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Am J Physiol Heart Circ Physiol 279: H2620-H2626, 2000;
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Vol. 279, Issue 6, H2620-H2626, December 2000

Human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and K+ channels

Ken Terata, Hiroto Miura, Yanping Liu, Fausto Loberiza, and David D. Gutterman

Cardiovascular Research Center, Medical College of Wisconsin and Zablocki Veterans Administration Medical Center, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adrenomedullin (ADM) is a vasodilator produced by vascular endothelium and smooth muscle cells. Although plasma ADM levels are increased in patients with hypertension, heart failure, and myocardial infarction, little information exists regarding the microvascular response to ADM in the human heart. In the present study we tested the hypothesis that ADM produces coronary arteriolar dilation in humans and examined the mechanism of this dilation. Human coronary arterioles were dissected and cannulated with micropipettes. Internal diameter was measured by video microscopy. In vessels constricted with ACh, the diameter response to cumulative doses of ADM (10-12-10-7 M) was measured in the presence and absence of human ADM-(22-52), calcitonin gene-related peptide-(8-37), Nomega -nitro-L-arginine methyl ester (L-NAME), indomethacin (Indo), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, SQ-22536, or KCl (60 mM). ADM dilated human coronary arterioles through specific ADM receptors (maximum dilation = 69 ± 11%). L-NAME or N-monomethyl-L-arginine attenuated dilation to ADM (for L-NAME, maximum dilation = 66 ± 7 vs. 41 ± 13%, P < 0.05). Thus the mechanism of ADM-induced dilation involves generation of nitric oxide. However, neither 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, SQ-22536, nor Indo alone altered dilation to ADM. High concentrations of KCl blocked dilation to ADM. The magnitude of ADM dilation was reduced in subjects with hypertension. We propose that, in human coronary arterioles, ADM elicits vasodilation in part through production of nitric oxide and in part through activation of K+ channels, with little contribution from adenylyl cyclase. The former dilator mechanism is independent of the more traditional pathway involving activation of soluble guanylate cyclase.

coronary disease; calcitonin gene-related peptide; hypertension; congestive heart failure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ADRENOMEDULLIN (ADM) is a novel vasodilator and natriuretic peptide consisting of 52 amino acids (17). It is a member of the calcitonin gene-related peptide (CGRP) family (17). Plasma ADM levels are increased in patients with hypertension (10, 19, 34), congestive heart failure (13, 14, 24), acute myocardial infarction (36), and chronic renal failure (10) in proportion to the clinical severity of the disease. Since ADM is produced from the vascular endothelium (9, 32) and smooth muscle cells (33), which also possess specific ADM receptors (4, 15), and since ADM has a potent and long-lasting hypotensive effect (11), it is plausible that ADM plays an important autocrine or paracrine role in regulating vascular tone. Animal studies suggest that the hypotensive activity of ADM is based on its ability to increase the concentration of cAMP (17); however, in some vascular beds, including human forearm resistance vessels, nitric oxide (NO) contributes to the response (5, 6, 8, 27, 35). Opening vascular smooth muscle cell K+ channels is also important to ADM-mediated dilation in dog coronary arteries (29) and rat cerebral arterioles (21). Thus significant heterogeneity in the mechanisms of dilation exist among species and vascular beds studied (26, 27). Part of this variability may reflect activation of CGRP receptors by ADM in certain vascular beds. The amino acid sequence of ADM has a large degree of homology to CGRP, and vasodilation to ADM can be blocked by a CGRP receptor blocker in the isolated rat mesenteric artery and cerebral arterioles (23, 28).

