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Anesth Analg 2006;102:779-785
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000194289.34345.63

ANESTHETIC PHARMACOLOGY

The Ventilatory Stimulant Doxapram Inhibits TASK Tandem Pore (K2P) Potassium Channel Function but Does Not Affect Minimum Alveolar Anesthetic Concentration

Joseph F. Cotten, MD, PhD, Bharat Keshavaprasad, MD, Michael J. Laster, DVM, Edmond I. Eger, II, MD, and C. Spencer Yost, MD

Department of Anesthesia and Perioperative Care, University of California San Francisco

Address correspondence and reprint requests to C. Spencer Yost, MD, Department of Anesthesia and Perioperative Care, 513 Parnassus Ave, Room S-261, Box 0542 San Francisco, CA 94143. Address e-mail to yosts{at}anesthesia.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
TWIK-related acid-sensitive K+-1 (TASK-1 [KCNK3]) and TASK-3 (KCNK9) are tandem pore (K2P) potassium (K) channel subunits expressed in carotid bodies and the brainstem. Acidic pH values and hypoxia inhibit TASK-1 and TASK-3 channel function, and halothane enhances this function. These channels have putative roles in ventilatory regulation and volatile anesthetic mechanisms. Doxapram stimulates ventilation through an effect on carotid bodies, and we hypothesized that stimulation might result from inhibition of TASK-1 or TASK-3 K channel function. To address this, we expressed TASK-1, TASK-3, TASK-1/TASK-3 heterodimeric, and TASK-1/TASK-3 chimeric K channels in Xenopus oocytes and studied the effects of doxapram on their function. Doxapram inhibited TASK-1 (half-maximal effective concentration [EC50], 410 nM), TASK-3 (EC50, 37 µM), and TASK-1/TASK-3 heterodimeric channel function (EC50, 9 µM). Chimera studies suggested that the carboxy terminus of TASK-1 is important for doxapram inhibition. Other K2P channels required significantly larger concentrations for inhibition. To test the role of TASK-1 and TASK-3 in halothane-induced immobility, the minimum alveolar anesthetic concentration for halothane was determined and found unchanged in rats receiving doxapram by IV infusion. Our data indicate that TASK-1 and TASK-3 do not play a role in mediating the immobility produced by halothane, although they are plausible molecular targets for the ventilatory effects of doxapram.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Doxapram stimulates the central nervous system (CNS) and is used to treat drug-induced ventilatory depression, chronic obstructive pulmonary disease, and apnea in premature infants. Actions upon the carotid bodies mediate the major ventilatory effects of doxapram, although larger concentrations stimulate ventilation through central effects (1,2). Doxapram's molecular site of action is unknown.

Type I (glomus) cells are the chemo-sensing cells of the carotid bodies. Hypoxia, acidic pH, and doxapram stimulate Type I cell calcium (Ca2+)-dependent neurotransmitter release by inhibiting a baseline membrane K conductance (3–5). One model for Type I chemo-sensing invokes inhibition (as by hypoxia, acidemia, or doxapram) of a membrane potassium (K) conductance. The inhibition causes membrane depolarization, l-Type Ca2+ channel activation, extracellular Ca2+ influx, and, finally, neurotransmitter release (5). Several K channels, including a voltage-dependent K channel in rabbits, the BK Ca2+-dependent K channel, and the TWIK-related acid sensitive K (TASK-1) tandem pore (K2P) channel in rats, may mediate the baseline conductance (6–8).

The K2P channel family (presently with 15 human members) mediates background "leak" K current, which regulates excitability in neurons. TASK-1 and TASK-3 proteins share approximately 60% amino acid sequence identity, and both pass a hypoxia- and acidic pH-inhibited and a halothane anesthetic-enhanced K current (9–13). Both are expressed in the brain, spinal cord, and carotid bodies (14). Buckler et al. (6) have identified a K conductance in carotid body cells with biophysical and pharmacological properties close to those of TASK-1 (14). Co-expressed TASK-1 and TASK-3 proteins interact functionally as a heterodimer, and the pKas for acidic pH inhibition of TASK-1, TASK-3, and the TASK-1/TASK-3 heterodimer function are 7.5, 6.8, and 7.3, respectively, within the physiological, extracellular pH range (15). Because the brain and spinal cord express TASK-1 and TASK-3, and because volatile anesthetics enhance their activity, they may contribute to some effects of volatile anesthetics including immobility during a noxious stimulus quantified as the minimum alveolar anesthetic concentration (MAC) (16).

