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Departments of 1 Medical Physiology and 2 Biochemistry and Genetics, University of Copenhagen, DK-2200 Copenhagen N; and 3 Department of Neurophysiology, Glostrup County Hospital, DK-2620 Glostrup, Denmark
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We examined the importance of nitric oxide (NO),
endothelium-derived hyperpolarizing factor (EDHF), and neurogenic
activity in agonist-induced vasodilation and baseline blood flow
[i.e., nerve microvascular conductance (NMVC)] in rat sciatic nerve
using laser Doppler flowmetry. Agonists were acetylcholine (ACh) and 3-morpholinosydnonimine (SIN-1). Vasodilation occurring despite NO
synthase (NOS) and cyclooxygenase inhibition and showing dependence on
K+ channel activity was taken as being mediated by EDHF.
NOS and cyclooxygenase inhibition with
N
-nitro-L-arginine
(L-NNA) + indomethacin (Indo) revealed two phases of
ACh-induced vasodilation: an initial, transient L-NNA + Indo-resistant vasodilation, peaking at 23 ± 6 s and
lasting 145 ± 69 s, followed by sustained
L-NNA + Indo-sensitive vasodilation. L-NNA
alone did not affect sustained ACh-induced vasodilation but decreased baseline NMVC by 55%. In the presence of L-NNA + Indo, the K+ channel blocker tetraethylammonium (TEA)
inhibited transient ACh-induced vasodilation by 58% and reduced
baseline NMVC by 25%. SIN-1-induced vasodilation increased fourfold in
the presence of L-NNA, whereas the specific guanylyl
cyclase inhibitor 1H-(1, 2,4)oxadiazolo(4,3-
)quinoxalin-1-one abolished it. However,
in homogenates of rat sciatic nerve, SIN-1-stimulated soluble guanylyl cyclase (sGC) activity was unaffected by L-NNA. TTX
affected neither SIN-1- nor ACh-induced vasodilation. In conclusion,
ACh-induced vasodilation consisted of two components, the first
partially mediated by EDHF and the second by a vasodilatory prostanoid + NO. Baseline NMVC was dependent on NO and EDHF. Although
L-NNA enhanced SIN-1-induced vasodilation, it had no effect
on sGC-activity.
nitric oxide synthase inhibition; tetrodotoxin; endothelium-derived hyperpolarizing factor; nerve microvascular conductance; acetylcholine
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACETYLCHOLINE
(ACh) has been used to determine endothelial function and dysfunction
since Furchgott and Zawadzki (5) introduced the concept of
endothelium-dependent vasodilation using this amine. In vitro studies
have shown that ACh-induced vasodilation is mediated by nitric oxide
(NO) and endothelium-derived hyperpolarizing factor (EDHF) (1,
2). EDHF produces hyperpolarization of vascular smooth muscle
and accompanying vasodilation via K+ channels, including
large conductance K+ (BK) channels (10),
whereas NO produces vasodilation via stimulation of soluble guanylyl
cyclase (sGC), giving rise to the second messenger cGMP (NO-cGMP
pathway) (16). Thus, in an in vitro study, dilatory responses to ACh were abolished by combined inhibition of NO synthase (NOS) and BK channels with
N-nitro-L-arginine
(L-NNA) and iberiotoxin (9). ACh has also been
shown to produce vasodilation independently of the endothelium by
inhibiting sympathetic vasoconstrictory tone (13, 27). This raises the possibility that blood flow responses to ACh in vivo
are dependent on all of these factors, i.e., that ACh-induced vasodilation in vivo is not strictly endothelium mediated.
The circulation of the sciatic nerve is well characterized and, roughly speaking, consists of an endoneurial capillary network supplied by longitudinally oriented arteries in the epi- and perineurium (14). These epi- and perineurial blood vessels are richly innervated by sympathetic nerve fibers, although the endoneurial capillary network is not (22). However, endoneurial blood flow is determined by the degree of vasoconstriction and vasodilation of the epineurial vessels (11). Thus basal sympathetic vasoconstriction corresponding to ~30% of resting endoneurial blood flow was demonstrated in sciatic nerve after chronic treatment with the ganglion blocker guanethidine (32). Basal NO production is also present in the sciatic nerve, because topical application and endoneurial microinjection of NOS inhibitors resulted in a 30-60% decrease in nerve blood flow (11, 28). Therefore, the sciatic nerve was chosen as a suitable tissue for the study of the factors mediating microcirculatory ACh-induced vasodilation.