Plasma levels of ADM are increased in cardiovascular diseases such as hypertension, heart failure, and myocardial infarction (14, 19, 36). However, ADM-mediated vasomotor control in resistance vessels is impaired in patients with heart failure (24). We hypothesized that ADM produces endothelium-dependent vasodilation of human coronary arterioles through activation of K+ channels in vascular smooth muscle. Furthermore, we predicted that dilation to ADM is reduced in patients with hypertension or congestive heart failure. In the present study we examined responses to ADM in human coronary arterioles where vasodilation to shear stress (7) and several pharmacological agonists is largely dependent on release of endothelium-derived hyperpolarizing factor and where NO plays a minor role in regulating coronary arteriolar dilation (22, 31).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue acquisition and general protocol. A piece of human right atrial appendage, removed for cannulation during cardiopulmonary bypass, was obtained at the time of cardiac surgery and placed in cold 4°C HEPES buffer solution. Arterioles (50-150 µm diameter, ~2 mm long) were dissected from adjacent tissue and cleaned of fat and connective tissue. In a 20-ml tissue chamber, both ends of the arteriole were secured to impedance-matched glass pipettes (internal tip diameter 40 µm) by use of 10-0 Ethilon monofilament nylon suture (Ethicon). Vessels were bathed continuously with a cold bicarbonate buffer (physiological saline solution) consisting of (in mM) 123.0 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 16 NaHCO3, 1.2 KH2PO4, and 11 glucose. The preparation was then transferred to the stage of an inverted microscope (magnification ×200, Olympus CK2). Attached to the microscope were a videocamera (model WV-BL200, Panasonic), a video monitor (Panasonic), and a calibrated video measurement system (model VIA-100K, Boeckeler Instruments). Internal diameter (resolution 2 µm) was measured by manually adjusting the video micrometer. The vessel was pressurized to a predetermined level by simultaneously adjusting the height of each reservoir attached to the pipettes. Vessels were incubated in oxygenated physiological saline solution (21% O2-5% CO2-74% N2) for 30 min at 20 mmHg pressure and 37°C. Pressure was slowly increased to 60 mmHg with a subsequent 30-min incubation period. A final pressure of 60 mmHg was selected on the basis of estimates of physiological pressure in 100-µm coronary arterioles (1). All chemicals were obtained in powdered form from Sigma Chemical (St. Louis, MO) except ADM, which was purchased from Bachem (Torrance, CA).

Experimental protocols. All pharmacological agents were added to the external bathing solution. After a 30-min equilibration period at 60 mmHg, vessels were constricted with 75 mM KCl. Vessels that constricted >30% from resting internal diameter were used for subsequent experiments. Inhibitors or vehicle was added to the chamber on warming, with any change in diameter recorded.

Most vessels demonstrated some myogenic tone when warmed at 60 mmHg pressure. ACh was added (average dose 50 ± 6 nM) to further constrict vessels to a goal of 30-50% of their passive diameters. To confirm that responses were not specific for the constrictor used, in some experiments endothelin-1 (10-10-10-9 M), instead of ACh, was used to constrict arterioles. Although the response of human coronary arterioles to ADM constricted with ACh or endothelin-1 was not significantly different (data not shown), we used ACh for the remainder of the experiments, since ADM has been shown to interact with endothelin-1 in vascular smooth muscle (20). The endothelium-independent dilator sodium nitroprusside (SNP, 10-4 M) was used to determine the maximal diameter at 60 mmHg (passive diameter). Since we performed two consecutive dose-response relationships to ADM (before and after antagonists), for purposes of paired comparisons, time-control experiments were carried out separately.

Mechanism of the dilation to ADM. Since ADM is a member of the CGRP superfamily and since ADM activates CGRP receptors in some vascular beds, we tested the effect of the putative ADM receptor antagonist fragment ADM-(22-52) (10-7 M) and the CGRP receptor antagonist fragment CGRP-(8-37) (10-6 M) on ADM- and CGRP-induced dilation.

In some experiments the endothelium was removed by injection of 2 ml of air through the lumen of the cannulated vessel. After this procedure, the dilation to ADP (10-5 M) was abolished, while dilation to the endothelium-independent agonist SNP was preserved, confirming the efficacy of the technique in denuding the endothelium.

In separate studies we examined the effect of Nomega -nitro-L-arginine methyl ester (L-NAME, 10-4 M), indomethacin (Indo, 10-5 M), and KCl on the dilation to ADM. In some cases, the specific inhibitor of guanylate cyclase 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 5 × 10-5 M) or the specific adenylyl cyclase inhibitor SQ-22536 (10-4 M) was used.

