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Unidad de Regulación Neurohumoral, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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To evaluate whether sympathetic activity
induces nitric oxide (NO) production, we perfused the rat arterial
mesenteric bed and measured luminally accessible norepinephrine (NE),
NO, and cGMP before, during, and after stimulation of perivascular
nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 ± 7 mmHg, accompanied by peaks of 23 ± 3 pmol NE, 445 ± 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 µM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and
duration. Endothelium denudation with saponin abolished the NO peak
without changing NE release. Inhibition of NO synthase with 100 µM
N
-nitro-L-arginine reduced basal
NO and cGMP release and blocked the electrically stimulated and
exogenous NE-stimulated NO peak while enhancing vasoconstriction.
Blocking either sympathetic exocytosis with 1 µM guanethidine or
1-adrenoceptors with 30 nM
prazosin abolished the electrically evoked vasoconstriction and NO
release. 2-Adrenoceptor
blockade with 1 µM yohimbine reduced both vasoconstriction and NO
peak while increasing NE release. In summary, sympathetically released
NE induces vasoconstriction, which triggers a secondary release of
endothelial NO coupled to cGMP production.
nitric oxide chemiluminescence; norepinephrine; guanosine 3',5'-cyclic monophosphate release; sympathetic neurotransmission; rat arterial mesenteric bed; endothelial activation; adrenoceptor blockade
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE HOMEOSTASIS of the vascular wall is complex and involves a growing number of humoral, neuronal, and endothelial factors (19, 41). The importance of the vascular endothelium was highlighted by the finding that nitric oxide (NO) plays a pivotal role in the minute-to-minute regulation of blood pressure (27, 29, 36). The best support for this notion derives from studies using drugs that act as selective blockers of NO synthase. Numerous reports demonstrate that guanidino-substituted analogs of L-arginine act as selective inhibitors of NO synthase (21). Administration of NO synthase blockers evokes an increase in local or systemic blood pressure in experimental subjects, including humans (36, 38, 39). The assessment of the direct role of NO in vascular wall homeostasis has been precluded by the technical difficulties in accurate determination of NO in body fluids. Such limitations hindered our understanding of the physiological and pathophysiological relevance of NO levels in the control of vascular tone.
Because physiological regulation mechanisms usually involve opposing signals, we assessed whether the vasoconstriction associated with electrical stimulation leads to the release of NO as an opposing vasodilating agent. We reasoned that a meaningful physiological role of the NO eventually released was its coupling to the synthesis of cGMP by the vascular smooth muscles. We anticipated, therefore, that if NO is released after a vasoconstrictor response, it should cause an opposing vasodilation dependent on cGMP.
To assess this working hypothesis, we conducted experiments in the arterial mesenteric bed of the rat, in which we earlier studied the physiology and pharmacology of the perivascular sympathetic fibers (14). We demonstrated that the electrical stimulation of these nerve fibers elicits vasomotor responses that are mediated by the release of norepinephrine (NE), neuropeptide Y (NPY) and, also likely, ATP, the sympathetic triad (14, 16, 34). In the present work, we investigated the correlation between changes in perfusion pressure elicited by either transmural electrical depolarization or exogenous NE and the release of NO and cGMP. The results suggest that NE-driven vasoconstriction is coupled to endothelial release of NO and associated with a cGMP surge.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Perfusion of Rat Arterial Mesenteric Bed
Rats were anesthetized with 40 mg/kg pentobarbital sodium, and the abdomen was opened by a midline incision. The superior mesenteric artery was cannulated and perfused with warm (37°C) Krebs-Ringer buffer solution, equilibrated with 95% O2-5% CO2, at a constant rate of 2 ml/min. The attachment of the mesentery to the intestinal wall was severed (28), and the isolated arterial mesenteric bed was transferred to a warm chamber. A pressure transducer was connected at the entrance of the mesenteric artery to monitor changes in the perfusion pressure in a recording polygraph. Platinum electrodes were placed surrounding the artery and were connected to a Grass S44 stimulator. A 30-min buffer perfusion period was allowed for equilibration. The perfusate was collected once every minute in test tubes for determination of the luminally accessible pool of NO and/or NE and cGMP in the tissue perfusate.Electrical Depolarization of Nerves Surrounding Mesenteric Artery