The identity of EDHF is not yet established, but in vivo vasodilation induced by agonists such as ACh, which persists in the face of NOS and cyclooxygenase inhibition, is attributed to EDHF (29). Thus we examined the effects of NOS inhibition with and without simultaneous cyclooxygenase inhibition as well as the effects of inhibition of voltage-dependent Na+ channels on vasodilation induced by ACh and the NO donor 3-morpholinosydnonimine (SIN-1) in rat sciatic nerve. We also attempted to elucidate the biochemical basis for the enhanced nitrovasodilator effect seen in the presence of NOS inhibition.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Male Wistar rats (372 ± 34 g) kept in plastic cages with wood shavings, given free access to tap water and Altromin 1314 rat chow, and acclimatized to a 12:12-h light-dark cycle were used in all experiments. Twenty-three animals were used for the in vivo hemodynamic studies and fifteen for the in vitro sGC enzyme assay.Hemodynamic Studies
Anesthesia was induced with halothane (3%) and maintained during surgery (1.8%). The left jugular vein was cannulated for the administration of drugs, and the left carotid artery was cannulated for blood sampling and measurement of arterial blood pressure. The rats were tracheotomized and artificially ventilated with 30% O2-70% N2O, ensuring PO2 > 100 mmHg and PCO2 = 39 ± 2 mmHg. The lumbar backbone was partially exposed for fixation purposes. The left sciatic nerve was exposed by carefully separating the biceps and quadriceps muscles of the femur, which then formed walls of a well around the sciatic nerve. After surgery, halothane was discontinued and anesthesia was maintained with continuous infusion of intravenous pentobarbital sodium (initial bolus 19.0 mg/kg, infusion 22.9-28.6 mg · kg
1 · h1).
Suxamethonium (initial bolus 12.9 mg/kg, supplements of 2.6 mg/kg every
30 min) was given through a peritoneal catheter to eliminate the
influence of muscle activity. Adequate levels of anesthesia were
ensured by regularly verifying the absence of blood pressure responses
to tail pinch throughout the experiment.
Methods
Variations in nerve blood flow (NBF) were measured with laser Doppler flowmetry (LDF). Four identical needle probes (Probe 411, Perimed) were connected to two PeriFlux 4001 monitors (PeriMed). The probes were designed with 150 µm between the transmitting and receiving fibers, and held red laser light with a wavelength of 780 nm allowing the measurement of NBF to a tissue depth of ~0.5 mm. All probes were calibrated using Perimed motility standard (Perimed). The needle probes were held by micromanipulators and placed over the sciatic nerve under microscopic control. All probes were located above the trifurcation within a total distance of 1.5 cm (Fig. 1). Once in place, the probes were kept in the same position for the duration of the experiment. NBF and blood pressure were measured continuously with sampling rates of 10 and 100 Hz, respectively. Blood flow was measured in arbitrary units of flux with a time constant of 0.2 s. Spike 2 software (Cambridge Electronic Design) was used for data acquisition and later off-line analysis. As NBF measured with the laser Doppler technique can vary greatly from point to point because of local differences in the nerve microcirculation, we averaged the measurements from two stimulations of each vasodilator recorded with the four LDF probes, giving eight sets of data to reduce variability.
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Protocol
The well was initially filled with Ringer solution (in mM: 145 NaCl, 3.5 KCl, 2 CaCl2, and 5 HEPES; pH adjusted to 7.4). Baseline NBF was established, and the NBF response to the topical application of two vasodilators, ACh and SIN-1, was recorded (see Fig. 2B). Initially, SIN-1 was applied repeatedly at increasing concentrations until the smallest possible concentration at which NBF increased 25-50% was achieved (range: 0.1-0.75 mM). Once this concentration of SIN-1 was found, it was used throughout the experiment. The well was washed out, and the baseline was reestablished after each 5-min application of vasodilator. SIN-1 and ACh (100 µM) were each applied twice at each step of the experimental procedure.
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Once the responses to SIN-1 and ACh in the presence of Ringer solution
were established (step 1), the animals in group 1 (n = 6) were given topically applied L-NNA
(1 mM), an irreversible NOS inhibitor (step 2). The
preparation was allowed to stabilize for 30 min, after which SIN-1 and
ACh were applied in the same manner as before. Step 3 involved the addition of a specific sGC inhibitor,
1H-(1,2,4)oxadiazolo(4,3-)quinoxalin-1-one
(ODQ; 0.1 mM), to the L-NNA already in the well. Because
the effect of ODQ diminishes with time, the ODQ solution in the well
was renewed every 10 min. As before, the preparation was allowed to
stabilize for at least 30 min before application of SIN-1 and ACh.
EDHF and K+ channel involvement in baseline NBF and
vasodilatory responses was evaluated in group 2 (n = 5 animals). After the vasodilatory responses to
ACh and SIN-1 were determined in the presence of Ringer solution,
L-NNA and indomethacin (1 mM and 105 M) were
applied to inhibit NOS and cyclooxygenase, respectively. Step
3 consisted of the addition of the K+ channel blocker
tetraethylammonium (TEA; 10 mM) to the L-NNA and
indomethacin already present. TEA, at the concentration used in the
present study, functions as a nonspecific K+ channel
blocker, affecting both voltage-sensitive and ATP-sensitive channels as
well as BK channels (18). NBF responses to the two vasodilators, SIN-1 and ACh, at the same concentrations as in the other
groups were measured at each step.
