Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545
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ABSTRACT |
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We utilized
[3H]prazosin saturation and competition radioligand
binding studies to characterize the expression of
1-adrenoceptors in preglomerular vessels. mRNA for
adrenoceptor subtypes was assayed using RT-PCR. The vessels were
isolated using an iron oxide-sieving method. [3H]prazosin
bound to a single class of binding sites (Kd
0.087 ± 0.012 nM, Bmax 326 ± 56 fmol/mg
protein). Phentolamine displaced [3H]prazosin (0.2 nM)
with a pKi of 8.37 ± 0.09. Competition
with the selective
1A-adrenoceptor antagonist
5-methylurapidil fit a two-site model (pKi
9.38 ± 0.21 and 7.04 ± 0.15); 59 ± 3% of the sites
were high-affinity, and 41 ± 3% were low-affinity binding sites.
Competition with the
1D-adrenoceptor antagonist
8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) fit a one-site model with low affinity (pKi 6.83 ± 0.03). The relative contents
of
1A-,
1B-, and
1D-adrenoceptor mRNAs were 64 ± 5, 25 ± 5, and 11 ± 1%, respectively. Thus there was a very good
correlation between mRNA and receptor binding for the subtypes. These
data indicate a predominance of the
1A-adrenoceptor subtype in rat renal resistance vessels, with smaller densities of
1B- and
1D-adrenoceptors.
renal circulation; vascular smooth muscle; reverse transcription-polymerase chain reaction; mRNA; renal nerve; catecholamine
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INTRODUCTION |
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THE IMPORTANCE OF THE
SYMPATHETIC nerves in the control of the cardiovascular system is
well known. Catecholamines of nervous and humoral origin exert their
effect by reversible activation of cell surface adrenoceptors
on vascular smooth muscle cells which produces changes in cytosolic
calcium concentration ([Ca2+]i). This in turn
leads to binding of Ca2+ to calmodulin followed by a change
in calmodulin structure, causing the activation of myosin light chain
kinase that is responsible for the increased tone in the smooth muscle
cells (38). The adrenoceptors are subdivided in several
major classes, namely, the 1-,
2-, and
-adrenoceptor subtypes (42). The renal resistance vessels are richly innervated by nerves of sympathetic origin (1). In the rat renal vasculature the
1-adrenoceptors have been shown to mediate the action of
sympathetic control of renal hemodynamics, glomerular ultrafiltration,
and both short- and long-term regulation of extracellular fluid volume
and arterial blood pressure (11). In addition, the release
of renin from the granular cells at the distal end of the afferent
arteriole may be partly controlled via renal
- as well as
-adrenoceptors (11). A possible role of the
1-adrenoceptors in the generation of hypertension is
suggested by the observation that the responsiveness of these
adrenoceptors in the kidney is augmented in spontaneously hypertensive
rats (SHR) compared with Wistar-Kyoto rats (WKY) (36).
According to current criteria, the vascular
1-adrenoceptors are pharmacologically further subdivided
into three known subtypes, the
1A-,
1B-,
and the
1D-subtypes (5, 20, 26, 31), all of
which have been cloned. Initially it was thought that activation of
1A-adrenoceptors elicits entry of Ca2+ from
the extracellular space via voltage-gated channels and that activation
of
1B-adrenoceptors causes mobilization of
Ca2+ from intracellular inositol trisphosphate
(IP3)-sensitive stores (21). However, this
notion has been challenged by more recent studies. For example,
activation of
1A-adrenoceptors is known to trigger
recruitment of Ca2+ from IP3-sensitive
intracellular stores, at least in some vessels (4). Little
is known about signaling pathways mediating effects of different
1-adrenoceptors in renal resistance vessels.
