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Am J Physiol Heart Circ Physiol 284: H1570-H1576, 2003. First published January 9, 2003; doi:10.1152/ajpheart.00772.2002
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Vol. 284, Issue 5, H1570-H1576, May 2003

Microinjection of a cannabinoid receptor antagonist into the NTS increases baroreflex duration in dogs

David J. Rademacher1, Sachin Patel1, Francis A. Hopp2,3, Caron Dean2,3, Cecilia J. Hillard1, and Jeanne L. Seagard2,3

Departments of 1 Pharmacology and Toxicology, and 2 Anesthesiology, Medical College of Wisconsin, Milwaukee 53226-0509; and 3 Zablocki Department of Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Baroreceptor afferent fibers synapse in the nucleus tractus solitarius (NTS) of the medulla. Neuronal cannabinoid (CB)1 receptors are expressed in the NTS and central administration of CB1 receptor agonists affect blood pressure (BP) and heart rate. In addition, there is evidence that endocannabinoids are produced in the brain stem. This study examined whether changes in CB1 receptor activity in the NTS modulated the baroreceptor reflex, contributing to changes seen in BP and heart rate. Baroreflexes were evoked in anesthetized dogs by pressure ramp stimulations of the isolated carotid sinus before and after microinjection of CB1 receptor agonist WIN-55212-2 (1.25-1.50 pmol) or antagonist SR-141716 (2.5-3.0 pmol) into cardiovascular regions of the NTS. Microinjection of the SR-141716 did not affect baseline BP or baroreflex sensitivity. However, SR-141716 significantly prolonged the time needed to return to the baseline level of BP after the pressure ramp. Microinjection of WIN-55212-2 had no effect on the baroreflex. These data suggest that endocannabinoids can modulate the excitability of NTS neurons involved in the baroreceptor reflex, leading to modulation of baroreflex regulation.

endocannabinoids; sympathetic outflow; glutamate; SR-141716; WIN-55212-2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS KNOWN THAT THE CANNABINOIDS (CBs) produce cardiovascular effects in vivo. For example, in anesthetized rats and dogs, Delta 9-tetrahydrocannabinol (Delta 9-THC) produces a transient pressor response followed by long-lasting hypotension and bradycardia (12, 16, 39). The hypotensive effect of Delta 9-THC is mimicked by various CBs with a rank order of potency that strongly correlates with the affinity of these CBs for the neuronal CB1 receptor (15). The administration of the endocannabinoid, N-arachidonylethanolamine (AEA; anandamide), produces a brief pressor response, followed by a prolonged decrease in blood pressure (BP) (37). The AEA-induced depressor response is inhibited by coadministration of the CB1 receptor antagonist, SR-141716 (37), and is absent in CB1 null mice (13).

The hypothesis was made decades ago that the hypotensive and bradycardiac effects of the CBs resulted from an inhibition of sympathetic outflow (10, 40). For instance, the hypotensive effect of the synthetic CB, 1-hydroxy-3(1,2-dimethylheptyl)-6,6,9-trimethyl-7,8,9, 10-tetrahydro-6-dibenzopyran (DMHP), was lost with spinal cord transections of the first cervical vertebra. Additionally, the administration of a low dose of DMHP to dogs resulted in a loss of the pressor reflex induced by common carotid artery occlusion. Because the pressor response to epinephrine was preserved in these animals, it was concluded that DMHP interrupted sympathetic innervation of blood vessels (10). Other researchers (40) arrived at the same conclusion and suggested that the site of CB action was at CNS cardioregulatory centers. In contrast, recent animal studies using AEA and the CB1 receptor agonist WIN-55212-2 suggest that CBs also act at CNS cardioregulatory centers, and the result of these effects is enhanced sympathetic outflow (21, 22, 37). AEA increases the activity of sympathetic premotor neurons in the rostral ventrolateral medulla, an obligatory outflow pathway for CNS-mediated sympathomodulatory effects (38). The administration of WIN-55212-2 into the cisterna cerebellomedullaris produces both sympathoexcitation and activation of cardiac vagal fibers resulting in an increase in BP and a decrease in heart rate (HR) (21).