Statistical analysis. Values are means ± SE. Percent dilation was calculated as the percent change from the constricted diameter to the maximal passive diameter (maximal diameter in the experiment at 60 mmHg luminal pressure), which was generally the diameter after SNP (10-4 M). Percent constriction was determined by calculating the percent reduction of maximal diameter after application of the constricting agent (ACh or endothelin-1).

Each experimental group (presence of pharmacological inhibitor or endothelial denudation) was compared with its corresponding paired control group. Separate models were constructed to compare the percent maximum dilation between the control and experimental group using two-factor repeated-measures ANOVA. The type of treatment (e.g., control vs. denudation) and the regularly spaced dosages were the two factors examined in a fixed-effect model fashion. Interaction between treatment and dose was first examined, and only if P < 0.05 was a specific dose-effect contrast done using Bonferroni's adjusted paired t-test. Secondary models were likewise constructed comparing dose-treatment responses between vessels from diseased and nondiseased subjects while adjusting for the presence of other significant covariate disease/variables (e.g., hypertension vs. no hypertension with control for diabetes and age). All two-factor repeated-measures modeling was done using autoregressive covariance structure after comparison of the model's Akaike's information criterion with several covariate models. All computations were done using the proc MIXED procedure in SAS version 6.12 for Windows.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Right atrial appendage tissue was obtained from 56 patients. One arteriole (average 81 ± 5 µm ID) was used from each patient. Patient demographic information is summarized in Table 1.

                              
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  		Table 1.   Patient information

Vasodilation to ADM. ADM (10-12-10-7 M) dilated human coronary arterioles in a dose-dependent fashion, with the dilation to a dose of 100 nM being 70 ± 10% (Fig. 1). No tachyphylaxis to repeated application (spaced 45-50 min apart) was observed. A similar dose-dependent and reproducible response was observed to graded doses of CGRP (10-12-10-7 M; data not shown).


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  		Fig. 1.   Repeated application of adrenomedullin (ADM). ADM reproducibly induced dose-dependent dilation on 2 consecutive applications 45-50 min apart, indicating no tachyphylaxis. Maximum dilation was 70 ± 10 vs. 63 ± 8% (not significant), and -log ED50 was 9.2 ± 0.3 vs. 9.2 ± 0.3 (not significant). Passive diameter was 78 ± 11 µm (n = 4).

Dilation to CGRP was inhibited by CGRP-(8-37), a selective blocker of CGRP1 receptors (Fig. 2). These data indicate that the dose and mode of application of CGRP-(8-37) (10-6 M) were sufficient to inhibit the dilation mediated by activation of the CGRP receptor. However, the same dose of CGRP-(8-37) (10-6 M) did not affect the dilation to ADM (Fig. 3).


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  		Fig. 2.   Effect of calcitonin gene-related peptide (CGRP) receptor antagonist on dilation to CGRP. CGRP produced potent vasodilation, which was inhibited by CGRP-(8-37). Maximum dilation was 95 ± 2 vs. 68 ± 8%; -log ED50 was 10.2 ± 0.4 vs. 8.5 ± 0.2 (n = 4, P < 0.05). Passive diameter was 102 ± 18 µm. These data suggest that 10-6 M CGRP-(8-37) was sufficient to inhibit dilation mediated by activation of the CGRP receptor. #P < 0.05 vs. control.



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  		Fig. 3.   Effect of CGRP receptor antagonist on dilation to ADM. CGRP-(8-37) (10-6 M) produced minor nonsignificant changes in baseline diameter (1.7 ± 1%) and did not affect dilation to ADM. Maximum dilation was 72 ± 5 vs. 68 ± 7%; -log ED50 was 9.2 ± 0.4 vs. 9.2 ± 0.4 (not significant). Passive diameter was 80 ± 17 µm (n = 5). Although ADM has sequential homology to CGRP and, in some cases, activates CGRP receptors, our results suggest that, in human coronary arterioles, ADM-induced dilation is mediated by specific ADM receptors and not by activation of receptors for CGRP.