The effect of neurogenic tone on NOS was evaluated in group 3 (n = 6 animals). After the responses to SIN-1 and ACh in the presence of Ringer (step 1) were established, the well was filled with TTX (20 µM), an inhibitor of voltage-dependent Na+ channels (step 2). Step 3 in this group was the addition of L-NNA (1 mM) to the TTX solution. As with the other groups, the vasodilatory responses to ACh and SIN-1 were measured at each step.
The constancy of NBF responses to SIN-1 and ACh over time was examined in group 4 (time controls; n = 6 animals). In effect, step 1, in which NBF responses to the vasodilators were measured in the presence of Ringer solution, was repeated three times. Between repetitions, the nerve was allowed to rest for 30 min corresponding to the stabilization periods after application of the inhibitors in groups 1, 2, and 3.
Homogenate Studies
NOS inhibition has been shown to accentuate the vasodilatory response to NO donors both in vitro and in vivo (17, 21, 23). Our present study revealed a fourfold increase in the vasodilatory response to SIN-1 during NOS inhibition with L-NNA (see RESULTS). Therefore, we attempted to elucidate the biochemical mechanism responsible for this enhancement using nervous tissue. The NOS inhibitor L-NNA, the specific inhibitor of sGC, ODQ, or both was added to homogenates of sciatic nerve (n = 15 animals for each treatment). Half of each group of homogenates was then stimulated with SIN-1. To confirm our results from the sciatic nerve, we repeated the procedure using homogenates of rat cerebellum (n = 10 animals), a tissue known to contain large amounts of neuronal NOS (4).Specifically, 15 animals were decapitated under halothane anesthesia
(3%). Biopsies were taken from both sciatic nerves and the cerebellum,
which were immediately frozen in liquid nitrogen and stored at 
80°C
until the enzyme assays were performed. The tissue samples were kept in
an ice bath during homogenization in 50 mM Tris buffer, 1.15 mM KCl, 1 mM EDTA, 5 mM glucose, 0.1 mM dithiothreitol, 200 units/ml superoxide
dismutase, 2 mg/l leupeptin, 2 mg/l pepstatin A, 10 mg/l trypsin
inhibitor, and 44 mg/l phenylmethylsulfonyl fluoride (PMSF). The
samples were first centrifuged for 10 min at 2,000 g to
remove cellular debris. Subsequently, the supernatant was centrifuged
for 2 h at 26,700 g to ensure that there was no contamination with membrane-bound guanylyl cyclase. All centrifugation occurred in a refrigerated centrifuge at 2°C. Protein content was
estimated according to the method of Groves et al. (7).
Aliquots (40 µl) of supernatant from the tissue homogenate were incubated for 30 min at 37°C with SIN-1 (final concentration 50 µM) in Tris buffer (150 µl) containing the sGC substrate GTP (final concentration 300 µM) in addition to 1.5 mM IBMX and 1 mM theophylline to inhibit degradation of cGMP, 3 mM creatine phosphate and 94 µg/ml creatine kinase to prevent the metabolization of GTP by other enzyme systems, and 5 mM MnCl2 to maximize sGC activity, as well as 94 µg/ml bovine serum albumin. Before incubation, 10-µl aliquots of L-NNA (final concentration 10 or 100 µM), ODQ (final concentration 10 µM), or solvent without inhibitor (DMSO) were added to the samples. L-NNA was added 30 min and ODQ was added 15 min before incubation; in samples with both L-NNA and ODQ, L-NNA was added 45 min before incubation to allow the NOS inhibitor 30 min of exposure to untreated sGC before the addition of ODQ for the remaining 15 min. The reaction was terminated by adding 10 µl of 1 mM EDTA and boiling for 3 min. After cooling on ice, the samples were centrifuged for 10 min at 10,000 g. The supernatant (25 µl), diluted appropriately, was added to radioimmunoassay buffer (475 µl). The RPA 525 cGMP-(125I) assay system (Amersham International) was used.
Drugs
Fresh solutions of all drugs using Ringer solution were made each day; fresh solutions of ACh were made immediately before each application. Substances that were not readily dissolvable in Ringer solution were ultrasonicated for 5 min. ACh, SIN-1, L-NNA, indomethacin, TTX, TEA, and ODQ were all administered topically in the hemodynamic studies.ACh chloride, SIN-1, L-NNA, GTP, PMSF, IBMX, theophylline, EDTA, and TTX were obtained from Sigma Chemicals. Superoxide dismutase, leupeptin, pepstatin A, trypsin inhibitor, creatine kinase, and creatine phosphate were obtained from Boehringer. ODQ was obtained from Tocris Cookson, and TEA was obtained from ICN Biomedicals. Indomethacin (Confortid) was obtained from Dumex, and suxamethonium (50 mg/l) was obtained from the pharmacy of the National Hospital of Denmark in Copenhagen.