Only a few studies (21, 22, 40) have been conducted to characterize the effects of CBs on cardiovascular regulatory centers of the medulla. One important problem with the studies to date is the method of CB administration. Because the administration of CBs into the lateral ventricles (40), into the cerebellomedullaris (21), or intracisternally (22) will affect CB1 receptors in several cardiovascular regulatory centers in the brain (19), the results of these studies are difficult to interpret mechanistically.

Activation of arterial baroreceptors by stretch due to increases in pressure in receptive areas results in reflex depressor and bradycardiac responses. The site of first termination of baroreceptor afferent fibers in the CNS is the nucleus tractus solitarius (NTS) (5, 7, 27, 35). CB1 receptors are expressed in the NTS (36), and central administration of CB1 receptor agonists affects BP and heart rate (6, 10, 15, 21, 22, 40). We report here that microinjection of the CB1 receptor antagonist SR-141716 into the NTS significantly prolonged the time to recover to the basal BP level and decreased the maximum BP value during recovery. These results suggest that the CB1 receptor is active during the reflex and functions to accelerate the return to a normal level of BP.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General procedures. The effects of microinjection of an inactive CB1 receptor agonist, R(+)-WIN-55212-3 (Research Biochemicals International, Natick, MA), an active CB1 receptor agonist, S(-)-WIN-55212-2 (Research Biochemicals International), or a CB1 receptor antagonist, SR-141716 (National Institute on Drug Abuse Drug Inventory Supply Program, Bethesda, MD) into baroreceptive regions of the NTS on responses evoked by controlled activation of carotid baroreceptors were examined in a total of nine 14-18 kg mongrel dogs, anesthetized with alpha -chloralose and urethane (initial dose 50 mg/kg alpha -chloralose and 500 mg/kg urethane, supplemental continuous infusion of 250 mg alpha -chloralose plus 2.5 g urethane/h iv). All experimental procedures were approved by the Animal Care and Use Committees of the Medical College of Wisconsin and the Zablocki Department of Veterans Affairs Medical Center. All experiments were carried out in accordance with the Declaration of Helsinki and with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Insitutes of Health Publication No. 85-23, Revised 1985).

The left femoral artery and vein were cannulated to permit measurement of arterial BP and infusion of anesthetic, respectively. Arterial blood gases were measured by using an blood gas analyzer (ABL 30 Radiometer; Copenhagen, Denmark) and kept within normal ranges by adjustment of ventilation and infusion of bicarbonate. Arterial BP was measured via a catheter in the left femoral artery connected to a Statham pressure transducer (Gould, Cleveland, OH) and a polygraph (model 7D; Grass, Quincy, MA). To evoke baroreceptor activity, an isolated carotid sinus preparation was prepared as previously described (29). Briefly, the left carotid sinus was vascularly isolated to permit either a flow-through pulsatile perfusion of the sinus region at constant mean conditioning pressures or a ramp increase in carotid sinus pressure (CSP) (1-2 mmHg/s). Buffered lactated Ringer solution was used as the perfusate that was oxygenated with 100% O2 to chemically denervate any chemoreceptors not physically eliminated by the isolation technique (31). CSP was measured via a catheter in the lingual artery and recorded by using a Statham pressure transducer and a Grass model 7D polygraph. Constant CSP was maintained by using a servocontroller developed in this laboratory (30). Arterial BP and CSP were also recorded on a pulse code modulation recording adapter (model 3000A; Vetter, Rebersburg, PA) for subsequent analysis of data. To limit carotid baroreceptor afferent input to that from the isolated sinus, the contralateral sinus was denervated and both vagosympathetic trunks were sectioned.

After isolation of the sinus, the animal was placed in a head holder (Kopf; Tujunga, CA) for stereotaxic placement of a four-barrel glass micropipette, allowing for the microinjection of four test compounds. With the animal in a stereotaxic frame an occipital craniotomy was performed, the dura opened, and the caudal portion of the fourth ventricle exposed by lifting the vermis cerebelli. Obex, the point at which the central canal opens into the fourth ventricle, was visible on the dorsal surface of the medulla and was used as a zero reference for micropipette penetrations. Penetrations were made into the region of the NTS from 1.0-mm caudal to 2.0-mm rostral to the obex, 0.0- to 2.0-mm lateral to the midline, and from the surface to 2.0 mm deep, targeting sites that have been found to express Fos-like immunoreactivity in response to carotid baroreceptor stimulation in an earlier study (7). A barosensitive site was located by microinjection of the glutamate receptor agonist, (±)-alpha -amino-3-hydroxy-5-methyl-isoxazole-4-proprionic acid (AMPA), as described below.