ADM-induced dilation was significantly attenuated by ADM-(22-52) (10-7 M), a putative ADM receptor antagonist (Fig. 4; P < 0.05 vs. control). Neither CGRP-(8-37) (10-6 M) nor ADM-(22-52) (10-7 M) changed baseline diameter.


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  		Fig. 4.   Effect of ADM receptor antagonist on dilation to ADM. Although it did not affect baseline diameter (0.4 ± 1%), ADM-induced dilation was significantly attenuated by ADM-(22-52) (10-7 M). Maximum dilation was 64 ± 12 vs. 30 ± 11%; -log ED50 was 11 ± 1 vs. 9 ± 1. Passive diameter was 120 ± 12 µm (n = 6). #P < 0.05 vs. control.

Effect of endothelial denudation and pharmacological antagonists. Dilation to ADM was abolished in vessels denuded of endothelium (Fig. 5; n = 4, P < 0.05). L-NAME (10-4 M) or L-NAME (10-4 M) + Indo (10-5 M) produced only minor, nonsignificant changes in baseline diameter (-4 ± 1 and -8 ± 3%, respectively). L-NAME, L-NAME + Indo, or Nomega -monomethyl-L-arginine (10-4 M) markedly reduced dilation to ADM (Fig. 6; P < 0.05 vs. control). However, the soluble guanylate cyclase inhibitor ODQ (5 × 10-5 M) did not affect dilation to ADM (Fig. 7A), whereas the same dose of ODQ reduced dilation to SNP (Fig. 7B). In separate vessels constricted with KCl (60 ± 5 mM) rather than ACh, dilation to ADM was prevented (Fig. 8; P < 0.05 vs. control). The magnitude of constriction to ACh and KCl was similar (39 ± 4 and 39 ± 2%, respectively, not significant).


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  		Fig. 5.   Endothelial denudation. The effect of ADM was significantly attenuated by endothelial denudation, which abolished the vasorelaxant effect of ADP (10-5 M) and preserved the vasodilation to sodium nitroprusside (SNP, 10-4 M; data not shown). Maximum dilation was 69 ± 2 vs. 20 ± 13% (P < 0.05 vs. control). Passive diameter was 114 ± 14 µm (n = 5). #P < 0.05 vs. control.



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  		Fig. 6.   Effect of nitric oxide (NO) synthase and cyclooxygenase inhibition on dilation to ADM. Dilation to ADM was attenuated by Nomega -nitro-L-arginine methyl ester (L-NAME, 10-4 M; A) or by Nomega -monomethyl-L-arginine (L-NMMA, 10-4 M; B) alone. L-NAME (10-4 M) + indomethacin (Indo, 10-5 M; D) also reduced dilation to ADM; however, Indo alone (C) did not. Maximum dilation (control vs. antagonist) was 66 ± 7 vs. 41 ± 13% (P < 0.05; A), 79 ± 8 vs. 27 ± 4% (P < 0.05; B), 75 ± 8 vs. 70 ± 7% (C), and 71 ± 9 vs. 55 ± 12% (P < 0.05; D); -log ED50 was 9.1 ± 0.3 vs. 8.1 ± 0.2 (P < 0.05; A), 8.8 ± 0.4 vs. 7.0 ± 0.2 (P < 0.05; B), 9.1 ± 0.5 vs. 8.7 ± 0.4 (C), and 9.3 ± 0.2 vs. 8.1 ± 0.2 (P < 0.05; D), respectively. Passive diameter was 82 ± 5 µm. These data indicate that NO contributes to dilation induced by ADM. #P < 0.05 vs. control.



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  		Fig. 7.   Effect of 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) on dilation to ADM. A: ODQ (5 × 10-5 M) did not affect dilation to ADM. Maximum dilation was 85 ± 5 vs. 88 ± 5%; -log ED50 was 9.2 ± 0.3 vs. 8.7 ± 0.3 (not significant). Passive diameter was 82 ± 14 µm. B: 5 × 10-5 M ODQ is sufficient to block dilation to the NO donor sodium nitroprusside (SNP) (10-4 M). Maximum dilation was 94 ± 2 vs. 18 ± 7% (P < 0.05). #P < 0.05 vs. control.