The basic protocol consisted of transmural depolarization of the perivascular mesenteric nerves with a train of 30 Hz (50 V, 1 ms) for 1 min (14, 16). Perfusion pressure and NE, NO, and cGMP content of perfusate were determined before, during, and after the stimulus. Other protocols were performed to assess the threshold of the frequency of stimulation required to evoke NO release (2.5, 5, 10, 20, and 30 Hz over 1 min). Additional experiments were aimed at determining the influence of the duration of the 30-Hz pulses on the recovery of NO in the mesenteric perfusate, giving train pulses of 30 Hz applied for 0.5, 1, 2, and 4 min.Source of NO and Influence of Adrenergic Mechanism on Release of NO
Six separate experimental protocols were performed to investigate the source of NO and to assess how the electrical depolarization of the sympathetic mesenteric nerve terminals evoked the release of NO to the perfusion media. In all these protocols, two 30-Hz, 1-min electrical stimuli were delivered. The first stimulus served as the control pretest value, and the second was performed after 30 min of perfusion with the test drug. Perfusate samples were collected before, during, and after each electrical stimulus. Drugs were dissolved in the perfusion buffer and maintained until completion of the second electrical stimulation protocol. Perfusion pressure was monitored over the whole experiment. In some experiments, NE release during the second stimulus was also measured.Krebs-Ringer controls. To determine whether in an intact preparation the second stimulus evoked an equivalent amount of NO release compared with the first stimulus, six arterial mesenteric beds were perfused with buffer between the first and second stimuli. This series served as the control for the second stimulus.
Saponin-induced endothelial shedding. To assess whether NO measured in the perfusion medium derived from the mesenteric endothelium, four preparations were perfused with 0.1% saponin for 55 s, and the second stimulus was delivered 30 min later, once the perfusion pressure returned to basal levels, as previously characterized (15, 33).
Blockade of NO synthase.
To assess whether the NO recovered in the perfusate was sensitive to
blockade of NO synthase, in a series of six experiments, the second
electrical stimulus was performed 30 min after perfusion with 100 µM
N-nitro-L-arginine to inhibit NO
synthase (1, 11, 23). Control measurements were performed to assess
whether this concentration of
N-nitro-L-arginine interfered
with NO determination.
Blockade of sympathetic exocytosis. In four preparations, the second stimulus was performed during and after perfusion with 1 µM guanethidine.
Blockade of
1-adrenoceptors.
The influence of
1-adrenoceptors was studied in
four mesenteries perfused with 30 nM prazosin.
Blockade of
2-adrenoceptors.
Five mesenteries were used to assess the effect of perfusion with 1 µM yohimbine, a well-characterized
2-adrenoceptor antagonist.
Perfusion With Exogenous NE
The cause-effect relationship between NE-induced vasoconstriction and NO and cGMP release was further studied in the following protocols. First, in six mesenteries the electrical stimulus was mimicked by perfusion for 1 min with buffer containing 10 µM NE, and the time course of perfusion pressure and luminal NO release was measured. To check whether NE-induced NO release was caused by NO synthase stimulation, four additional mesenteries were submitted to an identical protocol in the presence of 100 µM N-nitro-L-arginine. Furthermore,
to clarify the results obtained with the electrical stimulation during
adrenergic blockade, a similar 1-min exogenous NE stimulation was used
with 1 µM guanethidine (n = 3) and 1 µM yohimbine (n = 6).
Second, to get a better quantitative assessment of cGMP release, six
mesenteries were perfused for 10 min with 10 µM NE and perfusate was
collected in 10-ml samples just before, and at the end of, the NE
perfusion period. The total cGMP content of these samples was
determined. In addition, to assess whether cGMP release was caused by
NO stimulation of guanylyl cyclase, four mesenteries were submitted to
an identical protocol during NO synthase blockade by perfusion with 100 µM N-nitro-L-arginine.