Calculations and Statistics
Because NBF varies proportionately to mean arterial blood pressure (15), we converted NBF values into nerve microvascular conductance (NMVC) values to take into account any drift in blood pressure during the course of the procedure. The following formula was used
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Previous studies showed that in vitro vasodilation induced by ACh consists of a transient NO-independent phase that initiates vasodilation followed by a NO-dependent phase that maintains it (1, 3). Therefore, to avoid confounding the data by mixing information from two putatively different vasodilatory mechanisms, the NMVC responses in this study were measured during the last 60 s of each 5-min exposure to ACh in all groups (see Fig. 7) and at the start of vasodilation from 0 to 23 s in group 2 (see RESULTS). To be able to compare responses to both vasodilators with each other, NMVC responses to SIN-1 were also measured during the last 60 s. In this article, vasodilatory NMVC responses to ACh and SIN-1 refer to these 60-s intervals unless otherwise specified. NMVC responses to L-NNA with and without indomethacin, ODQ, TTX, and TEA were measured immediately before and after 30-min exposure to these inhibitors.
In the present study, the term "NMVC response" refers to the sum of the baseline NMVC and NMVC increment (see Fig. 2A). Ratios between the NMVC responses and the immediately preceding NMVC baselines were determined for both vasodilators and inhibitors and used for statistical calculations. All data were log-transformed to ensure normal distribution of the residuals. Statistical analysis was carried out using one-, two-, and three-way ANOVA, and when significance was found, paired (within groups) and unpaired (between groups) t-tests were performed. Bonferroni corrections were made in cases of multiple t-tests. The data are expressed as back-transformed means and confidence intervals (CI) unless otherwise specified. P < 0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hemodynamic Studies
Between-group comparisons of step 1. In all groups, systemic blood pressure was stable and uninfluenced by applications of topical vasodilators (Fig. 2B). The vasodilatory responses to topical ACh (0.1 mM) in the presence of Ringer solution during step 1 were similar in all four groups of rats, the ACh-induced NMVC increment being 61.1% (CI: 34.4-93.2%) in group 1, 44.7% (CI: 20.7-73.4%) in group 2, 58.5% (CI: 32.2-90.0%) in group 3, and 47.2% (95% CI: 22.8-76.5%) in group 4 (P = 0.7856, ANOVA; see Fig. 5). Likewise, the SIN-1-induced NMVC increment was 35.2% (CI: 16.3-57.3%) in group 1 vs. 40.0% (CI: 20.4-62.9%) in group 2 vs. 34.6% (CI: 15.7-56.6%) in group 3 vs. 29.3% (CI: 11.2-50.4%) in group 4 (P = 0.904, ANOVA; see Fig. 6). Thus the initial NMVC responses in step 1 were similar for all three experimental groups of rats, allowing comparisons between them.
Time controls (group 4).
In the time control group (n = 6), systemic blood
pressure remained stable throughout the experimental procedure
(P = 0.9973, ANOVA; see Fig. 3). The ACh-induced NMVC
increment declined gradually to 66.3% of the value in step
1 (P = 0.3077, ANOVA; see Fig. 5); this decrement
was statistically insignificant. In comparison, the SIN-1-induced NMVC
increment increased significantly to double the value in step
1 (P = 0.0026, ANOVA; see Fig. 6). During the two
periods of rest, baseline NMVC values tended to increase slightly by
15.2% (CI: 5.9 to 40.8%) and 17.1% (CI: 11.7 to 55.3%),
respectively (see Fig. 4); these increments were not statistically
significant (P = 0.1316 for 1st rest period and
P = 0.2109 for 2nd rest period). All results presented
below were statistically compared with the corresponding time control
values to determine the level of significance.
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Group 1: Rats receiving L-NNA followed by ODQ + L-NNA.
Blood pressure in group 1 (n = 6 animals)
was not affected by the topical application of either L-NNA
or ODQ (P = 0.9668 vs. time controls, ANOVA; Fig.
3). Thus there was no systemic effect of
these substances. In the presence of topical L-NNA (1 mM), baseline NMVC decreased by 54.8% (CI: 41.1-65.3%,
P = 1.619 × 104 vs. time controls;
Fig. 4), revealing a basal production of
NO. There was no further decrease in baseline NMVC after subsequent application of ODQ (0.1 mM; P = 1.000 vs. time
controls; data not shown).
