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1 Physiologisches Institut, It is unclear to what extent the
endothelium-derived hyperpolarizing factor (EDHF) contributes to the
control of microcirculatory blood flow in vivo. We analyzed, by
intravital microscopy in hamster muscles, the potential role of EDHF
along the vascular tree under stimulated (ACh) or basal conditions.
Experiments were performed in conscious as well as anesthetized
(pentobarbital, urethan) animals. Additionally, cellular effects of the
potential EDHF were studied in isolated small arteries. In
pentobarbital-anesthetized animals, treatment with
N
endothelium-dependent hyperpolarization factor; calcium
ion-dependent potassium ion channels; charybdotoxin; 17-octadecynoic
acid; P-450 monooxygenase; anesthesia
IN MANY BLOOD VESSELS
the endothelium-dependent dilation on stimulation with ACh is not
exclusively mediated by the "classical" autacoids, nitric oxide
(NO) and prostaglandins, but also by a NO- and
prostaglandin-independent hyperpolarization of vascular smooth muscle
cells. The latter is thought to be elicited by an endothelium-derived
hyperpolarizing factor (EDHF) (19). In studies on large-conductance
arteries, ACh induced an endothelium-dependent hyperpolarization that
was caused by activation of K+
channels and subsequent K+ efflux
(3).
Recent data obtained in a bioassay system performed on
large-conductance arteries in vitro suggest the existence of a
transferable EDHF (18) that displays characteristics of a cytochrome
P-450-dependent metabolite of
arachidonic acid (10). In accordance with this, EDHF-induced dilations
of isolated arteries were reduced by various barbiturates depending on
their efficacy to interfere with the cytochrome
P-450 pathway (13). As a result,
EDHF-mediated dilations in the microcirculation of a conscious animal
may differ considerably from those in a barbiturate-anesthetized
animal. Some studies demonstrated a NO synthase- and
cyclooxygenase-independent dilation at the microcirculatory level in
anesthetized animals (14, 24), but its relative significance compared
with that in conscious animals is rather unclear. Furthermore, the role
of EDHF in differently sized arterioles has never been studied
systematically in vivo, although in vitro studies suggest that
differences may exist between arteries of different calibers (23).
The aim of our study was to investigate EDHF-mediated dilation in vivo
and its relative significance in differently sized arterioles.
Moreover, we tested which K+
channels are involved in EDHF-induced hyperpolarization by the use of
specific blocking agents. To elucidate the effect of different anesthetics, EDHF responses were studied in animals before and after
the induction of anesthesia with agents exhibiting different potencies
to affect the P-450 pathway and
compared with the inhibitor of the
P-450 monooxygenase, 17-octadecynoic
acid (ODYA). Potential cellular mechanisms of the
anesthetic pentobarbital on the EDHF-mediated dilations were
additionally investigated in isolated arteries.
Preparation of cremaster muscle.
Golden Syrian hamsters (80- to 150-g body wt) were anesthetized by
intraperitoneal injection of pentobarbital sodium (75 mg/kg) followed
by continuous administration of the anesthetic via a jugular vein
catheter at a rate of 5-10
mg · kg Preparation of dorsal skinfold chamber.
Arteriolar responses in conscious hamsters were studied using the
dorsal skinfold chamber, containing the thin striated skin muscle
within an observation window. Implantation of the chambers was
performed under pentobarbital anesthesia as described previously (12).
One epidermal layer was completely removed to expose the underlying
skin muscle, which was thereafter protected by a coverslip. The animals
were then allowed to wake up, and a period of 72-96 h was allowed
before investigation of the microcirculation to eliminate the effects
of anesthesia and surgical trauma. For local application of vasoactive
substances, the coverslip was removed and the skin muscle was
superfused as described in Experimental Protocols. In this way, arterioles could
be monitored without the use of fluorescent dyes.
Experimental setup.