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  		Fig. 8.   Effect of KCl on dilation to ADM. ADM-induced dilation was completely inhibited in the vessel preconstricted with KCl (60 mM) instead of endothelin-1. Maximum dilation was 67 ± 11 vs. 4 ± 7% (P < 0.05). Passive diameter was 74 ± 10 µm. #P < 0.05 vs. control.

To determine whether inhibition of adenylyl cyclase reduced the coronary arteriolar dilation to ADM, we tested the effect of the specific adenylyl cyclase inhibitor SQ-22536 (10-4 M). This dose of SQ-22536 inhibited dilation to forskolin (data not shown) but had no effect on dilation to ADM (dilation in absence vs. presence of SQ-22536 at increasing concentrations of ADM: 10 ± 0.1 vs. 10.4 ± 0.6% at 10-12 M, 21 ± 10 vs. 21.5 ± 0.8% at 10-11 M, 37 ± 10 vs. 39.4 ± 2.3% at 10-10 M, 50 ± 10 vs. 51.3 ± 3.9% at 10-9 M, 56 ± 8.5 vs. 62.5 ± 1.5% at 10-8 M, and 78 ± 12.2 vs. 76 ± 0.8% at 10-7 M).

Effect of disease. Since plasma levels of ADM are increased in cardiovascular disease (13, 14, 18, 19, 36), we evaluated whether the presence of cardiovascular disease influenced the dilation to ADM. With the presence of known risk factors of coronary artery disease (CAD) taken into account, it was determined that, in patients with hypertension, ADM produced significantly less dilation (Fig. 9). Neither congestive heart failure, myocardial infarction, diabetes mellitus, nor hypercholesterolemia affected the dilation to ADM (data not shown).


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  		Fig. 9.   Effect of hypertension on dilation to ADM. ADM-induced dilation was significantly inhibited in vessels from patients with hypertension [HTN(+), n = 16] compared with nonhypertensive patients [HTN(-), n = 31]. Maximum dilation was 75 ± 5 vs. 51 ± 6% (P < 0.05). Statistics were analyzed using other factors, including coronary artery disease, diabetes mellitus, hypercholesterolemia, heart failure, age, and gender, as covariates. None of these other factors were independently associated with a reduced dilation to ADM. #P < 0.05 vs. HTN(-).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The key findings of this study are fourfold. First, ADM is an endothelium-dependent dilator in human coronary arterioles. Second, production of NO plays an important role in ADM-induced vasodilation but likely not through activation of cGMP. Third, ADM-induced dilation is not inhibited by blockade of CGRP receptors in human coronary microvessels. This observation is important, since ADM has ~20% sequential homology with CGRP (17) and activates CGRP receptors in some vascular beds (23, 28). Finally, dilation to ADM involves a change in K+ gradients in the sarcolemma.

The mechanism of ADM-induced vasodilation depends on the vascular bed studied. ADM was discovered as a peptide that increases cAMP levels in platelets (17). Furthermore, ADM stimulates cAMP formation in cultured vascular smooth muscle cells (3, 4, 12); thus activation of the cAMP pathway has been implicated in ADM-induced dilation. However, some studies have determined that a portion of the ADM-induced dilation is endothelium dependent and may involve formation of NO, possibly independent of adenylyl cyclase (30). ADM independently activates K+ channels in the canine coronary vascular bed (29) and in rat cerebral arterioles (21). Thus there are a variety of mechanisms by which ADM may induce vasodilation. In human coronary arterioles, up to 50% of the ADM-induced dilation was inhibited by an arginine analog, suggesting a prominent role for NO. The entire response could be eliminated by KCl, implicating membrane hyperpolarization. From the data presented here, it is difficult to make definitive conclusions regarding the interactions between NO and hyperpolarizing factors, although it is clear that both contribute to ADM-mediated dilation.