Microinjection of drugs. All CB1 receptor ligands were dissolved in DMSO due to their hydrophobic nature. The glutamate receptor agonist, AMPA, was dissolved in artificial cerebral spinal fluid. The four drugs and their concentrations were as follows (in µM): 5 WIN-55212-3, 5 WIN-55212-2, 10 SR-141716, and 25 AMPA. An incision was made in the pia through which an electrode was inserted at coordinates identified relative to obex, and advanced slowly with the use of a microdrive. To identify a barosensitive site in the NTS, the glutamate receptor agonist AMPA was microinjected as the electrode was placed at different sites until a depressor response was evoked by excitation of baroreceptive neurons by AMPA. This site was then used to test for the effects of CB1 receptor activation or blockade. WIN-55212-3 is the enantiomer of WIN-55212-2, has a very low affinity for and does not activate the CB1 receptor (14, 24), and was used as a vehicle control. The effects of DMSO alone were initially used as a vehicle control, but the effects of WIN-55212-3/DMSO were not found to differ from DMSO alone and this combination also provided a control for the inactive form of the CB1 receptor agonist. Microinjections of the solutions were performed via micropressure ejections utilizing a picoejector constructed in the laboratory. Total ejection volume for each drug was 250-300 nl. The total amount of drug administered per microinjection was as follows (in pmoles): 1.25-1.50 WIN-55212-3, 1.25-1.50 WIN-55212-2, 2.50-3.00 SR-141716, and 6.25-7.50 AMPA.

NTS microinjection of WIN-55212-3, WIN-55212-2, and SR-141716 were examined for effects on both baseline (resting) and dynamic (ramp CSP changes) BP control. To determine the effects on baseline BP control, a microinjection was performed during constant mean CSP perfusion at 140-150 mmHg. This CSP is above the pressure threshold of most baroreceptors and would therefore not limit activation during constant CSP perfusion to baroreceptors with lower thresholds. The effects of WIN-55212-3, WIN-55212-2, or SR-141716 on dynamic baroreflex control of changes in BP were determined by measuring BP responses to slow ramp increases in CSP before and after microinjection. Dynamic baroreflex responses were evoked by halting constant mean pressure perfusion of the carotid sinus, clamping the outflow cannula, temporarily making the sinus a closed pouch. With the use of a Harvard syringe pump in-line with the inflow cannula, lactated Ringer solution was infused into the sinus pouch at a constant rate, producing a linear increase in CSP at a rate of 1-2 mmHg/s from 0 to 250 mmHg (Fig. 1A). The BP response to the CSP ramp was used to construct baroreceptor stimulus-response curves by plotting CSP versus BP, the slope of which was used as a measure of dynamic baroreflex control.


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  		Fig. 1.   Diagrams of methods used to analyze the reflex changes in blood pressure (BP) in response to changes in carotid sinus pressure (CSP). A: BP response to a CSP ramp and consequent return to constant CSP. B: nonlinear sigmoidal curve fit to the reflex decrease in BP produced by the ramp increase in CSP shown in A, the slope of which is a measure of baroreflex gain or sensitivity. C: exponential curve fit of the recovery responses of BP produced by a return to constant CSP shown in A. The time constant of the exponential function provides the time constant for the rate of the recovery response (TCR). The TCR determines the rate at which BP increases after the baroreflex-induced decrease in BP. For each time constant, the BP will recover 63.2% of the remaining distance to the asymptotic value (baseline BP). Thus after 4 TCR, BP will have recovered to 98% of the baseline level of BP.