Analytic Techniques
NE quantification. Each perfusate sample (2 ml) was collected in prechilled 5-ml collection tubes containing 100 µl of 1 M perchloric acid and 15 µl of 5% sodium metabisulfite. The NE in the samples was concentrated and purified using activated alumina (2). One hundred microliters of each purified sample, including an added internal standard of dihydroxybenzyl amine, were injected into an HPLC system, using the Merck L-6200A intelligent pump as described by Donoso et al. (14). The NE was quantified using a 656 DE Metrohm electrochemical detection system (6). Results are expressed as either the time course of the luminally accessible NE recovered (pmol/ml) or as the total integrated, recovered release elicited by the electrical depolarization (pmol). When simultaneous measurements were performed, a 200-µl perfusate sample was used for NO determination.
Quantification of NO by chemiluminescence. Freshly obtained triple-distilled water was used to prepare all buffer and drug solutions. Samples were collected in test tubes and immediately sealed with Parafilm to avoid contamination from room air. The sample content of NO was quantified using a Sievers 280 NO analyzer within 1 h after the experiment was terminated (3). The reaction chamber of the equipment was filled with 8 ml of glacial acetic acid containing 100 mg of potassium iodide at room temperature to reduce nitrites to NO (30, 32). A 50-µl perfusate sample was injected into the reaction chamber, and a nitrogen stream carried the resulting NO gas to a cell in which the specific chemiluminescence generated by the NO-ozone reaction was detected by a photomultiplier. Calibration of the equipment was performed daily using standards of 10-1,000 nM sodium nitrite. The sensitivity of the equipment allows for a detection threshold of 0.5-1.0 pmol NO (10-20 pmol/ml). Background buffer readings were subtracted to determine mesentery NO release. Results are expressed either as the time course of luminally accessible NO recovered before, during, and after the stimulus (pmol/ml) or as the integrated NO recovered above basal values elicited by the electrical pulse (pmol). We chose to integrate NO release for 4 min because this period accounted for >90% of the NO peak.
cGMP determinations. The cyclic nucleotide was quantified with an RIA for acetylated cGMP. The sensitivity limit of this assay was 10 fmol. The procedure outlined by Boric and Croxatto (7) was followed. As radioactive tracer we used 2'-O-monosuccinylguanosine 3',5'-cyclic monophosphate tyrosyl methyl ester that was labeled locally with 125I. In a few electrical stimulus protocols, cGMP was measured directly from 100- to 200-µl perfusate samples. In the experiments with exogenous NE perfusion, 10-ml samples were concentrated by passage through C-18 Sep-Pak columns (Merck), eluted with 2 ml of methanol, evaporated, and resuspended in 1 ml of RIA buffer for determination of cGMP (31).
Animal and Drug Sources
Male Sprague-Dawley rats (250-300 g) were bred in the animal facilities of our faculty. Experiments were conducted in accordance with the Helsinki declaration on research involving animals and human beings. Protocols complied with the guiding principles in the care and use of laboratory animals endorsed by the American Physiological Society and were approved by the Internal Animal Care and Use Committee of the Pontifical Catholic University of Chile. Saponin, N-nitro-L-arginine, guanethidine
sulfate, NE, prazosin, and yohimbine hydrochloride were purchased from
Sigma (St. Louis, MO).
Data Analysis
Time-course experiments were analyzed using two-way ANOVA. Paired or unpaired Student's t-test and regression analysis were used to compare differences between groups. Dunnett tables for multiple comparisons with a common control were used when appropriate. Significance was set at a probability of P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Release of NE, NO, and cGMP After Electrical Depolarization of Perivascular Mesenteric Nerves
Baseline levels of 0.1 pmol/ml NE, 40-80 pmol/ml NO, and 80-150 fmol/ml cGMP were detected in the tissue perfusate; basal perfusion pressure was 20-25 mmHg. The time course of a representative experiment showing luminally accessible NE, NO, and cGMP in the perfusate is depicted in Fig. 1. A 30-Hz stimulus for 1 min evoked an abrupt and sustained increase in the perfusion pressure, which returned to baseline immediately after the stimulation was ended. The NE peak was sharp, reaching its maximum during the stimulus, and faded to baseline mainly within the next 2 min. There was a NO surge that rose for at least 2 min, and its maximum was usually detected the minute after the stimulation. The cGMP peak was slightly more prolonged compared with NO, suggesting that these mediators are released in sequence (Fig. 1).
