (0.1 and 0.3 mg/kg) were very similar to those of caffeine in nontolerant subjects. Both drugs produced significant, dose-related increases in f during exposure to air alone and during exposure to 3%, 4% and 5% CO2 in air (fig.4). Drug-induced increases in VE (data not shown) were caused primarily by increases in f, and closely paralleled drug effects on f shown in figure 4. Compared with nontolerant subjects, the acute effects of theophylline and Ro 20–1724 were less pronounced during chronic administration of caffeine. For example, effective doses of theophylline (10.0 mg/kg) and Ro 20–1724 (0.1 mg/kg) in nontolerant subjects had no significant effects on ventilation in caffeine-tolerant subjects.
A final series of experiments investigated potential changes in baseline sensitivity to CO2 during chronic administration of caffeine. To preclude acute drug effects during CO2 determinations, the daily dose of caffeine was administered 10 min postsession. Chronic administration of 10.0 mg/kg caffeine for 8 consecutive days had no significant effect on sensitivity to CO2; both nontolerant and caffeine-tolerant subjects exhibited similar CO2-induced increases in f and VE (fig. 5).
Behavioral Experiments
Responding during the FI 300-sec schedule was characteristic of performance maintained under FI schedules of stimulus termination (Morse and Kelleher, 1966). Little or no responding occurred early in the interval, and the response rate increased as the interval elapsed. Mean response rates during control sessions were 0.89 ± 0.34, 0.40 ± 0.14 and 0.37 ± 0.10 response/sec for subjects RJF, RBL and RLM, respectively. Caffeine (10.0 mg/kg) produced significant increases in response rate to more than 200% of control values for the group of three monkeys during the first day of chronic administration (fig. 6). However, the behavioral-stimulant effects of caffeine gradually diminished during 5 consecutive days, and by day 5, caffeine had no significant effect on FI responding. When chronic administration of caffeine was terminated on day 20, there was no obvious behavioral disruption characteristic of drug withdrawal.
In subsequent experiments, administration of cumulative doses of caffeine (1.0–30.0 mg/kg) in nontolerant subjects had modest behavioral-stimulant effects (fig. 7) that were less pronounced than those obtained when a single dose of caffeine (10.0 mg/kg) was administered 5 min presession (see fig. 6). A maximum rate increase of approximately 130% of control rate occurred after a dose of 3.0 mg/kg, and the highest dose (30.0 mg/kg) significantly decreased response rate below control values. When subjects received chronic, daily administration of 10.0 mg/kg caffeine for 2 consecutive weeks, there was a significant main effect of chronic treatment during the second week, shown as a downward displacement of the caffeine dose-effect curve (fig. 7, top). However, caffeine-induced suppression of responding after a dose of 30.0 mg/kg was relatively unchanged. When chronic administration was terminated and the acute effects of caffeine were redetermined 7 days later, modest rate-increasing effects were obtained, and the caffeine dose-effect curve did not differ significantly from prechronic conditions.
In another series of experiments, the behavioral effects of theophylline (1.0–30.0 mg/kg), rolipram (0.01–0.3 mg/kg) and cocaine (0.03–1.0 mg/kg) were compared with those of caffeine in nontolerant and caffeine-tolerant subjects. Cumulative doses of theophylline had effects that were very similar to those of caffeine in nontolerant subjects (fig. 8, top). A maximum rate increase of approximately 125% of control rate occurred after a dose of 3.0 mg/kg, and the highest dose (30.0 mg/kg) significantly decreased response rate below control values. In contrast, rolipram only decreased response rate over a wide range of doses (fig. 8, center). Cocaine produced dose-related increases in FI responding that were much more pronounced than those of caffeine, and the highest dose (1.0 mg/kg) decreased response rate below the maximum rate (fig. 8, bottom). When subjects received chronic, daily administration of 10.0 mg/kg caffeine for 2 consecutive weeks and the acute effects of theophylline were redetermined during the second week, there was no significant main effect of chronic treatment. However, no dose of theophylline increased FI responding above control rates during chronic caffeine administration. In separate experiments, there was no significant change in sensitivity to the behavioral effects of rolipram and cocaine during the second and third week of chronic caffeine administration, respectively. Although the maximum rate-increasing effect of cocaine was partially attenuated during chronic caffeine administration, pronounced rate-increasing effects were obtained in all subjects, and the cocaine dose-effect curve did not differ significantly from prechronic conditions. Hence, there was some indication of cross-tolerance to theophylline in caffeine-tolerant subjects, but there was little evidence of cross-tolerance to rolipram or cocaine.