9 vs. time controls, ANOVA). The NMVC increment above
baseline in response to SIN-1 increased from 35.3% (CI:
15.9-57.9%) in the presence of Ringer solution to 160.8% (CI:
123.5-204.4%) in the presence of L-NNA
(P = 0.0096 vs. time controls), demonstrating an
enhanced activity of the NO-cGMP cascade at or distal to sGC. With the
addition of ODQ, the NMVC increment caused by SIN-1 fell to 3.2% (CI:
11.6 to 20.4%; P = 1.648 × 104
compared with response in the presence of L-NNA with
respect to time controls; Fig. 6). Thus
ODQ completely inhibited SIN-1-induced vasodilation.
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Group 2: Rats receiving L-NNA + indomethacin
followed by TEA + L-NNA + indomethacin combined.
In group 2 (n = 5 animals),
L-NNA + indomethacin (1 mM and 105 M,
respectively), with or without TEA (10 mM), had no effect on mean
arterial blood pressure when applied topically (P = 0.8855 vs. time controls, ANOVA; Fig. 3). L-NNA + indomethacin caused baseline NMVC to decrease by 56.0% (CI:
46.2-64.0%; P = 8.416 × 105
vs. time controls; Fig. 4). This was of a magnitude similar to the
54.8% decrement in baseline NMVC caused by L-NNA alone
found in group 1. Subsequent addition of TEA resulted in a
further decrement of baseline NMVC of 27.7% (CI: 18.4-36.0%;
P = 0.0226 vs. time controls; data not shown).
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8.9% (CI: 18.6-1.9%) in the presence of
L-NNA + indomethacin and was unchanged,
remaining at 10.3% (CI: 19.8-0.3%), in the presence of
TEA + L-NNA + indomethacin (P = 0.0063 vs. time controls, ANOVA; Fig. 5). Thus, although blockade of
K+ channels did not affect the second phase of the ACh
response, K+ channel activity was responsible for 57.8% of
the initial phase of ACh-induced vasodilation.
In contrast to ACh, the NMVC increment caused by SIN-1 was potentiated
more than fourfold by treatment with L-NNA + indomethacin, being 40.0% (CI: 20.0-63.5%) in the presence of
Ringer solution, rising to 179.7% (CI: 139.7-223.5%) in the
presence of L-NNA + indomethacin, and remaining at
160.4% (CI: 123.1-204.0%) in the presence of TEA + L-NNA + indomethacin (P = 1.074 × 105 vs. time controls, ANOVA; Fig. 6). The
potentiation found with L-NNA + indomethacin was not
different from that found with L-NNA alone in group
1 (P = 1.000 vs. time controls). As shown above, subsequent application of TEA had no effect on the SIN-1 response, indicating that vasodilation caused by the NO-cGMP pathway is not
dependent on K+ channels.
Group 3: Rats receiving TTX followed by L-NNA.
The blood pressure profile throughout the experimental procedure in
group 3 (n = 6 animals) revealed a systemic
effect of topical TTX (0.02 mM), decreasing gradually and significantly to 69.6 ± 9.4% (mean ± SE) of the initial blood pressure
levels in step 1 (P = 3.935 × 107 vs. time controls, ANOVA; Fig. 3). Application of
topical TTX did not alter baseline NMVC significantly compared with
time controls (P = 1.000; Fig. 4). Combined treatment
with TTX + L-NNA (0.02 and 1 mM, respectively)
resulted in a decrease in baseline NMVC of 51.2% (CI:
13.9-72.4%; P = 0.0349 vs. time controls), which was of the same magnitude as the decrement in baseline NMVC caused by
L-NNA alone in group 1.
Homogenate Studies
In this part of the study, we tested the hypothesis that enhancement of the in vivo NMVC response to SIN-1 during NOS-inhibition was caused by potentiation of sGC activity by L-NNA.As Fig. 8 shows, SIN-1 (50 µM)
potentiated the activity of sGC in nerve homogenates weakly, by 35.1%
compared with controls (P = 0.0035, n = 15). ODQ (10 µM) inhibited the SIN-1-stimulated activity of sGC by
24.9% (P = 0.0007, n = 15) but did not
influence the basal activity of sGC in nerve homogenates
(P = 1.000, n = 15). In contrast,
L-NNA had no effect on either the basal or SIN-1-stimulated activity of sGC. The activity of basal sGC was 119.6% of control in
the presence of 10 µM L-NNA (P = 1.000, n = 10) and 87.7% of control in the presence of 100 µM L-NNA (P = 0.6489, n = 5; data not shown). Neither 10 µM or 100 µM L-NNA
enhanced SIN-1-stimulated activity, because the activity of sGC at
these concentrations of L-NNA was 98.0% (P = 1.000, n = 10) and 93.2% (P = 1.000, n = 5; data not shown), respectively, of the
SIN-1-stimulated levels.