The muscle was superfused with warm (34°C) bicarbonate-buffered
salt solution at a rate of 8 ml/min. The superfusion fluid had a pH of 7.4, a PO2 of ~30 mmHg,
and a PCO2 of ~38 mmHg as measured
in samples taken from the surface of the muscle. One or two arterioles
were studied in each animal. Arterioles studied in different animals
were of varying size and vascular generation and were monitored by
means of a microscope (Metallux, Leitz, Wetzlar, Germany) equipped with
a video camera. If more than one arteriole was studied, the other
vessels were investigated subsequently, repeating the same protocol.
The microscopic images were displayed on a video monitor at 720-fold
magnification and recorded on videotape (S-VHS, Sony). Arteriolar inner
diameters were measured off-line from digitized images (MVP-AT, Matrox, Dorval, PQ, Canada) using a laboratory computer program.
Experimental protocols.
After the hamsters were placed onto a microscope stage, the
preparations were allowed to recover for 30 min before the start of the
experiments. The vascular diameter of an arteriole was measured before
(1-2 min) and during the local superfusion (3-4 min) of the
endothelium-independent NO donor sodium nitroprusside (SNP, 1 or 10 µmol/l) or the endothelial stimulator ACh (10-100 µmol/l).
Increasing concentrations of vasoactive drugs were applied consecutively, with a recovery period of 5 min between washout and
application of the next concentration or drug. During this recovery
period, the arterioles regained their baseline diameter. The same
protocol was then repeated in the presence of the NO synthase inhibitor
N
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-nitro-L-arginine
(L-NNA; 30 µmol/l) and
indomethacin (3 µmol/l) reduced the dilation in response to 10 µmol/l ACh from 60 ± 6 to 20 ± 4%. This nitric
oxide/prostaglandin-independent dilation (NPID), which was of a similar
magnitude in large and small arterioles, was abolished by potassium
depolarization or charybdotoxin (ChTX, 1 µmol/l) but not by
glibenclamide. In conscious animals, NPID amounted to 33 ± 3%. The
inhibitor of the P-450 monooxygenase 17-octadecynoic acid (ODYA) reduced NPID further to 9 ± 4%. ChTX abolished the NPID and also reduced basal diameters (by 
11 ± 3%). The induction of anesthesia with pentobarbital reduced NPID (to
12 ± 6%), whereas urethan anesthesia was without effect.
Pentobarbital also reduced the ACh-induced hyperpolarization of
vascular smooth muscle in isolated arteries, whereas ChTX abolished it.
This study suggests that a considerable part of the ACh dilation in the
microcirculation is mediated by EDHF, which also contributes to the
control of basal tone in conscious animals. The direct inhibitory
effect of pentobarbital and ODYA supports the idea that
"microcirculatory" EDHF is a product of the cytochrome
P-450 pathway. The role of EDHF might
be underestimated in pentobarbital-anesthetized animals.
INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
1 · h1.
Carotid artery pressure was measured continuously by means of a
pressure transducer (Statham, Costa Mesa, CA), and data were stored on
computer disk. The animals were artificially ventilated (7025 Rodent
Ventilator, Hugo Sachs Elektronik, Freiburg, Germany) to maintain
PO2 and
PCO2 at physiological values (~100
and 40 mmHg, respectively), as determined by blood gas analysis. The
right cremaster muscle was prepared as previously described (27). The
care of the animals and the conduct of the experiments were in
accordance with the rules of the German animal protection laws.
-nitro-L-arginine
(L-NNA, 30 µmol/l) and the
cyclooxygenase inhibitor indomethacin (3 µmol/l). These inhibitors
were added to the superfusion 30 min before the protocol was repeated.
The concentrations of the blockers used have been shown to block basal
and ACh-induced NO- or prostaglandin-mediated dilation in this
preparation (27, 28). In the cremaster preparation, arteriolar
responses were additionally investigated in the presence of an elevated
extracellular concentration of potassium (50 mmol/l), which was
exchanged for sodium in the buffer so as not to alter its osmolarity.