Experimental procedure. The experimental protocol was as follows. During perfusion of the carotid sinus at constant CSP, a depressor NTS site was identified through microinjection of AMPA. Once identified, the animal was allowed to recover and then 30 s of a control baseline level of BP was recorded. The flow-through perfusion was then halted and the ramp increase in CSP was performed. After the ramp, the carotid sinus was again perfused at the original conditioning pressure until baseline pressure was restored (~5 min). After recovery, microinjection of WIN-55212-3 into the NTS was performed to test for the effects of the vehicle and mechanical stimulation of the NTS region. Three minutes after microinjection of WIN-55212-3, the baseline and ramp-evoked changes in BP were obtained as described above. After this ramp, BP was again allowed to return to stable baseline levels. The effects of microinjection of WIN-55212-2 and SR-141716 were then studied by using similar protocols. The order of WIN-55212-2 and SR-141716 were not randomized due to the long-lasting blockade produced by SR-141716. The effects of a single microinjection of each drug on baseline and dynamic changes in BP were obtained, with time allowed between WIN-55212-2 and SR-141716 to allow baseline BP to recover. In addition, test ramps were performed at two time intervals after SR-141716 microinjection. Ramps were performed at three and 10 min after SR-141716 to ensure full expression of CB1 receptor blockade. To ensure that the results were not due to time or degradation of the preparation, time was always given after SR-141716 to determine whether recovery was possible. An end control baseline and reflex response in BP was obtained after this recovery.

Data analysis. For data analysis, analog-to-digital conversion of recorded parameters was performed by using a computer (model 310; Hewlett-Packard, Palo Alto, CA). Arterial pressure and CSP were sampled at a frequency of 20 Hz for each control and microinjection procedure and stored on disk files for quantification and statistical analysis. Baseline values of BP were obtained by using 30-s averages of this parameter sampled during the period immediately before ramps for control and each microinjection. To determine dynamic baroreflex sensitivity, mean BP was plotted versus ramp changes in CSP to obtain baroreflex response curves. Nonlinear regression was used to curve fit the sigmoidal response curves and determine maximum slope (sensitivity) of the curves (32) (Fig. 1B). To examine the rate of recovery of BP after each baroreflex stimulus, BP was plotted versus time and nonlinear regression was used to curve fit the first order exponential recovery response (Fig. 1C). This analysis provided values for the time constant of the rate of recovery (TCR) and the asymptotic maximum value for the end recovery BP. As shown in Fig. 1, TCR evaluates the rate at which BP increases after the baroreflex-induced decrease in BP. For each TCR, the BP will recover 63.2% of the remaining distance to the asymptotic value (baseline BP). Thus, after four TCR, BP will have recovered to 98% of the baseline level of BP. The values for baseline BP, baroreflex sensitivity, TCR, and end recovery BP were calculated as percentages of the WIN-55212-3 response for each animal. One-way analyses of variance were used to compare the responses and differences were located by using the Duncan's multiple-range test. An alpha -level of 0.05 was used for all statistical tests.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of CBs on baseline BP and baroreflex sensitivity. Microinjections of WIN-55212-2 and SR-141716 (measured at 3 and 10 min after microinjection) had no significant effect on baseline BP or baroreflex gain (sensitivity) compared with the initial control or WIN-55212-3 administration (Table 1; P > 0.05). In addition, end control baseline BP and baroreflex gain were not significantly different from the initial control or WIN-55212-3 baseline BP and baroreflex gain (Table 1; P > 0.05).

                              
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  		Table 1.   Values for a 30-s average baseline BP before initiation of a ramp increase in CSP and for the gain of the baroreflex-induced change in arterial BP evoked by a ramp in CSP

Effects of CBs on the TCR. CB1 receptor blockade by microinjection of SR-141716 significantly reduced the rate of BP recovery after the ramp increase of CSP. The TCR determines the rate at which BP returns to the baseline level of BP in response to a return to constant CSP after the ramp change in CSP. The TCR at 10 min after SR-141716 microinjection was significantly increased compared with all other treatments except SR-141716 at 3 min postinjection (Table 2 and Fig. 2; P < 0.05). Microinjections of WIN-55212-2 had no significant effect on the TCR compared with the initial control or WIN-55212-3 administration (Table 2; P > 0.05).