Because rat Type I cells have a putative role in chemo-sensing and express the TASK-1 protein, and because doxapram inhibits a K current in Type I cells, we hypothesized that doxapram would inhibit heterologously expressed TASK-1 channel activity. To test this initial hypothesis, we studied the response of TASK-1 channels and several other K2P channels (TASK-3, TASK-1/TASK-3 heterodimer, TASK2 [KCNK5], TREK1 [KCNK2], and TRESK [KCNK18]) to extracellular doxapram.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The University of California, San Francisco, Institutional Animal Care and Use Committee approved all animal studies and care. Complimentary (c)RNA was prepared from a linearized DNA template using either T7 or T3 RNA polymerase (mMessage Machine; Ambion, Austin, Texas). Xenopus laevis oocytes were prepared using standard methods and injected with 1–15 mL of cRNA. One to four days after cRNA injection, oocytes were studied at room temperature using the two-electrode voltage clamp technique and a GeneClamp 500B amplifier (Axon Instruments, Foster City, California) controlled by a PowerMac computer (Apple Computer, Mountainview, California) running MacLab Scope software (ADInstruments, Colorado Springs, Colorado). All experiments were performed using frog Ringer's solution as perfusate (in mM): NaCl 115, KCl 2.5, CaCl2 1.8, and HEPES 10, with a pH value of 7.4. The high potassium frog Ringer's substituted 115 mM of KCl for 115 mM of NaCl. Oocytes were impaled with 0.1–0.3 M{Omega} glass electrodes and studied in a 25-µL recording chamber under continuous perfusion. Time course data were collected by clamping the transmembrane potential at –60 mV and averaging the outward current over a 1-s duration square-wave + 60-mV voltage pulse using a 1.5-s interpulse interval. Signals were low-pass filtered (50–100 Hz) and digitized (100–1000 Hz) before analysis. Halothane perfusate was prepared and applied, as previously described (17); halothane concentration was confirmed by gas chromatography. Doxapram was diluted in frog Ringer's to the target concentration and perfused directly onto the oocyte.

The following DNA coding sequences were used: rat TASK-1, rat TASK-3, human TASK-2, mouse TREK-1, and mouse TRESK. Except for the TASK-1/TASK-3 heterodimer, all coding sequences including chimeras were subcloned into the pcDNA3.1D-TOPO vector (Invitrogen, Carlsbad, California). The TASK-1/TASK-3 heterodimer was prepared by ligating a polymerase chain reaction (PCR) fragment encoding the entire TASK-1 gene minus its stop codon upstream of the TASK-3 coding sequence and its Kozak sequence in pOX-rTASK-3 vector. This introduced an additional amino acid sequence ‘Lys-Leu-Ala-Thr' between the last amino acid of TASK-1 and the start methionine of TASK-3. TASK-1/TASK-3 chimeras were prepared by PCR using the method of overlapping extension. All PCR was performed using AccuPrime Pfx or Elongase high-fidelity DNA polymerase preparations (Invitrogen), and the identity of each construct used was confirmed by bidirectional sequencing.

We selected halothane for study because it enhances all three channels (TASK-1, TASK-3, and TASK-1/TASK-3 heterodimer) in vitro with greater efficacy than isoflurane. Four adult male albino Sprague Dawley rats weighing 389 ± 5 g were used. Each had a tunneled, intrajugular venous catheter (PE10 tubing) placed 4 days before the beginning of the study. MAC for halothane was determined for all rats on the same day using methods previously reported (18). Rectal temperatures were monitored and maintained between 36°C and 38°C. Halothane (Halocarbon Laboratories, River Edge, New Jersey) was added to the system using a calibrated variable-bypass vaporizer, and halothane, carbon dioxide, and oxygen partial pressures were monitored by continuous sampling and analysis via an infrared gas analyzer (Datascope, Helsinki, Finland). Halothane partial pressures were confirmed by gas chromatographic analysis (18) (Gow-Mac Instrument Corporation, Bridgewater, New Jersey).