Reports from various laboratories reveal heterogeneity with
respect to relative 1-adrenoceptor subtype expression in
different peripheral vascular beds. For example,
1B- and
1D-receptors appear to be present in rat aorta
(21, 25, 29). At least two
1-adrenoceptors
have been reported to mediate catecholamine-induced constriction of the
main renal artery in vitro (21, 37). Regarding the
physiological regulation of the renal hemodynamics in vivo, a major
role for the
1A-adrenoceptors has been suggested by most (2, 14, 33) but not all studies (6). In the
rat interlobar artery, norepinephrine (NE) activation seems to
be mediated by
1A-adrenoceptors (8). With
regard to a major resistance vessel, we recently found that the
NE-induced [Ca2+]i response in isolated
afferent arterioles and the constriction of the renal vasculature
in vivo are attenuated by
8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378), an adrenoceptor antagonist selective for
the
1D-adrenoceptor subtype (18), and by
5-methylurapidil (5-MU), which preferentially blocks the
1A-adrenoceptor subtype (5, 30, 32). These
observations implicate mediation by the
1A-adrenoceptor
or a combination of
1A- and
1D-adrenoceptors in catecholamine-induced
vasoconstriction in the renal microcirculation. Furthermore, we did not
find any indications for a major role of
1B-adrenoceptors based on the observation that there was
no discernible long-term inhibition by chloroethylclonidine
(CEC), an agent that preferentially alkylates the
1B-adrenoceptors (21, 26, 27).
In the present study, we extended our investigation of
1-adrenoceptors in renal resistance vessels to determine
directly which subtypes are present in isolated rat afferent
arterioles. Relative reverse transcription-polymerase chain reaction
(RT-PCR) and radioligand ([3H]prazosin) binding were used
to characterize the message and expression of the
1-adrenoceptors. Pharmacological agents were used to
identify
1A- or the
1D-adrenoceptor
subtypes in competitive binding studies. Receptor density and mRNA
abundance correlate well; both data sets indicate a predominance of the
1A-adrenoceptor subtype in renal resistance arterioles
with
1B- and
1D-adrenoceptor present in
lesser abundance.
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METHODS |
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Radioligand Binding Studies
Isolation of preglomerular vessels. Preglomerular resistance arterioles (diameter < 50 µm) were isolated from kidneys of 250- to 300-g male Sprague-Dawley rats (Harlan, Indianapolis, IN) using the iron oxide-sieving technique previously described by Chatziantoniou and Arendshorst (7). Briefly, renal vascular tissue was obtained from two to three rats for each experiment. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt), and the aorta was cannulated below the renal arteries through a midline abdominal incision. The aorta was ligated above the renal arteries, the left renal vein was cut, and the kidneys were slowly perfused with cold (4°C) PBS (in mM: 125 NaCl, 17 K2HPO4, 3 NaH2PO4, and 5 MgCl2, pH 7.4) solution until the renal venous effluent was free of blood. Thereafter, the kidneys were perfused with ice-cold magnetized iron oxide suspension (1% Fe2O3) for 10-15 s, excised, and placed in cold PBS. All subsequent steps of isolation were performed at 4°C. After decapsulation of the kidneys and dissection of the cortex from the medulla, the cortical tissue was minced with a razor blade and homogenized in 5 ml of PBS using a Polytron Tissuemizer. Preglomerular vessels, glomeruli, and the surrounding connective tissue were separated from the homogenate using a magnet. The iron oxide-loaded tissue was passed through needles of decreasing size (22-23 gauge) and sieved through a 125-µm mesh sieve to detach connective tissue, remove iron oxide from the large vessels, and to separate afferent arterioles from glomeruli. The vascular tissue was recovered from the top of the sieve and resuspended in PBS. A magnet was used to remove the microvessels and separate them from tubular fragments. The recovered vascular tissue consisted of interlobular arteries and afferent arterioles >95% free of glomerular and tubular tissue. The microvessels were incubated with collagenase (0.1 mg/ml PBS, type 1A, Sigma) for 30 min with constant shaking at 37°C. After enzymatic treatment, the preglomerular arterioles were removed from the digested tissue with the aid of a magnet and resuspended in PBS. This purified suspension was shaken vigorously (to help disperse iron oxide from the vessels) while being passed through a 27-gauge needle and sieved through a 125-µm mesh. The recovered supernatant was sonicated for two 30-s intervals to disperse any remaining iron oxide from the arterioles; a magnet was used to remove the free iron oxide. The purified solution was transferred to a 50-ml polyethylene centrifuge tube and centrifuged at 6,000 g for 30 min in a free-angle rotor. The final pellet of vascular tissue was resuspended in assay buffer and used for the binding and RT-PCR studies.