To avoid a potassium-induced constriction, the L-type
Ca2+-channel blocker felodipine (1 µmol/l) was concomitantly applied. Furthermore, the effects of the
Ca2+-dependent
K+-channel blocker charybdotoxin
(1 µmol/l) and the ATP-dependent K+-channel blocker glibenclamide
(1 µmol/l) were studied. To minimize the amount of charybdotoxin that
had to be applied, a reservoir (consisting of a 30-mm-diameter rubber
ring covered with a glass) was created on the cremaster, into which
charybdotoxin as well as the vasodilators were injected as a single
bolus. The arteriolar responses on vasodilator application were tested
before and after charybdotoxin. The loss of the compound caused by
washout was thus minimized despite the ongoing flow of the superfusion buffer.
Preparation of isolated arteries. Female golden Syrian hamsters (120- to 140-g body wt) were anesthetized by intraperitoneal injection of pentobarbital. The method for the isolation of a small artery (diameter ~160 µm) and measurement of intracellular calcium and diameter is described in detail elsewhere (2). Briefly, the right femoral artery was quickly exposed and occluded proximal to the site of the isolation of a microvessel, thereby preventing its exposure to the high concentration of pentobarbital, which was used to kill the animal later. The isolated small artery was cannulated, pressurized (45 mmHg), and loaded with fura 2-AM (MOPS-buffered salt solution, 2 µmol/l fura 2-AM, 0.5% bovine serum albumin) or the potential-sensitive dye bis-oxonol {bis(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)]; 50 nmol/l}. Intracellular free calcium ([Ca2+]i) or membrane potential changes and diameter were measured by means of a modified inverted microscope (Olympus IMT-2) equipped with a ×20 lens (Olympus D-APO 20 UV) and a video camera system. Alternating excitation wavelengths of 340 and 380 nm were used for measurement of [Ca2+]i, and the fluorescence at a wavelength of 510 nm was recorded using a photomultiplier tube (Photomed, Wedel, Germany). The ratio of fluorescence at 340 and 380 nm was calculated after subtraction of the background fluorescence (obtained after fura 2 quenching with 8 mmol/l MnCl2). For membrane potential measurement, the dye was excited at 490 nm and the fluorescence was recorded at 516 nm. Additionally, the vessel was transilluminated at wavelengths >610 nm, which did not interfere with the fluorescence measurements, and diameter was measured as described above.
Protocol in isolated arteries. The vessels were allowed to develop spontaneous myogenic tone in response to the applied transmural pressure for 30 min before the start of the experiments, which were carried out in the presence of indomethacin (30 µmol/l). All vessels were preconstricted with norepinephrine (NE, 0.3 µmol/l) 2 min before the addition of ACh (1 µmol/l). This was done in the same vessels (n = 4) before and 10 min after addition of pentobarbital (1-2 mmol/l) to the organ bath. The changes in membrane potential induced by ACh were measured in a separate group of vessels (n = 6) and repeated after addition of charybdotoxin (1 µmol/l) or pentobarbital (2 mmol/l) to the organ bath.
Solutions and drugs.
The salt buffer used for superfusion was of the following composition
(in mmol/l): 143 Na+, 6 K+, 2.5 Ca2+, 1.2 Mg2+, 128 Cl, 25 HCO3, 1.2 SO24, and 1.2 H2PO4. The MOPS-buffered salt
solution used for experiments on isolated vessels consisted of (in
mmol/l) 145 NaCl, 4.7 KCl, 1.5 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4,
2.0 pyruvate, 0.02 EGTA, 3.0 MOPS, and 5.0 glucose. ACh, SNP, NE, ODYA,
and charybdotoxin were purchased from Sigma (Deisenhofen, Germany), L-NNA from Serva (Heidelberg,
Germany), indomethacin (Confortid) from Dumex (Bad Vibel, Germany),
fura 2-AM and DiBAC4(3) from Molecular Probes, and MnCl2 from
Merck (Darmstadt, Germany). Felodipine was a kind gift of Astra (Wedel,
Germany). Stock solutions of indomethacin (12 mmol/l), felodipine (1 mmol/l), and fura 2-AM (1 mmol/l) were prepared in pure water, ethanol,
or water-free dimethyl sulfoxide, respectively, and stored at
20°C until use. On the day of the experiment, SNP (10 mmol/l) was dissolved in 1 mmol/l Na-acetate and
L-NNA was dissolved in a
HEPES-buffered salt solution by vigorous stirring at 60°C for 30 min. For all other solutions and further dilutions, freshly prepared
superfusion buffer was used. All locally applied drugs (concentrated
100-fold over the final concentration) were added to the superfusion
fluid by means of a roller pump at 1/100th of the total superfusion rate (0.08 ml/min) to obtain the final concentrations indicated above
except for charybdotoxin (see Experimental
Protocols).
