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Am J Physiol Regul Integr Comp Physiol 281: R401-R407, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 2, R401-R407, August 2001

Ontogeny of humoral heart rate regulation in the embryonic mouse

George A. Porter Jr.1 and Scott A. Rivkees2

Department of Pediatrics, Divisions of 1 Cardiology and 2 Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Catecholamines, acetylcholine, and adenosine are known to influence cardiac function, yet the effects of these agents on mammalian embryonic myocardium are largely unknown. To address this issue, we compared the chronotrophic effects of adenosinergic, adrenergic, and muscarinic agents on cultured murine embryos from postcoital day (PC) 8.0, when the fusing heart tubes first begin to beat, to PC 14, when cardiogenesis is essentially complete. At PC 8.0 and older, A1-adenosine receptor (A1AR) activation significantly decreased heart rates. Adrenergic stimulation caused modest increases in heart rates (145-155% of baseline) beginning at PC 9.0. Muscarinic activation decreased heart rates only after PC 13. When receptor gene expression was examined, A1ARs and beta 1ARs were expressed in isolated hearts as early as PC 9.0, and beta 2ARs and m2-muscarinic receptor genes were expressed at PC 11.0. These results identify the adenosinergic system as the earliest and most potent regulator of embryonic cardiac function and show that prenatal responsiveness to catecholamines and acetylcholine develops at later embryonic stages.

adenosine; catecholamines; acetylcholine; embryo


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN MATURE AND DEVELOPING MAMMALS, cardiac output is influenced by heart rate, stroke volume, and contractility (1, 4, 5, 19, 30). However, in embryos, available evidence suggests that heart rate plays a particularly important role in regulation of cardiac output (1, 4, 5). Factors that alter heart rate may greatly influence cardiac output at early developmental stages.

Previous work has shown that A1-adenosine receptors (A1ARs) are among the earliest expressed G protein-coupled receptors in the heart, with expression first observed at postcoital day (PC) 8.0 in rat embryos (22). A1AR activation potently slows embryonic heart rates as early as PC 9.5, the earliest age examined (9). Theophylline, an adenosine receptor antagonist, increases embryonic mouse heart rates as early as PC 12 (31).

Adrenergic receptors have been detected in mouse hearts at PC 13 and in the rat at PC 12 (2, 25, 29). Increases in heart rates due to adrenergic stimulation have been observed as early as PC 9.5 in mice and at PC 10.5 in rats (8, 15, 23).

Muscarinic receptor mRNA has been detected in rat cardiac tissue at PC 18 (6), and receptor binding sites are present at PC 15 in the mouse (18, 25). Muscarinic stimulation alters heart rates at PC 12-13 in mice and at PC 11.5 in rats (8, 23, 25, 31).

Although available evidence shows that embryonic hearts can respond to extrinsic stimulation, we do not know when cardiac responsiveness to adenosinergic, adrenergic, and muscarinic agents first develops nor do we know the relative importance of these factors in regulating embryonic cardiac function. To further define the ontogeny of adenosinergic, adrenergic, and muscarinic influences on embryonic cardiac physiology, we examined the expression and influence of these receptor systems on embryonic heart rates from the inception of spontaneous contractility at PC 8.0 to the end of cardiac organogenesis at PC 14.0.


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

Animals. The Yale Animal Care and Use Committee approved all studies. C57J/BL mice were exposed to a 12:12-h dark-light cycle with free access to food and water. Males and females were paired and separated when a vaginal plug was observed. The morning a mating plug was observed was designated as PC 0.5.

Cultures. Dams were anesthetized with carbon dioxide and euthanized by cervical dislocation. To obtain embryos, hysterectomy was performed under sterile conditions, and the uteri were rinsed in Dulbecco's PBS with 2 mM MgCl2 at room temperature. Uteri were then transferred at 37°C into DMEM with 10% fetal bovine serum (Fetal Clone II, Hyclone Laboratories, Logan, UT) and 50 mM HEPES buffer, which was used for all subsequent incubations and studies.