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The accuracy and reproducibility of the measurements of the NO release
in response to electrical stimulation were confirmed when the values
obtained in all the experimental protocols were compared. The time
course of NO release is shown in Fig. 2.
The NO surge ended 5 min after the stimulus
(P < 0.07 vs. average baseline,
paired t-test,
n = 28). The average integrated
release of NE and NO is shown in Table 1;
the associated increase in cGMP was ~1 pmol
(n = 3). In controls, the
second electrical stimulus induced a vasomotor response and peaks of NE
and NO release similar to those attained during the first stimulation,
indicating the reliability of the preparations over time (Table 1).
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Influence of Frequency and Duration of Electrical Nerve Stimulation
The electrically evoked increase in perfusion pressure and the associated NO surge were proportional to the frequency of nerve stimulation. One-minute trains of 5-, 10-, 20-, and 30-Hz stimuli caused a graded increase in perfusion pressure of 5 ± 0, 20 ± 3, 61 ± 18, and 90 ± 16 mmHg, respectively, with a parallel rise of the integrated NO release recovered in the perfusate (Fig. 3). A 1-min, 2.5-Hz train did not change perfusion pressure or basal NO. A significant correlation was observed between the increase in perfusion pressure and the integrated peak of NO released (r = 0.73, P < 0.01).
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A 0.5-min, 30-Hz stimulation resulted in almost undetectable NO release compared with the 1-min stimulus that attained the maximum release. This stimulus induced a brief peak of 74 ± 13 mmHg rise in perfusion pressure. Interestingly, increasing the duration of the stimulus to 2 or 4 min did not increase further the total NO recovered (Fig. 3) or change the maximal rise in perfusion pressure (107 ± 14 and 100 ± 6 mmHg, respectively).
Role of Endothelial Cell Layer
Removal of the endothelium with saponin resulted in a significant increase in the baseline NE released to the lumen (P < 0.05, paired t-test; Fig. 4). However, NE release evoked by the electrical stimulus was comparable, both in time course and the integrated total, to that attained in the controls (Fig. 4, Table 1). Although baseline NO release showed a tendency to decrease, the integrated peak of NO elicited by the stimulus was abolished (Fig. 4, Table 1). The rise in perfusion pressure was not altered (Table 1). Light microscopy revealed that saponin induced endothelial shedding without muscle edema or other alterations (not shown), in agreement with our previous report (15).
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Inhibition of NO Synthase
In the electrical stimulation experiment, treatment with 100 µM N-nitro-L-arginine tended to
reduce baseline NO release and blunted the evoked luminal NO release
(Fig. 5, Table 1). In parallel trials, a
1-min electrical stimulation of buffer containing 100 µM
N-nitro-L-arginine produced a
large artifact signal, which was not detected in the control buffer.
This interference fully accounts for the residual NO peak elicited by
the stimulus (Fig. 5).
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The inhibitor did not change baseline perfusion pressure or the
electrically evoked vasoconstriction (Table 1). However, in the course
of the 4-min, 30-Hz stimulation protocols, we noticed that perfusion
pressure was not maintained. To assess the influence of NO release in
this effect, we conducted similar experiments during NO synthase
blockade. As shown in Fig. 5, the pressor response was prolonged in the
presence of 100 µM
N-nitro-L-arginine. More evident
effects of NO synthase inhibition on perfusion pressure and NO release
were observed in the experiments using exogenous NE.
Effect of NE
Mimicking the effect of the electrical stimulus, a 1-min perfusion with 10 µM NE produced a sustained rise in perfusion pressure associated with an increase in NO release (Table 2). The time course of pressure and NO changes was similar to that attained during 1-min electrical stimulation (Fig. 6).
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Perfusion with 100 µM
N-nitro-L-arginine resulted in a
significant reduction of baseline NO (57.6 ± 8.1 vs. 88.3 ± 3.2 pmol/ml, P < 0.02). In addition, the
NE-induced contractile response was significantly enhanced, whereas the
associated NO peak was completely suppressed (Fig. 6, Table 2).