[3H]prazosin binding assays.
Aliquots of microvessels (25 µg of protein) were incubated in a final
volume of 250 µl, 10 mM Tris buffer (pH 7.4) with multiple [3H]prazosin concentrations up to 1 nM under continuous
shaking for 60 min at room temperature. Protein concentrations, as well as binding conditions, and time for equilibrium were determined in
preliminary studies (3). Bound ligand was separated from the free ligand using centrifugation in a sucrose gradient: 0.18 ml of
the samples was gently layered over 0.2 ml of assay-binding buffer
containing 20% sucrose in 0.5-ml polyethylene microcentrifuge tubes
and were centrifuged at 5,000 g for 30 min in a free-angle rotor. Thereafter, the tubes were rapidly frozen in dry ice, the tips
and tops of the tubes were separated, and the tips, containing the
bound ligands, and the tops, containing the free ligands, were assayed
in a liquid scintillation counter (Wallace Win Spectral 1414).
Nonspecific binding, defined as binding in the presence of 10 µM
phentolamine, was less than 10% of total binding. In competition
studies, the 1-adrenoceptor antagonists 5-MU and BMY-7378 were used to compete against 0.20 nM
[3H]prazosin. 5-MU is considered to be selective for
1A- and
1D-adrenoceptors; BMY-7378 is a
selective
1D-adrenoceptor antagonist (18).
Phentolamine was used as a nonselective
1-adrenoceptor
agonist. All assays were performed in triplicate.
Reverse Transcriptase-Polymerase Chain Reaction
Isolation of preglomerular smooth muscle cells and RNA extraction. The vessels were isolated from Sprague-Dawley (SD), WKY, and SHR rats (200-250 g) using the iron oxide-sieving method as described above. However, in this series of experiments, the vascular tissue was recovered from the top of the sieve and homogenized in the RNA STAT-60 solution. Thus no enzymatic collagenase treatment was used in these preparations. Smooth muscle RNA was extracted using the RNA STAT-60 method (Tel-Test, Friendswood, TX) according to the product manual and as described elsewhere (41). Total RNA from tissues was subjected to a second round of RNA extraction with the guanadinium method (41), followed by treatment with RQ1 RNase-free DNase (1 unit per 10 µg RNA) for 45 min at 37°C to prevent genomic DNA contamination. RNA concentration was determined spectrophotometrically at 260 nm. Purity was assessed according to absorbance ratio at 260/280 nm and was found to be >1.8. Quality was evaluated by electrophoresis on a 1.2% agarose gel after formaldehyde treatment.
Oligonucleotide primers.