No effect on ADM-induced dilation was observed in the presence of the soluble guanylate cyclase inhibitor ODQ in concentrations sufficient to markedly attenuate the vasodilation to 10-4 M SNP. This was a rather surprising finding, since L-NAME inhibited a substantial portion of the dilation to ADM. These findings might be explained by alternative or invoked compensatory dilator mechanisms linked to activation of the ADM receptor. Such mechanisms would not be activated by administration of SNP. Collectively, these data are consistent with the previously demonstrated importance of vascular smooth muscle hyperpolarization in the dilation of human coronary arterioles (22). KCl nonspecifically blocks hyperpolarization-induced dilation. Future studies should be designed to identify the specific K+ channels or K+ exchange mechanisms involved in ADM-induced dilation.

In contrast to animal studies, where ADM elicits greater endothelium-independent relaxation in isolated basilar arteries from spontaneously hypertensive rats than from controls (25), hypertension reduced the vasodilation to ADM in humans, but congestive heart failure, myocardial infarction, diabetes mellitus, or hypercholesterolemia did not. This observation is tempered by the small sample of the study. Nakamura et al. (24) demonstrated that the potent and long-lasting NO-mediated dilation to ADM in normal human peripheral vessels was significantly attenuated in patients with heart failure. We cannot explain the difference in findings, but by design, our vessels were obtained from diseased subjects, most of whom had severe coronary atherosclerosis, possibly resulting in endothelial dysfunction. This could obscure an enhanced dilation to ADM, as observed by others. There have been conflicting reports as to the relative potencies of ADM and CGRP (5, 28). Cockcroft et al. (2) reported that ADM is more potent than CGRP in relaxing resistance and capacitance vessels of the healthy human forearm. However, in our subjects, ADM exhibited less potent vasodilator activity than CGRP (maximum dilation at 10-7 M = 69 ± 11 vs. 95.2 ± 2%, -log ED50 = 9.2 ± 0.8 vs. 10.2 ± 0.4). Possible reasons for this difference include the different vascular bed studied and the presence of coronary disease in our subjects.

Potential problems. A limitation of all studies involving human cardiac tissue is the lack of disease-free controls. All patients undergoing cardiopulmonary bypass have some disease that may influence the vascular response to ADM. We attempted to account for disease by statistical means. Although this subject population limits our ability to assess true physiological responses, it provides a unique opportunity to determine vascular responsiveness in an important pathological condition with direct clinical relevance. There was no apparent difference between studies on 8 vessels from subjects with no coronary artery disease (CAD) (3 vessels from children) and studies of 32 subjects with CAD (mean maximal dilation = 68 ± 3 and 76 ± 7% in subjects with and without CAD, respectively).

We studied only intermediate-sized arterioles. Larger or smaller vessels may respond differently, but the arterioles we studied are likely to contribute most to alterations in coronary vascular resistance (1).

In summary, the present study suggests that ADM binds a specific ADM receptor on the endothelium to release NO, which may directly activate K+ channels in human coronary arterioles. These findings will be important for considerations of therapeutic intervention with ADM in the coronary circulation.

Clinical implications. ADM is a potent vasodilator peptide originally isolated from pheochromocytoma cells but is also known to be produced in the vascular endothelium (32). It acts on a broad range of vascular beds (6), including the human coronary circulation. ADM levels have been reported to be elevated in patients with several cardiovascular diseases, including hypertension (16). This may be a compensatory mechanism to reduce the elevated pressure. We observed a reduced dilation to ADM in subjects with hypertension, possibly due to chronic desensitization (acute tachyphylaxis was not observed in our study). Alternatively, the reduced sensitivity to ADM in hypertension may contribute to elevations in arterial pressure in these patients. Better understanding of the mechanism of dilation in the human coronary circulation, which appears to involve NO and vascular smooth muscle hyperpolarization, may enable new treatments to improve myocardial perfusion in disease states.


    ACKNOWLEDGEMENTS

The authors thank Kari Beyer for secretarial assistance.


    FOOTNOTES

Grants from the American Heart Association-Wisconsin Affiliate, the Gutterman Foundation, the Cora and John H. Davis Foundation, the Veterans Affairs Medical Center, and the National Institutes of Health supported this work.

Address for reprint requests and other correspondence: D. D. Gutterman, CVRC Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: dgutt{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.

Received 20 March 2000; accepted in final form 3 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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