MAC was determined at three stages: Stage 1 = after vehicle-only infusion; Stage 2 = after small-dose doxapram infusion; and stage 3 = after large-dose doxapram infusion. Because doxapram redistributes, it must be delivered as a continuous infusion. For Stage 1, rats first received a vehicle-only infusion at 8 mL/h for 15 min and then 2 mL/h. After 15 min at 2 mL/h, a noxious stimulus was applied. Until movement was noted in all rats, halothane was decreased by 0.1% inhaled concentration, and after 30 min of reequilibration, the noxious stimulus was reapplied. MAC for each rat was the average of sequentially inhaled halothane concentrations at which "movement" and "no movement" were noted. For Stage 2, halothane was returned to the smallest inhaled concentration at which movement was suppressed in all rats in Stage 1. A doxapram infusion was started at 20 mg · kg–1 · h–1 (8 mL/h of vehicle) for 15 min and decreased to 5 mg · kg–1 · h–1 (2 mL/h of vehicle) for the remainder. After 15 min at 5 mg · kg–1 · h–1, MAC was determined as above. Total time for Stage 2 was 1 h 57 min. For Stage 3, halothane was returned to the smallest concentration at which movement was suppressed in all four rats in Stage 2. A doxapram infusion was started at 80 mg · kg–1 · h–1 (8 mL/h of vehicle) for 15 min and decreased to 20 mg · kg–1 · h–1 (2 mL/h of vehicle) for the remainder. After 15 min at 20 mg · kg–1 · h–1, MAC was determined as above. Total time for Stage 3 was 2 h 17 min. Dosing was guided by Schroeder et al. (19) who determined escalating doxapram serum levels of 10 and 25 µM at 10 and 45 minutes, respectively, during continuous 18-mg · kg–1 · h–1 infusion.

Doxapram hydrochloride injection (Dopram® Injectable; A.H. Robins Company, Richmond, Virginia) (1-ethyl-4-[2-(4-morpholinyl)ethyl]-3,3-diphenyl-2-pyrrolidinone monohydrochloride, monohydrate) is supplied 20 mg/mL in water with 0.9% benzyl alcohol added as preservative. Pure doxapram was obtained from ChemPacific Corporation (Baltimore, Maryland). All other chemicals were from Sigma Corporation (St. Louis, Missouri).

Statistical significance was determined using an analysis of variance test with a Bonferroni posttest (Prism 3.0a Software; Prism, San Diego, California). Significance was defined as P < 0.05, and results are reported as mean ± sd.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Doxapram inhibits TASK-1, TASK-3, and the TASK-1/TASK-3 heterodimer function. Doxapram inhibited both TASK-1 and TASK-3 function in a dose-dependent manner (half-maximal effective concentrations [EC50s] of 410 nM and 37 µM, respectively; Fig. 1). As a point of reference, doxapram induces ventilatory effects in humans and rodents at small micromolar or small microgram/milliliter total serum concentrations (19,20). TASK-1 inhibition was slowly reversible (more than 10 min), whereas TASK-3 inhibition reversed more rapidly (approximately 5 min; Figs. 1 and 2). Because TASK-1 and TASK-3 may function as a heterodimer in vivo, we also studied the effects of doxapram on the heterodimer. To assure that TASK-1 and TASK-3 were coexpressed with a 1:1 stoichiometry, TASK-1 and TASK-3 subunits were fused and expressed as a single protein, as has been performed in another study (15). Doxapram inhibited the heterodimer with an EC50 of 9 µM (Fig. 1, C and D), intermediate to that of TASK-1 and TASK-3. Next, to approach a more physiologic situation, we examined the effect of doxapram on the resting transmembrane potential (RMP) of oocytes expressing TASK-3 or the TASK-1/TASK-3 heterodimer (Table 1). Doxapram depolarized the hyperpolarized RMP of these oocytes, suggesting that its effects are not limited to the depolarized potential we selected for routine analysis (+60 mV); oocytes regained much of their hyperpolarized RMP upon doxapram washout (5 min). Doxapram was without effect on the RMP of uninjected oocytes.