Pharmacokinetic Experiments
Standard curves for caffeine and its metabolites were linearly related to peak areas over a range of 1.0 to 50.0 μg/ml. Chromatograms were completed in less than 10 min with reliable separation of peak retention times for caffeine, theophylline and paraxanthine at approximately 7.9, 5.3 and 4.9 min, respectively. The percent yield obtained during extractions of standards varied little among xanthines and ranged from 86.9% to 95.6% with a mean (± S.E.M.) value of 92.4% ± 1.7%.
Before chronic administration, 10.0 mg/kg caffeine resulted in a peak plasma concentration (± S.E.M.) of 8.6 ± 0.6 μg/ml at 30 min postinjection for the group of six monkeys (fig.9). The calculated half-life of caffeine was 10.8 ± 0.6 hr, and as caffeine plasma levels decreased, there was a corresponding increase in theophylline levels. No significant amount of paraxanthine was detected. At 24 hr postinjection, only small amounts of caffeine (<1.0 μg/ml) were detected, whereas significant amounts of theophylline (3.9 μg/ml) were still present. When caffeine pharmacokinetics were redetermined during the second week of chronic, daily administration of 10.0 mg/kg caffeine, neither the peak plasma concentration (8.0 ± 0.2 μg/ml) nor the calculated half-life (10.3 ± 0.9 hr) of caffeine differed significantly from prechronic conditions (fig. 9). However, theophylline levels were significantly greater throughout the 24-hr postinjection period because of accumulation of theophylline from prior daily injections of caffeine. The results indicate that chronic caffeine administration had little effect on caffeine metabolism or clearance.
Discussion
The present study established a nonhuman primate model of caffeine tolerance to investigate neurochemical mechanisms that mediate the physiological and behavioral effects of caffeine. Chronic daily administration of caffeine resulted in tolerance to its respiratory- and behavioral-stimulant effects that developed gradually over several days, and was reversible when chronic administration was terminated. Caffeine tolerance appeared to be pharmacodynamic because no significant changes in caffeine metabolism or clearance were evident in caffeine-tolerant subjects. Although the time course for the development and loss of tolerance was similar for the respiratory and behavioral effects of caffeine, important characteristics of caffeine tolerance differed for respiration and behavior. Tolerance to the respiratory-stimulant effects of caffeine was surmountable and extended to the type IV PDE inhibitors, rolipram and Ro 20–1724. A high dose of caffeine produced significant increases in ventilation in caffeine-tolerant subjects, and the acute effects of rolipram and Ro 20–1724 were less pronounced during chronic caffeine administration. In contrast, tolerance to the behavioral-stimulant effects of caffeine was insurmountable and did not extend to rolipram. No dose of caffeine increased responding above control rates in caffeine-tolerant subjects, and the acute effects of rolipram were unchanged during chronic caffeine administration. The results indicate that different neurochemical mechanisms mediate the effects of caffeine on respiration and behavior, and that inhibition of type IV PDE plays a prominent role in caffeine-induced respiratory stimulation.