Statistics and calculations. Vascular tone is expressed as the quotient of vessel resting diameter divided by its maximal diameter. Changes in inner diameter of the vessels were normalized to the maximal possible constriction or dilation according to the relationship
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![% change = [(<IT>V</IT><SUB>Tr</SUB> /<IT>V</IT><SUB>Co</SUB>) × 100] − 100](/content/vol276/issue5/fulltext/H1527/img002.gif)
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DISCUSSION
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Arteriolar dilations in cremaster muscle of anesthetized animals.
A total of 28 arterioles with maximal luminal diameters between 34 and
99 µm (mean: 57.6 ± 3.1 µm) were studied in 24 animals. Arterial blood pressure was 86 ± 4 mmHg at the start of
the experiment and was virtually stable throughout the experiment (end:
91 ± 4 mmHg, P = 0.42). Similarly,
heart rate did not vary significantly during the experiment (305 ± 14 beats/min). The vessels exhibited varying degrees of spontaneous
tone, i.e., the quotient of resting to maximal diameter ranged from
0.37 to 0.68 (mean: 0.53 ± 0.02). Local superfusion of 10 and
100 µmol/l ACh dilated the arterioles by 59.5 ± 6.2 and
75.0 ± 5.1%, respectively. The dilation on ACh superfusion lasted
as long as ACh was applied (4 min) without an attenuation of the
dilation during this period of observation (Fig.
1). The addition of
L-NNA and indomethacin reduced
arteriolar diameters from 28.9 ± 1.6 to 23.9 ± 1.8 µm
(18.1 ± 3.8%) and led to a significant attenuation of the
ACh-induced dilation. However, ACh still induced a significant dilation
(Fig. 1). This L-NNA- and
indomethacin-resistant dilation decreased with time after ACh
application (1 vs. 3 min: 29.9 ± 4.8 vs. 17.1 ± 4.2% at 10 µmol/l, P < 0.05; 43.6 ± 4.6 vs. 30.1 ± 5.9% at 100 µmol/l, P = 0.09). However, a significant
dilation was found even after 4 min (Fig. 1). Under control conditions
as well as in the presence of
L-NNA and indomethacin the
normalized ACh-induced dilations were of a similar magnitude
in small (maximal diameter <50 µm) and large (maximal diameter
>50 µm) arterioles (Table 1).
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Effect of anesthesia, ODYA, and charybdotoxin on arteriolar
dilations in skin muscle.
Twenty-three arterioles with maximal luminal diameters between 52 and
125 µm (mean: 76.4 ± 3.5 µm) were studied in the skin muscle of
23 conscious animals. The spontaneous resting tone of these vessels
ranged from 0.39 to 0.85 (mean: 0.66 ± 0.02).
L-NNA and indomethacin reduced
the arteriolar diameter from 56.5 ± 3.1 to 47.7 ± 3.2 µm (12.0 ± 5.3%) and the dilation on application of ACh
(10 µmol/l: 56.2 ± 4.8 vs. 33.4 ± 3.2%,
P < 0.05; 100 µmol/l: 68.5 ± 4.3 vs. 47.9 ± 4.6%, P < 0.05), whereas the dilation in response to SNP (10 µmol/l) remained unaffected (64.2 ± 3.8 vs. 63.3 ± 3.8%,
P = 0.87, n = 21). The induction of anesthesia
in the animals by pentobarbital itself did not alter basal diameters (44.9 ± 7.9 vs. 51.5 ± 8.3 µm;
P = 0.08) but led to a significant attenuation of the L-NNA- and
indomethacin-resistant portion of the ACh dilation (Fig.