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To verify our method, we repeated this procedure using homogenates of
rat cerebellum (Fig. 9), a tissue known
to contain large amounts of NOS (4). In cerebellar
homogenates, SIN-1 (50 µm) potentiated the activity of sGC by 598.7%
(P = 0.0000, n = 10). ODQ (10 µm)
inhibited the SIN-1-stimulated activity of sGC by 66.0%
(P = 0.0000, n = 10) but did not
inhibit the activity of basal sGC. In contrast, ODQ significantly
potentiated the activity of basal sGC by 15.6% in this assay
(P = 0.0434, n = 10). Again, L-NNA had no effect on either the SIN-1-stimulated or basal
activity of sGC in cerebellar homogenates. In the presence of 10 µM
L-NNA, the activity of sGC was 105.3% of control
(P = 1.000, n = 5) and 103.2% of
SIN-1-stimulated values (P = 1.000, n = 5). In the presence of 100 µM L-NNA, the corresponding
values were found to be 88.8% (P = 1.000, n = 5; data not shown) and 94.3% (P = 1.000, n = 5; data not shown), respectively. Thus ODQ
and L-NNA produced similar, but much more extreme, effects
in the cerebellar homogenates compared with the sciatic nerve
homogenates. In conclusion, the activity of sGC was not enhanced by
L-NNA at any concentration in either basal or in
SIN-1-stimulated homogenates of sciatic nerve or cerebellum.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study has focused on the regulation of the microcirculation in peripheral nervous tissue, both during baseline conditions and in response to the vasodilators ACh and SIN-1. Our findings include demonstration of a basal release of NO, accounting for ~50% of baseline NMVC. Subsequent treatment with TEA reduced the remaining baseline NMVC by ~25%. The NMVC increment caused by the NO donor SIN-1 was enhanced by NOS inhibition with L-NNA and subsequently totally inhibited by the sGC inhibitor ODQ. However, the in vitro enzyme assay did not reveal any potentiation of sGC activity during NOS inhibition to account for this enhanced in vivo SIN-1 response. In vivo vasodilation in response to ACh was found to consist of two components: an initial, transient vasodilation partially mediated by EDHF followed by a phase of sustained vasodilation. ACh-induced sustained vasodilation was not affected by L-NNA alone but was abolished entirely by the combination of L-NNA with the cyclooxygenase inhibitor indomethacin. Finally, neither K+ channel blockade with TEA nor inhibition of voltage-dependent Na+ channels with TTX was found to have any effect on the sustained vasodilatory responses to ACh and SIN-1.
In the following discussion, the effects of the inhibitors L-NNA, TTX, and TEA on baseline NMVC are considered. The in vivo potentiation of the vasodilatory response to SIN-1 during NOS inhibition is then related to the effects of SIN-1 and L-NNA on the biochemical activity of sGC found in the in vitro assay. Finally, mechanisms responsible for ACh-induced in vivo vasodilation as demonstrated in this study are discussed.
In our study, NOS inhibition by topically applied L-NNA with or without indomethacin resulted in a 55% decrease in baseline NMVC, indicating that NO is continuously released in the sciatic nerve microcirculation. This finding is in good agreement with recent studies in which superfusion of the nerve and endoneurial injection with NOS inhibitors caused the baseline NBF to decrease by 35% and 64%, respectively (11, 28). Thus resting blood flow in the rat sciatic nerve is largely dependent on NO production and release. Subsequent addition of TEA resulted in a 28% decrease of the baseline NMVC found in the presence of L-NNA + indomethacin, indicating that EDHF plays a role in maintaining basal nerve blood flow.
We found that inhibition of neurogenic activity with TTX did not alter baseline NMVC. In contrast, ganglion blockade with guanethidine was previously shown to increase NBF by 35% (32). However, in the present study, topically applied TTX had a systemic effect on mean arterial blood pressure, causing a substantial decrease over time. Thus a putative increase in baseline NMVC caused by blockade of neurogenic activity by TTX could conceivably have been counteracted by the falling blood pressure because peripheral nerves have no autoregulation (15, 25).
There is some controversy regarding the interaction between sympathetic tone and NO release. Some in vivo studies have shown that ganglion blockade almost abolished the hypertensive response to systemic NOS inhibition (12), and sectioning the lumbar sympathetic trunk greatly reduced vasoconstriction in skeletal muscle induced by systemic NOS inhibition (8). The conclusion was reached that NO release is partially dependent on sympathetic activity. Conversely, other in vivo studies measuring blood pressure before and after systemic NOS inhibition, as well as arterial norepinephrine and epinephrine levels in sympathectomized, ganglion-blocked, and normal rats, found NO-dependent vasodilation preserved in spite of varying sympathetic and humoral adrenergic stimuli between the groups (20). We found that the combination of local neurogenic and NOS inhibition with topically applied TTX + L-NNA had no effect on baseline NMVC other than that caused by topical L-NNA alone. Thus, at the microcirculatory level, basal NO release is not dependent on neurogenic activity in the rat sciatic nerve.