The pharmacological profile of caffeine in the present study is consistent with previous studies reporting the involvement of type IV PDE in the acute respiratory-stimulant effects of xanthines (Howellet al., 1990; Howell, 1993a). Studies in rhesus monkeys have compared the effects of caffeine, theophylline, 8-phenyltheophylline, 8-cyclopentyltheophylline, 3-isobutyl-1-methylxanthine and enprofylline, as well as the nonxanthine adenosine antagonist, CGS 15943, and the selective PDE inhibitors, rolipram and Ro 20–1724. All drugs except 8-phenyltheophylline and CGS 15943, potent adenosine antagonists lacking PDE inhibitory effects, were found to have prominent respiratory-stimulant effects. Enprofylline and rolipram, both of which have prominent PDE-inhibitory effects but low affinity for adenosine receptors, had respiratory effects similar to those of caffeine. When the effects of prototypic xanthines were compared with nonxanthines that inhibit different molecular forms of PDE, only rolipram and Ro 20–1724, which selectively block the type IV (cAMP-specific, cGMP-insensitive) PDE, had respiratory effects in common with xanthines, and the drug potencies as respiratory stimulants correlated with their potencies as PDE inhibitors (Howell et al., 1997). The results obtained in the present study further support a prominent role of type IV PDE in the respiratory effects of xanthines. Theophylline, rolipram and Ro 20–1724 had acute respiratory effects that were similar to those of caffeine in nontolerant and caffeine-tolerant subjects. Moreover, the demonstration of cross-tolerance to theophylline and the type IV PDE inhibitors in caffeine-tolerant subjects provides convincing evidence that these drugs share a common mechanism of action.
Caffeine had pronounced effects on ventilation during conditions of normocapnia, and produced dose-dependent increases in f and VE during exposure to elevated levels of CO2 mixed in air such that the CO2 response curve was shifted upward in a parallel manner. The effects observed during hypercapnia are consistent with previous studies reporting that xanthines act by affecting central mechanisms controlling respiration (Howell et al., 1990;Lundberg et al., 1981; Trippenbach et al., 1980;Wessberg et al., 1985). For example, caffeine increases the ventilatory response to CO2 in cats at levels of CO2 equal to or below the apneic range (Mazzarelli et al., 1986), and theophylline increases the ventilatory response to CO2 in dogs during hypercapnia (Olson and Schlitt, 1981). Accordingly, a series of experiments investigated potential changes in baseline sensitivity to CO2 during chronic administration of caffeine to determine whether physiological adaptation resulted from repeated exposure to hypercapnia. When the chronic daily dose of caffeine was administered postsession to preclude acute drug effects during CO2 determinations, caffeine-tolerant subjects exhibited CO2-induced increases in ventilation that were similar to those obtained in nontolerant subjects. The latter results indicate that caffeine tolerance was pharmacological and did not result from changes in baseline responsiveness to CO2.
The pharmacokinetics of caffeine and its primary metabolites have been well characterized in humans and in a variety of laboratory animals, but the direct involvement of pharmacokinetic factors in caffeine tolerance has not been demonstrated. The major route of caffeine metabolism is through liver biotransformation to several dimethylxanthine metabolites including theophylline, paraxanthine and theobromine (Bonati et al., 1985; Lelo et al., 1986), and significant species differences in xanthine metabolism are related to multiple isoforms of the P450 enzyme (Berthou et al., 1992). For example, N-3 demethylation to paraxanthine is the major metabolic pathway in humans, whereas N-7 demethylation to theophylline is predominant in cynomolgus monkeys (Berthou et al., 1992) and rhesus monkeys (Howell, 1995). In the present study, the calculated half-life of caffeine in nontolerant subjects was approximately 11 hr, and as caffeine plasma levels decreased, there was a corresponding increase in theophylline levels. When caffeine pharmacokinetics were redetermined in caffeine-tolerant subjects, neither the peak plasma concentration nor the calculated half-life of caffeine differed from nontolerant subjects. The latter results indicate that chronic administration of caffeine had little effect on caffeine metabolism or clearance and that caffeine tolerance resulted from pharmacodynamic rather than pharmacokinetic factors. Moreover, the long duration of action of caffeine in the rhesus monkey may explain how a single daily dose of caffeine could result in caffeine tolerance during the course of a week. The accumulation of theophylline, an active metabolite of caffeine, during chronic caffeine administration suggests that caffeine tolerance was actually underestimated. Similarly, the gradual decline in caffeine blood levels with a corresponding increase in theophylline may account for the absence of overt withdrawal signs in the present study.