The following oligonucleotides were used (BRL; Life Technologies,
Grand Island, NY): 1A-sense,
5'-CGAGTCTACGTAGTAGCC-3';
1A-antisense,
5'-GTCTTGGCAGCTTTCTTC-3';
1B-sense,
5'-ATCGTGGCCAAGAGGACC-3';
1B-antisense,
5'-TTTGGCTGCTTTCTTTTC-3';
1D-sense,
5'-CGCGTGTACGTGGTCGCAC-3';
1D-antisense,
5'-CTTGGCAGCCTTTTTC-3'; cyclophilin sense, 5'-ATCCTGAAGCATACAGGTC-3'; and cyclophilin antisense, 5'-AGTGAGAACAGAGATTAC-3'; which amplify 204-, 201-, 218-, and 340-bp fragments of the
1A,
1B,
1D, and cyclophilin genes,
respectively. The single-tube method with rTth DNA polymerase
(Promega, Madison, WI) was used for both reverse transcription and PCR
amplification as described by the manufacturer. A quantity of 100 ng
total RNA was used for
1A,
1B, and
1D detection, and 20 ng total RNA was used for
cyclophilin as an internal control. Reverse transcription of RNA was
performed in a 20-µl volume consisting of 200 µM dNTP, 1 mM
MnCl2, 10 mM Tris · HCl, pH 8.3, 90 mM KCl, 75 µM
antisense primer, and 5 U of rTth DNA polymerase. The reactions were
performed in a GeneAmp model 2400 thermocycler amplifier for 20 min at
60°C utilizing thin-walled MicroAmp reaction tubes (Perkin-Elmer)
without mineral oil overlay. The reaction was stopped by placing the
tubes on ice. PCR amplification was carried out in the same tube in a
volume of 100 µl containing 1.5 mM MgCl2, 10 mM
Tris · HCl, pH 8.3, 100 mM KCl, 0.05% (wt/vol) Tween 20, 0.75 mM EGTA, 5% (vol/vol) glycerol, and 15 µM sense primer. PCR
was preceded with one step at 95°C for 60 s, followed by 35 cycles for
1-adrenoceptors (30 cycles for cyclophilin) at 95°C for 60 s and 62°C for 30 s, and a final extension
step of 8 min at 62°C. Each 20 µl of PCR product was
subjected to electrophoresis in 1.5% agarose gel containing 0.5 µg/ml ethidium bromide for electrophoresis at 120 V. The ethidium
bromide-stained products were then photographed and analyzed with
densitometry. Adrenoceptor mRNA values were normalized to the RT-PCR
product of cyclophilin, which was amplified for each RNA sample as an
internal control. The absence of genomic DNA contamination was
demonstrated by control samples without reverse transcriptase.
Data analysis. The LIGAND program was used to estimate maximum specific binding (Bmax) and dissociation constant (Kd) under equilibrium conditions. Competition binding data were analyzed with Graph-Pad PRISM for best fit to a one- or two-site model. P < 0.05 was considered statistically significant. pKi values were determined according to Cheng and Prusoff (9).
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RESULTS |
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Radioligand Binding Data
Saturation binding of [3H]prazosin.
[3H]prazosin bound to isolated renal preglomerular
vessels in a concentration-dependent manner between 0.03 to 1 nM (Fig.
1). Nonspecific binding in the presence
of excess phentolamine (10 µM) was less than 10% of total binding.
Specific binding increased until the radioligand concentration reached
a saturating value of ~0.25 nM. [3H]prazosin bound to a
single class of binding sites, with a Kd of
0.087 ± 0.012 nM and a Bmax of 326 ± 56 fmol/mg
protein within the concentration range examined (n = 6).
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Effects of competitive antagonists on
[3H]prazosin binding.
To examine subtypes of 1-receptors, competition binding
studies were performed initially using the nonselective
1-adrenoceptor antagonist phentolamine. Subsequent
studies were conducted using the
1A- and
1D-subtype-specific antagonist 5-MU and the
1D-adrenoceptor subtype-selective antagonist BMY-7378,
to displace the binding of 0.2 nM [3H]prazosin. As is
shown in Fig. 2, all three compounds
displaced [3H]prazosin in a dose-dependent manner.
Phentolamine competed with [3H]prazosin binding in a
manner consistent with a one-site model; the pKi
was 8.37 ± 0.09 (n = 5). In contrast, 5-MU
evidenced significant two-site competition, with
pKi values of 9.38 ± 0.21 and 7.04 ± 0.15, respectively (n = 7). The high-affinity binding
sites amounted to 59 ± 3% of the total, with 41 ± 3%
representing the low-affinity site. On the other hand, the
1D-receptor antagonist BMY-7378 displayed one-site
competition and a lower potency [pKi 6.83 ± 0.03 (n = 6)], indicative of a small population of
1D-receptors.