While visualized using a dissecting microscope (Zeiss), embryos were separated from uteri and transferred to fresh media for further dissection. Embryonic age was determined by morphology and somite number (12). Embryos younger than PC 10.0 were incubated intact. For older embryos, isolated hearts were studied, as this is required to maintain a consistent heart rate. Embryos or embryonic hearts were transferred to individual wells with 3 ml of media and incubated at 37°C in a 5% CO2-room air incubator. Dose-response experiments were performed after 1-4 h to allow for stabilization of the specimens in culture medium and for dissection of multiple litters. Preincubation for different times between 1 and 4 h did not appear to change our results. In addition, when we examined A1AR, adrenergic, or muscarinic receptor expression during the experiments we found no changes in receptor expression >5 h (not shown).

Heart rate assessments. Heart rate measurements were performed using visualization with a dissecting microscope. Temperature was maintained between 35 and 38°C using a heated stage. Individual plates containing 4-12 embryos were removed from the incubator and placed on the stage for 5 min. The baseline heart rate of each embryo was measured, and drugs or vehicle was added to each well. Heart rates were determined by direct visual counting for 15 s; two measurements were recorded per sample.

To assess the effects of receptor activation on heart rates, receptor agonists, antagonists, and reuptake blockers were applied to the culture media. Except when noted, the doses ranged from 1 nM to 10 µM. For studies of A1AR activation, the agonist N6-cyclopentyladenosine (CPA) and the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were used. Dipyridamole was used to block the reuptake of endogenous adenosine. Adrenergic receptors were stimulated with isoproterenol and inhibited with alprenolol. Tyramine was used to increase levels of endogenous catecholamines. Bethanecol was used to stimulate muscarinic receptors at doses from 100 nM to 10 mM. Atropine was used to antagonize muscarinic receptors, and neostigmine to inhibit acetylcholinesterase and increase endogenous levels of acetylcholine.

To generate dose-response curves, individual embryos were treated with increasing doses of drug without changing the media. Stock solutions of each drug were made fresh daily (isoproterenol, neostigmine) or kept frozen at -20°C per the recommendations of the manufacturer.

Data analysis. Heart rate measurements were transferred to a spreadsheet (Microsoft Excel, Microsoft, Redmond, WA), and average heart rates in beats per minute were calculated. To standardize heart rates from all studies, heart rates were then recorded as a percent change from baseline. Data from specimens were combined, and the mean values for each dose at each age were plotted. Dose-response curves for each drug at each age were generated using the sigmoidal fit function for a dose-response curve in Microcal Origin software (version 6.0, Microcal Software, Northampton, MA). From these curves, the drug concentrations that caused a decline in heart rate to 50% of the baseline (EC50) were obtained. To determine statistically significant changes in the maximal response to a drug (Emax), the maximal responses from each individual specimen were gathered and the mean maximal responses between treatment groups at a given age were compared by ANOVA with Dunnett's post hoc testing using GraphPad Prism (version 2, GraphPad Software, San Diego, CA). A significant difference was defined as P < 0.05.

RT-PCR. Embryos and embryonic hearts were obtained as described above. Hearts from neonatal mice were dissected to separate atria from ventricles. Samples were washed in PBS and frozen on dry ice. Frozen specimens were thawed in Trizol (Life Technology, Grand Island, NY), and RNA was extracted per the manufacturer's instructions. First-strand cDNA synthesis was performed with 1 µg of total RNA using random decamers as primers for Moloney murine leukemia virus reverse transcriptase (Retroscript, Ambion, Austin, TX). PCR was performed using 20 pmol of each gene-specific primer, 0.5 µl of cDNA, and 2.5 U of Hot Star Taq in a final volume of 25 µl as per the manufacturer's instructions (QIAGEN, Valencia, CA). Amplification of the mouse beta 2-adrenergic primers required the use of 20% QIAGEN Q solution. The amplification sequence consisted of 95°C for 15 min; 34 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min; and 72°C for 10 min. The gene-specific primer pairs used were A1AR (5'-ACATTGGGCCACAGACCTACTTCC-3', 5'-GGCGGCAGCACCCAGACGA-3'), beta 1AR (5'-CTCACCAACCTCTTCATCATG-3', 5'-GAAGCGGCGCTCGCAGCTG-3'), beta 2AR (5'-TCCTTAACTGGTTGGGCTAC-3', 5'-AGTCTGGTTAGTGTCCTGTC-3'), and M2 muscarinic receptor (M2MR) (5'-CCGGGCGAGCAAGAGCAGAATAA-3', 5'-GGCGCCCACGTGATGATGAAAG-3'). Primer sequences were based on published data. As a control for total RNA levels, amplification of 18s rRNA was performed. With the use of a ratio of primer to competimer of 1:8, per the manufacturer's instructions (Ambion Alternate 18s Internal Standards), 18s rRNA amplification was exponential in these samples (not shown). PCR products were separated on 2% agarose gels, and images were obtained in a Gel Doc 1000 with Molecular Analyst Software (Biorad, Hercules, CA) and saved as TIFF files. Images were analyzed using National Institutes of Health Image (version 1.62).