In contrast to the respiratory-stimulant effects of caffeine and related xanthines, the behavioral-stimulant effects have been linked more closely to adenosine receptor antagonism than to PDE inhibition (Bruns et al., 1980; Katims et al., 1983; Snyderet al., 1981). In previous studies conducted in nonhuman primates, only xanthines with prominent adenosine-antagonist actions had significant behavioral-stimulant effects, and there was a close correspondence between the drug potencies for increasing response rate and for antagonizing the behavioral-suppressant effects of the adenosine receptor agonist, NECA (Spealman, 1988; Howell, 1993a; Howell and Byrd, 1993; Coffin and Spealman, 1989). Selective PDE inhibitors that lacked adenosine-antagonist effects either suppressed behavior or had no behavioral effect (Howell, 1993a). Consistent with these findings, rolipram only decreased response rate in the present study, and subjects tolerant to the behavioral-stimulant effects of caffeine were not cross-tolerant to rolipram. The pharmacological specificity of caffeine tolerance was exemplified further by the lack of cross-tolerance to the nonxanthine psychomotor stimulant, cocaine. In contrast to rolipram, acute administration of theophylline, a xanthine with adenosine-antagonist effects, had modest behavioral-stimulant effects similar to those of caffeine. Although there was no significant main effect of chronic caffeine treatment on the acute effects of theophylline, no dose of theophylline increased responding above control rates during chronic caffeine administration. Hence, there was some indication of cross-tolerance to theophylline in caffeine-tolerant subjects. However, the role of adenosine antagonism as a mechanism underlying tolerance to caffeine-induced stimulation of behavior remains undefined. Some investigators have reported an increase in the number of adenosine binding sites in brain during chronic administration of caffeine (Ahlijanian and Takemori, 1986; Hawkinset al., 1988), whereas others have found no appreciable changes in the potency of caffeine as an adenosine antagonist in caffeine-tolerant animals (Holtzman et al., 1991; Katz and Goldberg, 1987).
In summary, chronic administration of caffeine resulted in tolerance to its respiratory- and behavioral-stimulant effects which developed gradually and was reversible. Caffeine pharmacokinetics were unchanged during chronic administration, which indicated that caffeine tolerance was pharmacodynamic. Tolerance to the respiratory-stimulant effects of caffeine was surmountable and extended to the type IV PDE inhibitors, rolipram and Ro 20–1724, whereas tolerance to the behavioral-stimulant effects was insurmountable and did not extend to rolipram. The results suggest that different neurochemical mechanisms mediate the effects of caffeine on respiration and behavior and that inhibition of type IV PDE plays a prominent role in caffeine-induced respiratory stimulation.
Acknowledgments
The authors gratefully acknowledge the technical assistance of J.E. Majors, P.M. Plant and F.H. Kiernan, and the participation of L.B. Oettinger, J. Siegfried and M.C. Landrum.
Footnotes
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Send reprint requests to: Dr. Leonard L. Howell, Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30322.
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↵1 This research was supported, in part, by U.S. Public Health Service grants DA-05346, DA-01161, DA-06264 and RR-00165 (Division of Research Resources, National Institutes of Health). The Yerkes Primate Research Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care.
- Abbreviations:
- VE
- minute volume
- VT
- tidal volume
- f
- respiratory frequency
- FI
- fixed interval
- PDE
- phosphodiesterase
- cAMP
- cyclic AMP
- CNS
- central nervous system
- HPLC
- high-performance liquid chromatography
- Received January 14, 1997.
- Accepted June 9, 1997.
- The American Society for Pharmacology and Experimental Therapeutics