Using SIN-1 as a pharmacological probe to determine the level of functional activity of the NO-cGMP cascade, we found that inhibition of NOS with topical L-NNA (1 mM) increased the NMVC increment caused by SIN-1 more than fourfold from 35.3% in the presence of Ringer solution to 160.8% in the presence of L-NNA. This corresponds well with previous studies, in which NOS inhibition in vivo resulted in a supersensitivity to nitrovasodilators, e.g., the hypotensive response to NO donors was enhanced after systemic NOS inhibition (17) and nitroglycerin-induced vasodilation of the iliac artery was potentiated by local NOS inhibition (23). By using topically applied L-NNA and SIN-1 and measuring blood flow responses in peripheral nerve tissue, we have shown that this potentiation found systemically and in conduit arteries also occurs at the microcirculatory level.
Furthermore, we found that this L-NNA-enhanced NMVC increment to SIN-1 was abolished after subsequent treatment with the specific sGC inhibitor ODQ. These results are in good agreement with earlier studies using the mouse cortex, in which ODQ inhibited blood flow increments caused by NO donors by 80-90% (26). Because the vasodilatory action of the NO donor SIN-1 is solely dependent on sGC (19), this response pattern to SIN-1 could indicate that NOS inhibition potentiates the functional activity of this enzyme. Using homogenates of rat sciatic nerve and cerebellum, we then examined the effects of NOS inhibition with L-NNA and sGC inhibition with ODQ on the biochemical activity of sGC. In both tissues, the SIN-1-stimulated activity of sGC was significantly inhibited by ODQ, whereas the basal activity of sGC was not. These results are in good agreement with previous studies that showed that stimulated activity of the enzyme was more easily inhibited than basal activity (30). However, we were not able to demonstrate that L-NNA, at concentrations of either 10 µM or 100 µM, enhanced basal or SIN-1-stimulated activity of sGC in vitro. In contrast, an earlier study using aortic rings did demonstrate enhanced sGC activity during NOS inhibition (17). Because this earlier study did not examine the activity of the enzyme when saturated with substrate, its findings may have reflected other factors, such as substrate availability. Therefore, we speculate that the mechanism by which L-NNA potentiates the NMVC response to SIN-1 and the mechanism by which ODQ abolishes it are not the same, i.e., L-NNA may potentiate the NO-cGMP cascade downstream of sGC.
Inhibition of NOS and cyclooxygenase with L-NNA + indomethacin unmasked two phases of ACh-induced NMVC response, i.e., an initial, transient vasodilation peaking at 23 ± 6 s and lasting 145 ± 69 s followed by a second phase that exhibited no vasodilation at all, lasting for the duration of the exposure to ACh. The time-to-peak interval of the first phase (from 0 to 23 s) found in the presence of L-NNA + indomethacin corresponded to the initial rise in NBF induced by ACh in the presence of Ringer solution, whereas the second phase corresponded to a period of sustained vasodilation to ACh, found in the presence of Ringer solution. These findings are in good agreement with recent studies using isolated arterioles from the skeletal muscle and cheek pouch, in which ACh-induced vasodilation was shown to consist of a NO-independent component that initiated vasodilation peaking between 10 and 35 s and lasting ~2 min followed by a NO-dependent component that maintained vasodilation for the duration of the stimulus (1, 3). Thus the initial and steady-state components of ACh-induced vasodilation seen in vitro correlate timewise to the first and second phases found in vivo in the present study.
Regarding ACh-induced, sustained vasodilation found in the presence of Ringer solution, our present data showed a complete lack of attenuation of this response during NOS inhibition alone. Likewise, no statistically significant effect on this response was seen after subsequent addition of ODQ. Assuming that both ACh and SIN-1 use the NO-cGMP cascade to induce vasodilation in the microcirculation of peripheral nerve, any potentiation of the cascade at or beyond the level of sGC would be expected to affect the blood flow responses to both substances equally. Because topical L-NNA caused the NMVC increment to SIN-1 to increase more than fourfold without affecting the NMVC response to ACh, the conclusion could be drawn that this response pattern represents a 75% inhibition of NOS. However, a supramaximal concentration of L-NNA of 1 mM was chosen to ensure total NOS inhibition (4), whereas ODQ was shown to entirely inhibit sGC, as reported above. Therefore, at these concentrations of L-NNA and ODQ, NO-dependent vasodilation was practically abolished. Despite combined treatment with L-NNA and ODQ, a substantial NMVC response to ACh during the sustained phase was seen. Thus, although the steady-state component of the in vitro response to ACh was NO dependent (1, 3), the corresponding sustained phase of ACh-induced vasodilation in the sciatic nerve appeared to be NO resistant in vivo.