Drug and chemicals. PBS, DMEM, and HEPES were obtained from Life Technologies. Adenosine deaminase (ADA) was purchased from Boehringer Manheim (Indianapolis, IN). All other drugs and chemical were obtained from RBI/Sigma (Natick, MA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heart rates. To assess heart rate responses to different pharmacologic agents, we cultured mouse embryos at different stages of cardiogenesis. Before any drugs were applied, baseline heart rates were obtained 1-4 h after dissection, and the length of this preincubation did not appear to affect heart rates. Heart rates ranged from 62.5 beats/min at PC 8.0-8.5 to 119 beats/min at PC 10.0-11.0 (Fig. 1). Heart rates at PC 10.0-12.0 were similar to those reported for mouse embryos studied in vivo using fetal Doppler ultrasonography or in situ via hysterotomy (7, 13, 16, 30). In control experiments, heart rates were found to be stable throughout the time required to perform a dose-response curve, as demonstrated by the lack of significant variation of vehicle-treated specimens (Fig. 2, Table 1).


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  		Fig. 1.   Baseline heart rates of cultured embryos at different ages. Mean heart rates at baseline ± SE are shown vs. age. Between 44 and 82 embryos were studied per group. Mean heart rates rose from 62.5 beats/min (bpm) at postcoital day (PC) 8.0 to 119 beats/min at PC 10.0. After PC 10.0, heart rates fell to 76.1 beats/min at PC 13.0-14.0.



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  		Fig. 2.   Dose-response curves of adenosinergic agents at PC 8.0-8.5. Specimens were treated with increasing doses of vehicle, N6-cyclopentyladenosine (CPA), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), and dipyridamole. Heart rates were normalized to baseline, and curves were fitted to the data using a sigmoidal fit function. Individual data points and SE used to derive these curves are also presented. CPA caused a dose-dependent decrease in heart rate to a minimum of 65.2% of baseline. Treatment with vehicle, DPCPX, and dipyridamole had no effect at this age.


                              
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  		Table 1.   Maximal responses to each agent at each postcoital age

Responses to adenosinergic agents. Our previous studies demonstrated that A1ARs are expressed in the heart at the inception of rhythmic cardiac contractions and that activation of these receptors slows heart rates as early as PC 9.5 (9, 22). To extend these functional studies, embryonic heart rate responses to CPA (A1AR agonist), DPCPX (A1AR antagonist), and dipyridamole (adenosine reuptake inhibitor) were tested. Embryos were studied from PC 8.0 to PC 14.0. During initial experiments, the Emax to CPA increased markedly between PC 8.0 and PC 10.0. Therefore, specimens were divided into age groups by half-day increments (PC 8.0-8.5, PC 8.5-9.0, PC 9.0-9.5, and PC 9.5-10.0). Older specimens were divided into full-day age groups (PC 10.0-11.0, PC 11.0-12.0, PC 12.0-13.0, PC 13.0-14.0).

Treatment of embryos with 1 nM to 10 µM CPA decreased heart rates in a dose-dependent manner at all ages. However, Emax to CPA treatment increased with age. At PC 8.0-8.5, CPA decreased heart rates to 65.2% of baseline (Fig. 2, Table 1). Over the next 2 days, dose-dependent responses increased until complete asystole was observed after PC 11.0 (Fig. 3, Table 1). At all ages tested, the EC50 was between 9 and 50 nM.