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RT-PCR Data
Rat renal afferent arterioles expressed mRNA for all three known
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DISCUSSION |
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Catecholamine-induced vasoconstriction of the renal
microcirculation is mediated by -adrenoceptor adrenoceptors. Several in vivo and in vitro studies have established that function of the
1-adrenoceptor predominates over
2-adrenoceptors in renal resistance vessels (10,
12, 34, 39). Despite the considerable amount of work that has
been done regarding expression of
1-adrenoceptor subtypes in different vascular beds, there is no clear general consensus (for reviews see Refs. 5 and 30). In the large renal artery, all three subtypes appear to be present (21, 29, 37). Our previous study of rat preglomerular
1-adrenoceptor subtypes suggested that NE-induced
vasoconstriction most likely is mediated by
1A- or
1D-adrenoceptors, but not
1B-adrenoceptors (32). We observed that an
NE-induced increase in isolated afferent arteriolar
[Ca2+]i is resistant to CEC, which is well
known to preferentially alkylate and inactivate this subtype (21,
26, 27). Furthermore, we found excellent agreement between both
the NE-induced [Ca2+]i response and increases
in renal vascular resistance in vivo. Both responses were attenuated by
BMY-7378 and 5-MU, indicating contributions of both
1A-
and
1D-adrenoceptors. From the results of our previous
study, however, it was not possible to fully delineate the relative
contribution of these two subtypes. In the present study, we therefore
sought to define the relative abundance of
1-adrenoceptor subtypes utilizing two different in vitro
techniques, namely, radioligand binding and densitometric evaluation of
the RT-PCR product of
1-adrenoceptor mRNA extracted from
isolated rat preglomerular vessels.
For renal resistance vessels, a dominant role for the
1A-adrenoceptor subtype has been suggested in the
regulation of vasomotor tone (2, 14, 33). Likewise, in the
larger interlobar artery, which might contribute slightly to the
regulation of renal hemodynamics, 5-MU inhibited NE-induced contraction
with high potency, whereas the
1D-adrenoceptor-selective
antagonist BMY-7378 and CEC had minor attenuating effects
(8). An apparent exception to this notion is a study in
which receptor binding and RNase protection assay suggest a
predominance of the
1B-adrenoceptor in rat renal preglomerular microvessels (6). In the present study, the
high sensitivity to prazosin in the saturation studies confirmed the presence of
1-adrenoceptors. The
Kd value for prazosin obtained in our studies
agrees well with those reported for kidney cortex (16) and
cloned
1-adrenoceptors (23). Furthermore,
the relatively high sensitivity to 5-MU and absence of CEC-induced
alkylation in our previous study utilizing
[Ca2+]i measurements and renal blood flow are
consistent with these results (32).
Using [3H]prazosin binding in preparations from
different locations in the rat kidney, Feng et al. (16)
concluded that 1A- and
1B-adrenoceptors
are equally expressed in renal cortex, largely based on the observation
that CEC decreased Bmax roughly 50% from 186 to 75 fmol/mg
(16). It should be noted that their cortical tissue
consisted of both vascular and tubular structures. Relevant is the
observation of Gopalakrishnan et al., that 50% of the
[3H]prazosin binding to a pure proximal tubule
preparation is sensitive to CEC (19). Thus it is
reasonable to conclude that the large fraction of renal cortex
sensitive to CEC is primarily due to the predominance of tubular receptors.
The pKi value of the high-affinity 5-MU site in
the present study (9.38), representative of 59% of the total number of
1-adrenoceptors, and the pKi for
phentolamine (8.37) are in agreement with values previously reported
for the cloned
1A-adrenoceptor (17, 23, 22,
24). This indicates a predominance of the
1A-adrenoceptor, which is in agreement with our RT-PCR
results establishing that 64% of the total
1-adrenoceptor mRNA codes for
1A-adrenoceptors. This value is similar to the 67%
observed for whole rat kidney mRNA (35). There is also a
predominance of the
1A-adrenoceptor mRNA in human renal
artery and in branches thereof as assessed by RNase protection assay
and in situ hybridization (28). The pKi value (7.04) of the low-affinity site,
representative of 41% of the total receptor population, approximates
average values for cloned
1B- (6.87 ± 0.1) and
1D-adrenoceptors (7.46 ± 0.2) (15). The pKi value of 6.83 for
BMY-7378 in our afferent arterioles is considerably lower than values
for cloned
1D-adrenoceptors (8.44 ± 0.2) but in
the same range as the average value for cloned
1B-adrenoceptors (6.51 ± 0.2) (15).