Examining this phenomenon, we hypothesized that this NO-resistant component of the sustained NMVC response to ACh could be of neurogenic origin, because previous in vitro studies have shown that ACh can produce vasodilation independently of the endothelium by inhibiting sympathetic vasoconstriction (13;27). Using topical TTX to block neurogenic activity, we found, however, no effect of TTX on second-phase NMVC responses to ACh. These results are in good agreement with those found in hamster skeletal muscle, in which maximal vasodilation induced by microiontophoretically applied ACh was not influenced by either TTX or L-NNA (24). Similarly, in another study, denervated and innervated rat skeletal muscle responded equally to ACh (6). Thus our studies indicate that sustained ACh-induced vasodilation in the microcirculation of peripheral nerve is independent of neurogenic activity.
As mentioned above, we found the sustained phase of the ACh-induced NMVC increment entirely abolished in the presence of L-NNA + indomethacin. This in vivo finding is in good agreement with a recent in vitro study using spinal resistance arteries, in which combined treatment with L-NAME and indomethacin completely reduced ACh-induced vasodilation (31). Because treatment with L-NNA alone had no effect on this response in the rat sciatic nerve, the second phase of ACh-induced vasodilation appears to be mediated by a vasodilatory prostanoid.
In contrast, the NMVC increment measured during the time-to-peak interval of the ACh response was not attenuated by NOS and cyclooxygenase inhibition. However, nonspecific K+ channel blockade with TEA together with L-NNA + indomethacin halved this phase of ACh-induced vasodilation. ACh-induced vasodilation, occurring in the presence of simultaneous NOS and cyclooxygenase inhibition and dependent on K+ channel activity, has been attributed to EDHF (29). Thus we found that the time-to-peak phase of ACh-induced vasodilation is mediated to a large extent by EDHF, which is supported by the in vitro findings of Bakker and Sipkema (1). We also found that sustained ACh-induced vasodilation appears to be NO resistant and mediated to a large extent by a vasodilatory prostanoid. This is in contrast to the in vitro studies, in which steady-state vasodilation has been shown to be highly inhibitable by NO (1, 3). The reason for this difference may lie in the types of tissue examined in these studies, because Yashiro and Ohhashi (31) found in their in vitro study using neural tissue from the spinal cord that ACh-induced vasodilation was mediated by prostanoids as well as NO.
In conclusion, we found that the basal release of local NO, which accounts for ~50% of baseline NMVC, was not dependent on neurogenic activity. EDHF was also found to contribute to the regulation of baseline NMVC, although to a smaller degree. NOS inhibition with L-NNA potentiated the functional activity of the NO-cGMP cascade in the microcirculation of peripheral nerve in vivo without potentiating the biochemical activity of sGC, indicating that the site of potentiation lies distal to cGMP. Most importantly, ACh-induced vasodilation in vivo in rat sciatic microcirculation appears to consist of two phases, a transient phase initiating vasodilation mediated partially by EDHF followed by a second phase of sustained vasodilation that was not affected by NO alone but was abolished by NOS and cyclooxygenase inhibition, suggesting mediation by a vasodilatory prostanoid.
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ACKNOWLEDGEMENTS |
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The authors thank Lillian Grøndahl and Lillian Lund Hansen for cheerful and expert technical assistance.
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FOOTNOTES |
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This study has been supported by the Medical Research Council of Denmark, the Novo Nordisk Foundation, NeuroScience PharmaBiotech, and Bdr. Hartmanns Foundation.
Address for reprint requests and other correspondence: K. Thomsen, Inst. of Medical Physiology, Panum Institute, Bldg. 12.5, Univ. of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark (E-mail: kthomsen{at}mfi.ku.dk).
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 29 March 1999; accepted in final form 15 March 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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), group 1 (
, animals
receiving L-NNA and ODQ), and group 2 (
, animals receiving L-NNA + Indo and
TEA; P = 0.9986, ANOVA). There is a significant
difference in the trend in blood pressure levels between time controls
and group 3 (
, animals receiving TTX and
L-NNA; P = 3.935 × 10
, n = 15). sGC responded with a 35.1%
increase in activity when stimulated with SIN-1. L-NNA
affected neither the basal activity of soluble guanyl cyclase (sGC)
(L-NNA) nor the SIN-1-stimulated activity (SIN-1 + L-NNA). ODQ did not affect the basal activity of sGC (ODQ)
but did inhibit the activity of the enzyme after stimulation with SIN-1
by 24.9% (SIN-1 + ODQ). **P < 0.005 vs. control;
+++P < 0.001 vs. SIN-1. The data are given
as means and 95% confidence intervals.