                              
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  		Table 2.   Values for the time constant of the rate of recovery of blood pressure following a ramp change in CSP and the maximum value of arterial BP reached during that recovery, as determined using an exponential curve fit



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  		Fig. 2.   Example of the effects of treatments on baroreflex-induced decreases in arterial BP starting with constant pressure in the carotid sinus, the reflex decrease in BP in response to a ramp increase in CSP, and a return to a baseline level of BP in response to a return to constant pressure in the carotid sinus (i.e., recovery response). An example of the CSP stimulus paradigm and the accompanying reflex-induced changes in BP are shown in the inset. Treatments include initial control (control); nucleus tractus solitarius (NTS) microinjection of an inactive CB1 receptor agonist, WIN-55212-3, an active CB1 receptor agonist, WIN-55212-2, or the CB1 receptor antagonist/inverse agonist SR-141716 (3 and 10 min after microinjection); and end control. There was little effect of the baroreflex-induced decrease in BP, but there was a significant delay in recovery toward baseline BP levels as a result of treatment with SR-141716 at 10 min postinjection. A partial recovery was seen with the end control response, assuring that the response seen for SR-141716 at 10 min postinjection was not due to time or a decay of the preparation.

Effects of CBs on the asymptotic value for the end recovery BP. The asymptotic value for the end recovery BP is the calculated maximum BP value obtained during recovery after a ramp change in CSP, on the basis of nonlinear regression analysis. The maximum BP value during recovery was significantly decreased for SR-141716 at 10 min postinjection and for the end control condition relative to the initial control condition (Table 2 and Fig. 2; P < 0.05). Microinjections of WIN-55212-2 had no significant effect on the maximum BP value during recovery compared with the initial control or WIN-55212-3 administration (Table 2 and Fig. 2; P < 0.05).

Effects of WIN-55212-2 and SR-141716 on BP recovery rate. To ensure that the effects of SR-141716 were due to alteration of CB1 receptor activity, and not due to nonspecific effects, the receptor specificity of the effects of SR-141716 were tested by observing the effects of coinjection of WIN-55212-2 with SR-141716 on the effects of BP recovery rate in a limited study in three dogs. Microinjection of WIN-55212-2 attenuated the prolongation of TCR due to SR-141716 and reduced the time needed to return to control TCR after SR-141716 microinjection (data not shown). These results suggest that the effects of SR-141716 were due to blockade of CB1 receptors and not due to nonspecific effects.

Averaged curves for the recovery response. Figure 3 shows the curves obtained by averaging all parameters obtained by the exponential curve fits for each animal for each treatment. As seen in the figure, microinjection of SR-141716 has the greatest effects, particularly at 10 min after injection. The most pronounced effect was a decrease in the TCR, as well as a decrease in the maximum BP reached during the recovery phase.


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  		Fig. 3.   Curves obtained by averaging all parameters obtained by the exponential curve fits for each animal for each treatment. Treatments include initial control (control); NTS microinjection of an inactive CB1 receptor agonist, WIN-55212-3, an active CB1 receptor agonist, WIN-55212-2, or the CB1 receptor antagonist/inverse agonist, SR-141716 (3 and 10 min after microinjection); and end control. This figure reflects the finding that the largest effects were obtained 10 min after microinjection of SR-141716 resulting in a decrease in TCR as well as a decrease in the maximum BP value reached during the recovery phase.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The primary finding of this study is that microinjection of the CB1 receptor antagonist, SR-141716, into the NTS resulted in a delay in recovery to baseline levels of BP in response to a return to constant pressure in the carotid sinus. SR-141716 is a potent and selective antagonist of the CB1 receptor when administered in low doses (11) and readily prevents or reverses agonist activation of the CB1 receptor (25). Therefore, these data suggest that CB1 receptor activation in the NTS is necessary for a normal rate of recovery to a baseline level of BP after baroreflex activation. Interestingly, microinjection of the CB1 receptor agonist, WIN-55212-2, into the NTS did not modulate the rate of recovery. This finding suggests that CB1 receptors in the NTS were maximally activated endogenously during the baroreceptor stimulation protocol. Neither SR-141716 nor WIN-55212-2 microinjected into the NTS affected baseline BP, which suggests that the role of CB1 receptor activity in the baroreceptor reflex is modulatory and depends on the presence of reflex activation. In addition, neither agent was found to alter baroreflex sensitivity. This is similar to the response to exercise, in which baroreflex resetting is induced without an accompanying change in gain. It is not clear why there is a lack of effect on baroreflex gain, but it is possible that CB1 effects are directed more to modulation of type II baroreceptor input, which plays a greater role in setting baseline BP rather than baroreflex gain. In a recent study (22), intracisternal administration of the CB1 receptor agonists WIN-55212-2 and CP-55940 increased renal sympathetic nerve activity, plasma norepinephrine concentration, and BP, effects that were attenuated by SR-141716. The lack of effect of WIN-55212-2 microinjected into the NTS indicates that these sympathomimetic effects occur at other medullary cardioregulatory center(s).