Taken together, these results suggest that
1D-adrenoceptor protein is weakly expressed in isolated rat preglomerular vessels. This conclusion is reinforced by our PCR
data which show that
1D-adrenoceptor mRNA constitutes
only 11% of the total
1-adrenoceptor mRNA. The average
pKi value of 6.28 ± 0.1 of BMY-7378 for
the cloned
1A-adrenoceptor also supports the idea of
1A-adrenoceptor dominance in the afferent arterioles (15). Furthermore, our semi-quantitative RT-PCR
results demonstrate that
1B-adrenoceptor mRNA
constitutes 25% of the total
1-adrenoceptor mRNA. The
functional role for the
1B adrenoceptor in the renal vasculature is, however, uncertain. In this regard, we found in a
previous study that pretreatment with CEC (50 µM) for 15 min at
28°C had no effect on the ability of NE to elicit an increase in
[Ca2+]i in isolated preglomerular resistance
vessels (32). If
1B-adrenoceptors were
present and functional, then we would expect at least 50% effect of
alkylation of
1B-adrenoceptors under the prevailing conditions (40). Also, since it has recently been
suggested that the
1D-adrenoceptor may be alkylated by
CEC almost as rapidly as the
1B-adrenoceptor
(40), the absence of attenuation by CEC might indicate
limited participation of
1D-adrenoceptors (32). Other investigators have also reported only minor
effects of CEC on the renal circulation (2, 14).
Our analysis of mRNA clearly shows that there is potential for
synthesis of all known 1-adrenoceptor subtypes in the
renal vasculature. It might be that some receptors are expressed only during certain conditions. Support for this notion derives from the
finding that
1B-adrenoceptor expression is increased by
chronic hypoxia (13). Therefore, a contribution of other
1-adrenoceptor subtypes cannot be excluded even if it
seems obvious from this study, as well as a majority of studies in this
area, to conclude that the
1A-adrenoceptors predominate
in the renal microvasculature. It is possible in certain developmental
stages, conditions, or diseases that other
1-adrenoceptor subtypes may play a role in the control
of renal microcirculation.
In summary, our study presents the results of
[3H]prazosin binding and competition inhibition by agents
known to possess different selectivity for different
1-adrenoceptor subtypes (i.e., BMY-7378 and 5-MU) in
renal preglomerular resistance vessels. In addition, we present results
from an evaluation of the gene product expression for
1-adrenoceptor subtypes using RT-PCR. In agreement with
earlier observations by us and by others, the present results indicate that the predominant
1-adrenoceptor subtype in the rat
preglomerular vasculature is the
1A-subtype, although to
a smaller extent, the
1B and
1D-adrenoceptors are also expressed at the mRNA and protein levels. The predominance of the
1A-adrenoceptor
is consistent with this dominance as the major subtype controlling
renal vascular resistance.
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ACKNOWLEDGEMENTS |
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These studies were supported by National Heart, Lung, and Blood Institute Research Grants HL-02334 (to W. J. Arendshorst) and HL-62584 (to J. E. Faber). M. Salomonsson's visit was sponsored in part by the Swedish Medical Research Council, Medical Faculty Lund University, Maggie Stephens Foundation, and the Berth von Kantzow's Foundation.
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FOOTNOTES |
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Present address of M. Salomonsson: Dept. of Medical Physiology, The Panum Institute, Blegdamsvej 3, Copenhagen N 2200, Denmark.
Address for reprint requests and other correspondence: W. J. Arendshorst, Dept. of Cell and Molecular Physiology, CB #7545 School of Medicine, Rm. 152, Medical Sciences Research Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (E-mail: arends{at}med.unc.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 13 December 2000; accepted in final form 19 March 2001.
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