3). This pentobarbital effect was specific for ACh, because the dilation on SNP application remained unaffected (57.1 ± 7.1 vs. 59.6 ± 6.7%,
P = 0.67, n = 7). In contrast, if urethan was
used to anesthetize different hamsters
(n = 4), the dilation in response to
ACh remained unaltered (Fig. 3). Anesthesia induction using urethan
also did not alter the arteriolar resting diameter (56.0 ± 5.2 vs.
54.2 ± 5.0 µm) or the dilation on application of SNP (data not
shown).
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10.8 ± 3.8%; P < 0.05)
in conscious animals.
Effect of pentobarbital and charybdotoxin in isolated arteries of
gracilis muscle.
After addition of NE (0.3 µmol/l) vascular smooth muscle
[Ca2+]i
increased rapidly (by 16.3 ± 3.3% after 2 min). This was
accompanied by constriction of the arteries (by 34.3 ± 1.9%). This
constriction was not significantly altered in the presence of
pentobarbital (1 or 2 mmol/l). From this preconstricted level,
dilations were initiated by the application of ACh (1 µmol/l). In
controls, ACh rapidly decreased
[Ca2+]i
and induced a strong dilation. However, after the addition of
pentobarbital (1 mmol/l) the ACh-induced
[Ca2+]i
decrease was significantly attenuated and the diameter increase tended
to be smaller. When a higher concentration of pentobarbital was used (2 mmol/l), the ACh-induced
[Ca2+]i
reduction was virtually abolished and the concomitant relaxation was
significantly attenuated (Fig. 4). Similar
observations were found in this model after blockade of
Ca2+-dependent
K+ channels with
tetrabutylammonium or charybdotoxin (data not shown). In
different arteries loaded with the potential-sensitive dye bisoxonol,
ACh hyperpolarized vascular smooth muscle cells as reflected by a 12 ± 1% reduction of the fluorescence signal. This hyperpolarization
was reduced in the presence of pentobarbital (Fig.
5) and virtually abolished in the
presence of charybdotoxin (4 ± 2% of the fluorescence
signal, n = 4).
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METHODS
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DISCUSSION
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These experiments support the hypothesis that ACh-induced dilation in the microcirculation is partially an EDHF-mediated response. This view is substantiated by the fact that the dilation was abolished by an elevation of [K+]e or by the Ca2+-dependent K+-channel blocker charybdotoxin, which also abolished the ACh-induced hyperpolarization of vascular smooth muscle in isolated vessels. Of note, this NO- and prostaglandin-independent dilation was partially sustained in the continuous presence of ACh, which is in contrast to most in vitro findings. The dilation was strongly attenuated by ODYA, a blocker of the cytochrome P-450 pathway, and by anesthesia with pentobarbital but not with urethan, suggesting that microcirculatory EDHF is a cytochrome P-450-related metabolite.
In conscious as well as anesthetized animals a major part of the arteriolar dilation induced by ACh was mediated by NO and/or prostanoids. This is in accordance with a previous study using the same model (27). In that study using ACh concentrations up to 1 µmol/l, only a small fraction of the dilation was resistant to NO and cyclooxygenase blockade. Because the skeletal muscle that surrounds the arterioles contains high acetylcholinesterase activity, the concentration (1 µmol/l) used in that study was obviously too low to induce the release of substantial amounts of EDHF. This assumption is supported by observations in isolated hamster arterioles and in other tissues in which the stimulation of EDHF release required higher concentrations of ACh than the stimulation of NO release (2). This difference might be related to different receptor types involved in the release of the respective endothelial dilator. For instance, different types of muscarinic receptors exhibit striking differences in their respective EC50 (11). As shown in the present study, high concentrations of ACh induced a dilation that could not be prevented by NO synthase and cyclooxygenase inhibitors but was completely blocked by the elevation of [K+]e (50 mmol/l) (Fig. 1). In contrast, dilations induced by the NO donor SNP remained unaffected by high potassium (Table 2). The divergent inhibitory potency of the elevation of [K+]e excludes the possibility that blockade of the NO synthase was insufficient at higher concentrations of ACh. Moreover, the inhibitory effect of the elevated [K+]e suggests that the remaining part of the ACh dilation was caused by a hyperpolarization accomplished by the activation of K+ channels. In fact, ACh induced a hyperpolarization of vascular smooth muscle cells in isolated, preconstricted arteries.