Figure 117
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  		Figure 1. Doxapram inhibits TWIK-related acid-sensitive K+ (TASK)-1 (A), TASK-3 (B), and the TASK-1/TASK-3 heterodimer (C) channel activity. All data were collected in complimentary (c)RNA injected oocytes using the two-electrode voltage clamp technique. The bars indicate extracellular doxapram application or acidic pH application. Each data point represents the average outward current during the 1-s + 60-mV pulse. (D) Compilation of data from multiple time course experiments. Because of poor washout, no more than one doxapram dose was used per oocyte. n = 1–6 ± sd. Error bars omitted when smaller than the symbol. (E) Doxapram molecular structure. *n = 1 for this data point.

 

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  		Table 1. Doxapram Reversibly Depolarizes the Resting Transmembrane Potential (RMP) of TASK Expressing Oocytes

 


Figure 217
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  		Figure 2. Doxapram inhibition of TWIK-related acid-sensitive K+ (TASK)-1 is slowly reversible. Percentages are of current inhibition by doxapram at steady-state and 5 min after doxapram washout for TASK-1, TASK-3, and the TASK-1/TASK-3 chimeras. Data were collected in Xenopus oocytes, as in Figure 1. n = 3–4 ± sd. *P < 0.001. ND = no statistical difference.

 

To further explore the mechanism of doxapram inhibition, we examined the effect of extracellular pH, high extracellular potassium, and halothane on doxapram inhibition using the TASK-1/TASK-3 heterodimer (Table 2). None of these variables changed the extent of inhibition by doxapram. The halothane data suggest that the accessibility and affinity of the doxapram interaction site is unaffected by halothane.


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  		Table 2. Doxapram (5 µM) Inhibition of TASK-1/TASK-3 Heterodimer Function is Unaffected by Extracellular pH, Halothane, or High Extracellular Potassium

 

Our studies were performed using doxapram supplied for clinical use as a 20-mg/mL (46.2 mM) solution in water with 0.9% benzyl alcohol as a preservative. Benzyl alcohol, when applied by itself at 0.02% (the concentration applied were 1 mM of doxapram used), slightly enhances TASK-1 (25.6% ± 6.0%; n = 3) and TASK-3 (8.8% ± 0.2%; n = 3) function. Importantly, pure doxapram (in the total absence of benzyl alcohol) inhibited the TASK-3-1 chimera (1 µM; 69.5% ± 5.1%; n = 3) and the TASK-1/TASK-3 heterodimer (10 µM; 54.7% ± 5.6%; n = 3) with potency similar to the benzyl alcohol-containing preparation.

The potency of doxapram inhibition of TASK-1 versus TASK-3 function differed significantly. We used this observation to understand better the structural determinants of doxapram inhibition. TASK-1 and TASK-3 share approximately 60% amino acid identity with the greatest amino acid sequence dissimilarity in their intracellular, carboxy terminal domains. Exchange of carboxy terminal domains conferred TASK-1 sensitivity onto the TASK-3 channel and vice versa. The TASK-3-1 chimera (amino acids Met-1 through Phe-246 from TASK-3 and amino acids Met-247 through Val-411 from TASK-1) had an EC50 of 800 nM, similar to TASK-1 (Fig. 3, A and C) despite baseline currents similar to TASK-3 (baseline currents: TASK-1, 1.9 ± 0.9 µA; n = 14; TASK-3, 8.2 ± 2.9 µA; n = 20; TASK-3-1, 10.5 ± 3.8 µA; n = 14). The TASK-1-3 chimera (amino acids Met-1 through Phe-246 of TASK-1 and amino acids Leu-247 through Iso-396 of TASK-3) had an EC50 of 5 µM, which is intermediate between TASK-1 and TASK-3 (Fig. 3, B and C). These data suggest that the carboxy terminal domain of TASK-1 is important to doxapram inhibition; however, other parts of the channel may also contribute. Not surprisingly, doxapram depolarized the RMP of oocytes expressing the TASK-1/TASK-3 chimeras (Table 1). TASK-1/TASK-3 chimeras had washout rates intermediate to those of TASK-1 and TASK-3, although not significantly different from TASK-3 (Fig. 2).