The effect of SR-141716 administration into the NTS suggests that CB1 receptor activation has an effect on baroreflex duration that is opposite to the effect of SR-141716, i.e., decreases baroreflex duration. Increased activity of baroreceptor afferents as a result of an elevation in CSP increases glutamate release by the sensory afferent onto baroreceptor-sensitive neurons in the NTS. Moreover, in this view, glutamate increases the synthesis and release of endocannabinoids in the NTS consistent with data from other brain regions (18, 23). The source of endocannabinoid that targets presynaptic CB1 receptors may be the postsynaptic baroreceptor-sensitive neuron or glutamate-activated astrocytes (8, 41). With regard to the target receptor, it is known that activation of CB1 receptors acutely depresses glutamatergic synaptic transmission through a presynaptic mechanism in the cerebellum (17, 34), prefrontal cortex (2), nucleus accumbens septi (26), and striatum (9). One plausible mechanism is that activation of presynaptic CB1 receptors reduced glutamate release from the sensory neuron that results in decreased baroreflex duration. Because central glutamatergic neurotransmission is terminated predominantly by the rapid uptake of synaptically released glutamate into astrocytes (1), an alternative hypothesis is that endocannabinoids enhance glutamate removal from the synapse by astrocytes through activation of astrocytic CB1 receptors. It is known that astrocytes express CB1 receptors (2, 28) as well as the high- affinity glutamate transporters glutamate-1 and glutamate-aspartate (33). Further studies are needed to differentiate among these possibilities.

In light of the broad distribution of CB1 receptors in the neuronal network that regulates sympathetic outflow, it is difficult to ascribe a specific role for the NTS CB1 receptor in the cardiovascular effects of systemically administered CB1 receptor agonists and antagonists. Our data suggest that this receptor is "silent" unless the baroreceptors are activated. Thus it is likely that the CB1 receptor does not play a role in the hypotensive effects of the CBs in a recumbent animal or human. However, it has been shown that marijuana produces orthostatic hypertension in humans during a rapid change of posture from supine to standing (20). This physiological effect is consistent with an effect of Delta 9-THC on the NTS CB1 receptor, as described in this study.

In summary, microinjection of SR-141716 into the NTS resulted in a delay in recovery to baseline levels of BP in response to a return to constant pressure in the carotid sinus. These data suggest that CB1 receptor activation in the NTS is necessary for a normal rate of recovery to a baseline level of BP. Because microinjection of the CB1 receptor agonist, WIN-55212-2, into the NTS did not modulate the baroreflex, we suggest that CB1 receptors in the NTS are maximally activated during the baroreceptor stimulation protocol. It is possible that physiologically produced endocannabinoids dampen activation of baroreceptor-sensitive neurons in the NTS, perhaps through inhibition of glutamate release or acceleration of glutamate removal. The obligatory nature of the sensory synapse on NTS neurons makes this initial synaptic transmission process particularly important in the baroreflex circuit. In this regard, the development of pharmacotherapies capable of modulating central endocannabinergic neurotransmission may be of value in treating disorders characterized by dysregulation of the baroreflex circuit.


    ACKNOWLEDGEMENTS

The authors acknowledge the generous support of Northwestern Mutual Life Insurance.


    FOOTNOTES

This study was supported by Veterans Affairs Medical Center Funds (to J. L. Seagard), an American Heart Association Grant-in-Aid 0150518 (to J. L. Seagard), and National Institutes of Health Grant DA-09155 (to C. J. Hillard).

Address for reprint requests and other correspondence: C. J. Hillard, Medical College of Wisconsin, Dept. of Pharmacology and Toxicology, 8701 Watertown Plank Rd., Milwaukee, WI 53226-0509 (E-mail: chillard{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 9, 2003;10.1152/ajpheart.00772.2002

Received 2 September 2002; accepted in final form 7 January 2003.


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DISCUSSION
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