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  		Fig. 3.   Dose-response curves of CPA-treated embryos at different ages. Dose responses to treatment with CPA were generated by a sigmoidal fit function at each age interval. The curves demonstrate increasing maximal effect (Emax) with age but a relatively constant half-maximal drug concentration (EC50). All Emax values are significantly different from vehicle control (P < 0.01).

We next studied if adenosine released into the culture medium by the embryo affects heart rates. First, some specimens were treated with ADA, which degrades endogenous adenosine. The baseline heart rates were not different in the presence or absence of ADA (not shown). Second, we treated specimens at all ages with DPCPX. We found no consistent response, although a significant increase in heart rates was observed at PC 12.0-13.0 (Fig. 4, Table 1). Third, we treated embryos with dipyridamole, which decreased heart rates at all ages except PC 8.0-8.5 (Fig. 4, Table 1). Thus basal concentrations of endogenous adenosine do not appear to affect embryonic heart rates. Yet, increasing the local levels of adenosine with reuptake inhibitors can decrease heart rates.


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  		Fig. 4.   Emax to adenosinergic agents vs. age. The effects of treatment with CPA and dipyridamole increase over the entire period of organogenesis. DPCPX has a significant effect only at PC 12.0-13.0. *Significant effects compared with vehicle control at the same age (P < 0.05).

Responses to adrenergic and muscarinic agents. We then determined the effects of adrenergic and muscarinic receptor stimulation on heart rate. To assess the ontogeny of adrenergic responsiveness, we treated embryos with the nonspecific beta -adrenergic agonist isoproterenol. From PC 8.0 to 9.0, no significant alterations in heart rates were observed, even with maximal doses of isoproterenol (Fig. 5, Table 1). From PC 9.0-10.0 and 11.0-14.0, isoproterenol treatment increased heart rates to Emax of 145-155% of baseline (Fig. 5, Table 1) with an EC50 of 10-50 nM. Interestingly, from PC 10.0 to 11.0 no significant changes in heart rates were observed in 10 embryos from five different experiments.


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  		Fig. 5.   Emax to adrenergic agents vs. age. The increase in heart rate in response to treatment with isoproterenol becomes stable after PC 9.0, except at PC 10.0-11.0. Alprenolol and tyramine had no effect at any age. *Significant effects compared with vehicle control at the same age (P < 0.05).

We next treated embryos with the beta -adrenergic antagonist aloprenolol to determine if endogenous catecholamines alter heart rates in our culture system. Alprenolol had no significant effect on embryonic heart rates at any age tested (Fig. 5, Table 1). Treatment with tyramine, which increases endogenous catecholamine levels (11), also had no effect at any age (Fig. 5, Table 1). These observations suggest that endogenous catecholamines do not exert significant effects on heart rates during the embryonic period.

Finally, we examined the ontogeny of muscarinic responsiveness using a muscarinic agonist (bethanecol), a muscarinic antagonist (atropine), and an acetylcholine esterase inhibitor (neostigmine). Treatment with bethanecol caused significant decreases in heart rates only at PC 13.0-14.0 (Fig. 6, Table 1). Neither atropine nor neostigmine caused significant alterations in heart rates at any age (Fig. 6, Table 1). Therefore, muscarinic receptor activation appears to have little affect on murine heart rate until late in cardiac development.


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  		Fig. 6.   Emax to muscarinic agents vs. age. The only significant alteration in heart rate is observed in PC 13.0-14.0 embryos with bethanecol treatment. *Significant effects compared with vehicle control at the same age (P < 0.05).

Receptor expression assays. Our physiological measurements of receptor activation demonstrated that adenosinergic, adrenergic, and muscarinic receptors become functional at PC 8.0, 9.0, and 13.0, respectively. To assess if receptor expression correlated with these functional data, gene expression was qualitatively examined with PCR. RNA was obtained from specimens at the ages when changes in the physiological responsiveness were observed in the experiments above. In addition, expression in older embryos (PC 17) and neonatal atria and ventricles was performed for comparison. Using primers specific for mouse A1AR, beta 1AR, beta 2AR, and M2MR, we observed that each receptor subtypes were expressed in whole murine embryos at PC 8.0 (Fig. 7). Because of their small size, it was not possible to isolate hearts at this age. However, for PC 9.0 and older embryos, we examined RNA obtained from isolated hearts. These experiments demonstrated that A1ARs were expressed from PC 9.0 and later. beta 1AR was present in very low levels at PC 9.0, and at higher levels in older embryos. beta 2ARs were expressed in low levels at PC 11.0 and later. M2MR message was detectable at PC 11.0 and at later ages.