Figure 317
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  		Figure 3. The TWIK-related acid-sensitive K+ (TASK)-1 carboxy terminus confers doxapram sensitivity upon TASK-3. Data were collected in Xenopus oocytes as in Figure 1. (A) TASK-1-3 chimera. (B) TASK-3-1 chimera. (C) Compilation of data from multiple time-course experiments. n = 3–7 ± sd. Error bars omitted when smaller than symbol.

 

Doxapram also inhibits TRESK, TASK-2, and TREK-1 but at significantly larger concentrations (EC50s of 240 µM, 460 µM, and > 1 mM, respectively; Fig. 4). TASK-2 is an alkaline pH-activated channel, as illustrated in Figure 4B. The large concentration requirement makes it unlikely that these channels contribute to doxapram's ventilatory effects. As above, benzyl alcohol (0.02%) minimally affected baseline currents for these channels (TASK-2, +16.8% ± 5.4%; n = 5; TRESK, +23.8% ± 4.4%; n = 4); TREK-1, –10.1% ± 9.3%; n = 3).


Figure 417
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  		Figure 4. TWIK-related acid-sensitive K+ TASK-2 (B), TRESK (A), and TREK-1 (C) tandem pore channel activity are inhibited by doxapram at large concentrations. Data were collected in Xenopus oocytes as in Figure 1. (D) Compilation of data from multiple time-course experiments. Because of easier washouts, one or two doxapram doses were applied per oocyte. n = 2–3 ± sd. Error bars omitted when smaller than symbol.

 

Enhancement of TASK channels may be one mechanism by which volatile anesthetics mediate immobility during a noxious stimulus (quantified as MAC) (16). If true, doxapram, which inhibits TASK-channel activity, should be antagonistic and should decrease volatile anesthetic potency. However, we determined that doxapram had no effect on MAC for halothane (n = 4 rats ± sd): 1.05% ± 0.11% (Stage 1), 1.00% ± 0.12% (Stage 2), and 0.96% ± 0.15% (Stage 3) (P = 0.65 by analysis of variance).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we determined that doxapram potently inhibits TASK-1, TASK-3, and TASK-1/TASK-3 heterodimer channel function. Inhibition occurs in the clinical concentration range and is unaffected (at least for the heterodimer) by changes in proton or halothane concentration. Doxapram inhibition at both hyperpolarized (Table 1) and depolarized potentials (Figs. 1–3), as well as effects independent of extracellular potassium concentration, argue against a pore-blocking mechanism of inhibition. TASK-1 is approximately two orders of magnitude more sensitive to doxapram inhibition than TASK-3; other K2P channels, such as TRESK, TASK-2, and TREK-1, are even less sensitive. Using molecular chimeras, we determined that the intracellular carboxy terminus of TASK-1 mediates some of doxapram's effects. This suggests an intracellular site of action; however, we do not know if doxapram inhibits TASK through a direct or indirect mechanism.

Doxapram has an initial redistribution phase that rapidly decreases serum concentrations after a bolus injection. Mitchell and Herbert (1), studying phrenic nerve and carotid sinus nerve activity, determined that a 10-fold larger intraarterial dose into the descending aorta was required to reproduce the effects of a given IV dose, suggesting rapid clearance from the blood. However, of particular relevance to our study, they also demonstrated that a small dose of doxapram (a dose too small to stimulate carotid sinus nerve activity were it given IV) injected directly into the carotid artery produces prolonged effects (two to three minutes). They proposed that doxapram has a prolonged interaction with its effector site or in the tissue surrounding it. In agreement with the observations of Mitchell and Herbert (1), we observed that doxapram has a slowly reversible effect on, particularly, TASK-1, TASK-1/TASK-3 chimeric channels, and TASK-1/TASK-3 heterodimeric channels.