The findings of our study are in agreement with several studies on conductance arteries of different vascular beds and species. In most of these studies a L-NNA- and indomethacin-resistant dilation was demonstrated, which was mediated by hyperpolarization (8, 15) as confirmed by electrophysiological studies (16, 22). Different types of K+ channels have been identified in vascular smooth muscle. Data obtained on small arteries or arterioles in vitro suggest that the type of the K+ channel involved seems to vary depending on the vascular bed and species (6, 20, 30). Because charybdotoxin, but not glibenclamide, abolished the NO synthase- and cyclooxygenase-independent dilation (Figs. 2 and 3) as well as the ACh-induced hyperpolarization, our study supports the concept that Ca2+-dependent, but not ATP-dependent, K+ channels are involved in the dilation mediated by EDHF, at least in the hamster microcirculation.
It must be noted, however, that the design of this study (microcirculation in vivo) did not allow differentiation between K+ channels located in vascular smooth muscle and those located in the endothelium. In the case of the latter, endothelial hyperpolarization induced by the activation of endothelial K+ channels could affect the synthesis or release of a potential EDHF. Furthermore, endothelial hyperpolarization could be a modulator of myoendothelial signal transmission, which has recently been proposed to underlie "EDHF"-dependent dilation (5). However, the endothelial Ca2+ increase on application of ACh remained unaffected by charybdotoxin (S. S. Bolz, unpublished observation). This supports the view that charybdotoxin does not act on the endothelium but rather on the vascular smooth muscle cell.
It has been suggested that EDHF-mediated dilations are of a greater magnitude in smaller arteries (23) and that the role of EDHF could be more important in those vessels further downstream. This hypothesis cannot be extrapolated to the even smaller vessels of the microcirculation. As shown in this study, the L-NNA- and indomethacin-resistant dilation was of a similar magnitude in small (diameter <50 µm) and large (diameter >50 µm) arterioles.
To prevent the vasoconstriction induced by depolarization (caused by elevation of [K+]e), voltage-dependent L-type Ca2+ channels were blocked by felodipine (9). It can be assumed that the concentration used (1 µmol/l) is sufficient to block these channels, because the resting diameter increased on addition of felodipine. Moreover, addition of felodipine abolished the constriction induced by the elevated K+ concentration. Felodipine might act as a calmodulin inhibitor (25). Therefore, the blockade of the L-NNA- and indomethacin-resistant dilations in the presence of elevated [K+]e might be attributed to a calmodulin-inhibiting effect of the concomitantly applied felodipine. A direct inhibitory effect of felodipine on the release or action of the putative EDHF can, however, be excluded because felodipine alone did not abolish the L-NNA- and indomethacin-resistant dilation induced by ACh. Rather, the elevation of [K+]e was necessary to exert the blockade. It has been proposed that a hyperpolarization (induced by EDHF) elicits vasodilation by closing voltage-dependent L-type Ca2+ channels (19). The fact that the dilation remained unaffected by the L-type Ca2+-channel blocker suggests that EDHF exerts a dilation independent of these Ca2+ channels. Alternative mechanisms by which a hyperpolarization might exert vasodilation have been proposed (7), e.g., the interaction with the synthesis of inositol 1,4,5-trisphosphate (29). Nevertheless, the action of EDHF depends critically on the hyperpolarization induced by the activation of K+ channels.