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  		Fig. 7.   RNA expression of A1-adenosine receptor (A1AR), beta -adrenergic receptor (AR), beta 2AR, and M2 muscarinic receptor (M2MR). RT-PCR was performed using RNA from whole embryos at PC 8.0 (lane 1); from embryonic hearts at PC 9.0 (lane 2), 11.0 (lane 3), 13.0 (lane 4), and 17.0 (lane 5); or from neonatal atria (lane 6) or ventricles (lane 7). All receptors are expressed in whole embryos at PC 8.0, but only A1AR and low levels of beta 1AR are present in PC 9.0 hearts (as demonstrated by increased exposure of lane 2). All receptors are expressed at PC 11.0 and later, but at different relative concentrations. RT-PCR of 18s rRNA in each specimen is presented to compare relative levels of RNA between samples.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is generally believed that humoral heart rate control is dependent on the stimulation of adenosinergic, adrenergic, and muscarinic receptors (19, 28). However, the role that these receptor systems play during early development is not known. Our results identify the adenosinergic system as the earliest expressed and most potent humoral regulator of embryonic cardiac function. In addition, we demonstrate that adrenergic and muscarinic receptor activation influences heart rate later in gestation.

At all ages tested, A1ARs were functional. At older ages, A1AR activation more potently influenced heart rates than at younger ages, with a decline in heart rate to 65.2% of baseline at PC 8.0-8.5 followed by a steadily decreasing heart rate until PC 11.0-12.0, when asystole occurred. These data agree with and extend previous observations (9) and demonstrate that heart rates can be regulated from the inception of spontaneous cardiac contractions. These functional data also agree with studies of receptor expression showing A1AR gene expression in isolated hearts as early as PC 9.0. We also found that A1ARs are expressed in whole embryos at PC 8.0. In agreement with our observations, previous studies using in situ hybridization demonstrated that A1ARs are expressed in rat hearts at a developmental stage equivalent to mouse PC 8.5 (22).

Currently, the cellular mechanisms for the change in responsiveness to A1AR activation over time are not known. Our RT-PCR data suggest that increases in A1AR expression may explain some of the increase in responsiveness, but these experiments were not designed to obtain absolute concentrations of A1AR message. Additional developmental changes in the functional coupling of adenosine receptors to their secondary messengers, secondary messenger systems themselves (e.g., G proteins, adenylyl cyclase, or protein kinases), or effector systems (e.g., ion channels or transporters) may also be responsible.

Responsiveness to adrenergic stimulation has been previously demonstrated in the mouse heart as early as PC 9.5 (15). Our data extend the earliest known age of adrenergic receptor expression and function to PC 9.0. Adrenergic control of heart rate from PC 9.0 to 11.0 is most likely due to activation of beta 1AR, as no beta 2AR mRNA was detected before PC 11.0. Although our RT-PCR data were not quantified, the suggestion of lower levels of beta 2AR expression compared with that of beta 1AR is also consistent with studies in mature mammals (24).

Muscarinic receptor activation decreased heart rates only after PC 13.0, although M2MR gene expression was detected in hearts as early as PC 11.0. It appears unlikely that the discrepancy between receptor expression and functionality would be due to insensitivity in our measurements of heart rates. Therefore, these data suggest that the coupling of these receptors to their secondary messenger systems is also developmentally regulated, as indicated in previous studies (17). Alternatively, before PC 13, cardiac muscarinic receptors might perform functions other than heart rate control.