TASK-1 has been implicated as a chemo-sensing molecule important to both central and peripheral ventilatory regulation. TASK-1 and TASK-3 messenger RNA and proteins are expressed in rat Type I cells, and Buckler et al. (6) have identified a Type I cell K conductance with properties similar to that of TASK-1 (14). Electrophysiologic and antisense DNA techniques have implicated TASK channels, particularly TASK-3, in mediating chemo-sensing in neuroepithelial cells of the lung, chemo-sensing cells with properties similar to that of carotid body Type I cells (5). Our data do not prove that TASK-1 (or TASK-3) is involved in chemo-sensing, although they are consistent with such a role. Lahiri et al. (21) determined that 4-AP and TEA, two classic K channel inhibitors, had no effect on carotid body Type I cell chemo-sensing arguing against a role for some K channels in chemo-sensing. However, TASK-1 K channels are resistant to block by 4-AP and TEA (9,11). Although TASK-1 is expressed in the CNS and the carotid bodies, doxapram's peripheral site of action may be caused by pharmacokinetics after a bolus administration. Continuous infusion may be required to achieve more central effects. We do acknowledge that doxapram has effects on other K channels that may contribute to its in vivo mechanism of action and may have a mechanism of ventilatory stimulation, at least in part, different from hypoxia (3,22).

Doses of doxapram in and beyond the clinical range did not change the MAC of rats anesthetized with halothane. These results suggest that TASK-1 and TASK-3 channels do not mediate halothane-induced immobility (MAC). As discussed, halothane and isoflurane enhance TASK-1, TASK-3, and TASK-1/TASK-3 heterodimer channel. Immobility to a noxious stimulus, quantified as MAC, is believed secondary to volatile anesthetic effects on the spinal cord where, among other places, TASK-1 and TASK-3 are expressed. Activation of TASK channels by volatile anesthetics could cause spinal cord neuronal hyperpolarization and quiescence, and such an effect presents a plausible mechanism by which volatile anesthetics might induce immobility. We used doxapram to test this hypothesis and found it to be without effect on MAC for halothane at a dosage that should provide TASK inhibition. Our in vitro data suggest that doxapram inhibits the TASK-1/TASK-3 heterodimer to the same extent in the presence or absence of halothane. It is important to note that we did not measure cerebrospinal fluid (CSF) levels of doxapram in our study and can only assume that they reached a concentration capable of producing TASK-1 inhibition. However, previous work by others has demonstrated that doxapram does cross the blood-brain barrier. For example, during small-dose infusion in human infants, doxapram is detectable in the CSF at a significant fraction of its serum concentration, and its CSF levels correlate with its serum levels (23). Additionally, seizures, a CNS effect, occur in rats and mice after a single IV dose of 75 mg/kg and in cats and dogs after a dose of 40–80 mg/kg (Dopram®, package insert). Halothane is an anticonvulsant, which is probably why we did not observe seizures in our MAC study. Finally, some of doxapram's ventilatory stimulant effects are proposed to occur centrally (1). Results from other studies support our negative result. Eisele et al. (24) demonstrated that acidification of the CSF (less than a pH value of 7.0) decreases MAC for halothane. However, if TASK-channel activity were fundamental to MAC, acidification, which inhibits TASK function, should increase MAC not decrease it. We recognize that acidification of the CSF may cause many effects beyond inhibition of TASK channels. The TASK-2 K2P channel, although possessing limited homology, like TASK-1 and TASK-3, is expressed in the spinal cord and conducts a pH-sensitive and volatile anesthetic-enhanced current. The TASK-2 knockout mouse, despite missing this gene, retains a normal MAC to desflurane, halothane, and isoflurane (25). Besides immobility, which is all that we studied, volatile anesthetics produce other clinical effects including amnesia, analgesia, unconsciousness, and ventilatory depression. TASK-channel activity may yet contribute to some of these other effects.

Further work is required to completely define doxapram's mechanism of inhibition and to establish TASK-1 and TASK-3 as mediators of doxapram's in vivo ventilatory effects, including studies in primary mammalian neurons and knockout or knockdown animals. However, doxapram will be a useful tool for discriminating TASK-1 currents from other native currents, and a more complete understanding of its molecular site of action will foster development of better, more selective ventilatory stimulants.

We thank Mark Liao, John Popovich, and Jesus Quintos for excellent technical support, and Drs. Phil Bickler, John Feiner, Lily Jan, and Jim Sonner for helpful discussions.


   Footnotes
 
Supported, in part, by grants from the Foundation for Anesthesia Education and Research (J.F.C.) and the National Institute for General Medical Sciences – GM58149 (C.S.Y.) and 1P01GM47818 (E.E.).

Accepted for publication September 16, 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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