Studies performed in the coronary vascular bed of the rat (1) and porcine and bovine arteries (4, 10, 26) suggest that EDHF is a metabolite of the cytochrome P-450 pathway. In accordance with this, the action and/or synthesis of this factor is attenuated by certain barbiturates (e.g., thiopental) (13). Our comparison of ACh responses in conscious and anesthetized animals supports these observations. The L-NNA- and indomethacin-resistant dilation in conscious animals was significantly attenuated by ODYA, a relatively specific blocker of the P-450 pathway. Pentobarbital anesthesia depressed this part of the ACh dilation to a similar degree (Fig. 3). In contrast, the dilations in response to an exogenous NO donor were not attenuated by pentobarbital, which excludes a nonspecific effect on the vascular smooth muscle function. This effect of pentobarbital is not caused by general anesthesia per se, because urethan did not affect the L-NNA- and indomethacin-resistant portion of the ACh dilation. The additional data obtained in isolated microvessels (Fig. 4) further confirm that pentobarbital blocks EDHF-mediated dilations at the vascular level. The rapid decrease of [Ca2+]i, the hyperpolarization of the vascular smooth muscle cell, as well as the concomitant rapid dilation of the arteries are typical for EDHF responses in these vessels (2). All these responses were strongly attenuated by pentobarbital and virtually abolished by charybdotoxin. Because the effect of pentobarbital was found in vivo as well as in isolated vessels, we conclude that the attenuation of the putative EDHF release and/or action is caused by a direct effect of the compound. Moreover, because pentobarbital has the potential to inhibit cytochrome P-450 monooxygenase (21) and ODYA exhibited a similar pattern of inhibition, it might be speculated that EDHF is in the microcirculation, similar to larger arteries, a metabolite of the cytochrome P-450 pathway.
In conclusion, our data are consistent with the concept that ACh-induced dilation in the microcirculation is partially caused by a hyperpolarization, which is elicited by an EDHF activating charybdotoxin-sensitive K+ channels. Its role may be underestimated in studies on barbiturate-anesthetized animals because its action or, even more likely, synthesis is attenuated by pentobarbital. EDHF seems also to contribute to the control of the vessel's resting tone. This contribution is solely observed in conscious animals because charybdotoxin constricts the arterioles only in the absence of pentobarbital. The inhibitory effects of ODYA and pentobarbital strongly suggest that "microcirculatory" EDHF is a product of the cytochrome P-450 pathway.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Stefan Weicherding for the implantation of observation windows in hamsters and Sarah Neuhaus for scientific editing. This paper contains data from the doctoral thesis of N. Esser.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB 553, Teilprojekt B2).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Cor de Wit, Physiologisches Institut, Ludwig-Maximilians-Universität, Pettenkoferstrasse. 12, D - 80336 München, Germany (E-mail: dewit{at}lrz.uni-muenchen.de).
Received 20 July 1998; accepted in final form 22 January 1999.
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REFERENCES |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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).
Dilation is expressed as % of maximal response. Resting (r) and
maximal (m) vessel diameters (in µm) are as follows. For 10 µmol/l
ACh, control (n = 20): 29.9 ± 1.9 (r), 57.2 ± 3.9 (m); L-NNA + Indo (n = 17): 21.5 ± 2.0 (r),
52.5 ± 3.3 (m); L-NNA + Indo + [K+]e
(n = 10): 23.5 ± 2.4 (r), 50.6 ± 3.9 (m). For 100 µmol/l ACh, control
(n = 13): 24.9 ± 2.2 (r), 52.8 ± 3.9 (m); L-NNA + Indo (n = 17): 22.9 ± 2.2 (r),
52.5 ± 3.3 (m); L-NNA + Indo + [K+]e
(n = 11): 24.2 ± 2.1 (r), 51.8 ± 3.7 (m). Values represent means ± SE. 
,
P < 0.05 vs. 
; 
,
P < 0.05 vs. 