To determine if endogenous adenosine, catecholamines, and acetylcholine activate their respective receptors during early development, we also tested the effects of receptor antagonists and of agents that increase levels of these endogenous compounds. We saw no consistent alterations in heart rates due to treatment with any antagonist, indicating that local adenosine, catecholamines, and acetylcholine have no effect on basal heart rates under the conditions used. However, profound declines in heart rates were seen in all but the youngest embryos (PC 8.0-8.5) after treatment with dipyridamole. Therefore, although basal levels of intracellular adenosine have little effect on heart rates under these conditions, increases in the concentration of endogenous adenosine have dramatic effects. In contrast to adenosine reuptake studies, the absence of response to treatment with tyramine and neostigmine suggests that there are insufficient stores of catecholamines and acetylcholine present to alter heart rates at these stages of development.

We recognize some limitations of our approach. The embryonic culture system used allowed us to reliably assess changes in heart rats but was not an in vivo model. From PC 8.0 to 10.0, embryos were also cultured without an intact placenta, and from PC 10.0 to 14.0, cultures of isolated hearts were studied to maintain rhythmic beating. Under these conditions, the preload and afterload placed on the embryonic heart are nonphysiological. In addition, these isolated hearts lack any sympathetic or parasympathetic input. Although the effects of these conditions on heart rate control in the developing embryo are unknown, there is evidence that such conditions did not significantly affect our results. First, we observed that baseline heart rates using this culture system were very similar to those obtained in vivo in mice (7, 13, 16, 30). Second, although heart rate is dependent on stroke volume and thus preload, afterload, and contractility via neural feedback loops in mature mammals, the sympathetic and parasympathetic neurons first innervate the heart much later in gestation than the ages we studied (6, 20). Furthermore, we are unaware of other systems in which one may treat individual murine embryos with specific concentrations of drugs for a prolonged period without either affecting maternal or placental physiology.

In summary, we demonstrate that A1ARs influence heart rates beginning at PC 8.0, adrenergic receptors affect heart rates after PC 9.0, and muscarinic receptors affect heart rates only after PC 13. We also report that adenosine receptor activation has a more profound effect on embryonic heart rates than that of either adrenergic or muscarinic receptor activation. These studies identify the adenosinergic system as the most potent humoral regulator of mammalian embryonic cardiac function.

Perspectives

These observations emphasize the importance of local control of heart rate in the early embryo. The maintenance of cardiac output is vital to embryonic growth and survival (13, 26, 30). In chicken and mouse embryos, cardiac output appears to be largely dependent on heart rate (4, 5, 13, 30). Thus local, adenosine-mediated changes in heart rates would be expected to have a more profound effect on cardiac output than activation of adrenergic or muscarinic receptors, which depends on external sources of agonist (6, 20, but see Ref. 10).

In comparison with catecholamines and acetylcholine, adenosine also is an ideal regulator of embryonic cardiac function. Unlike catecholamines and acetylcholine, intracellular adenosine concentrations are not dependent on intercellular stores. All cells constantly produce this nucleoside, and conditions that favor the breakdown of ATP, such as hypoxia or increased cellular metabolism, lead to an increase in extracellular adenosine concentrations. It is well recognized in large mammals that fetal heart rates fall during hypoxic stress (21), when levels of both adenosine and catecholamines rise (3, 14, 27). Similar effects might be expected in small mammals, but we are unaware of such experiments. Our observations in cultured embryos, though, indicate that this phenomenon reflects the responsiveness of heart rate to adenosine and show the dominance of adenosine action in the embryonic period. These data may also explain the mechanism of stress-induced fetal bradycardia.


    ACKNOWLEDGEMENTS

G. A. Porter, Jr., is a Pfizer Postdoctoral Fellow. This work was supported by National Heart, Lung, and Blood Institute Grant HL-58442 to S. Rivkees. S. Rivkees is a Donaghue Medical Research Foundation Investigator.


    FOOTNOTES

Address for reprint requests and other correspondence: S. A. Rivkees, Dept. of Pediatrics, Division of Endocrinology, Yale Univ. School of Medicine, 239 YCHRC, 464 Congress Ave, PO Box 208081, New Haven, CT 06520-8081 (E-mail: scott.rivkees{at}yale.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.

Received 2 November 2000; accepted in final form 15 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 281(2):R401-R407
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