Similar to the electrical stimulation, exogenous NE also increased
luminal release of cGMP, the second messenger of NO. Perfusion with 10 µM NE for 10 min produced a sustained pressure increase and doubled
cGMP release (Table 3). NO synthase
inhibition resulted in a significant reduction of baseline cGMP
release. Moreover, the cGMP rise induced by 10 µM NE was abolished,
whereas the vasomotor response elicited by NE was significantly
increased (Table 3).
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Guanethidine-Induced Acute Chemical Sympathectomy
Luminal NE recovery during perfusion with guanethidine was undetectable (Table 1), confirming our previous report (14). Consequently, the elevation in perfusion pressure evoked by electrical stimulation was obliterated (Table 1). Baseline NO release was unchanged, but the NO surge after electrical stimulation was abolished (Fig. 7 and Table 1). In contrast, guanethidine did not affect vasoconstriction or luminal NO release induced by 1-min perfusion with 10 µM NE (Table 2), indicating that this drug does not interfere with NO synthase activity.
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1-Adrenoceptor Blockade
2-Adrenoceptor Blockade
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The main finding of this investigation is that electrical stimulation of the perivascular arterial mesenteric nerves of the rat results in endothelial NO release followed by a time-integrated increment in cGMP release. The temporal sequence of the changes in perfusion pressure and NE, NO, and cGMP release allows us to invoke a cause-effect cascade of chemical signals that are triggered by the electrical stimulus. We conclude that the NO release is most likely a NE-driven, vasoconstriction-mediated response, because the increases in perfusion pressure and NO release are elicited by exogenous NE and suppressed by sympathetic blockade.
Technical Considerations
We chose to investigate the possible relations between nerve stimulation and NE, NO, and cGMP release in the isolated arterial mesenteric bed because this preparation allows for monitoring vascular tone and direct access to chemicals released to the vascular lumen (14, 16). In addition, neurotransmitter release in response to electrical stimulation of sympathetic nerves has been characterized previously (14, 25). The term luminally accessible NE, NO, and cGMP was used to indicate that our measurements account for only a fraction of the total mediators released by the vascular wall to the lumen. The recovered fraction represents the sum of mediators released directly into the lumen plus diffusion from the interstitial space to the lumen.Measurements of NO are burdened with multiple technical problems. To date, chemiluminescence is the most specific, sensitive, and accurate method available for NO detection (3). However, the short half-life of NO in oxygen-rich biological fluids requires the measurement of NO oxidized products, nitrites and nitrates. To minimize background noise, we opted to use mild reducing conditions (KI/CH3COOH) that only reduce nitrites to NO (30, 32). In our hands, the use of strong reducing conditions (12) resulted in a low signal-to-background ratio because of the presence of chemicals containing amino groups that interfered with the measurement. Therefore, our measurements underestimate the total amount of NO released, because we did not detect the NO fraction further oxidized to nitrate.
The reproducibility of our measurements is highlighted by the small standard error obtained in baseline and stimulus-evoked NO determined in a large series of separate preparations. This indicates that our measurements accurately account for the changes of NO generated by the vascular wall within a minute-to-minute temporal resolution. Furthermore, the basal and stimulated luminal NO values we report in this study seem to be physiologically meaningful, because endothelium-dependent vasodilators induce NO increases of similar magnitude (18). Pilot experiments using authentic NO dissolved in the perfusing medium show that threshold arterial relaxation can be observed when 10-20 pmol/ml of NO are infused.
Perfusate cGMP levels were near detection threshold, making it difficult to regularly assess the minute-to-minute changes in the nucleotide simultaneously with NO. However, an equivalent cGMP production rate was detected after we extracted 10-ml perfusate samples. Thus our cGMP measurements likely correspond to the outflow fraction of this intracellular nucleotide produced before, during, and after activation of the vascular wall soluble guanylyl cyclase by NO (5).
NE recovery is influenced by the endothelial diffusion barrier and by specific cellular uptake mechanisms. Bitran and Tapia (6) recently showed that in the sympathetic neurons of the vas deferens, close to 80% of the released NE is recaptured by the sympathetic varicosities. In the present experiments, we avoided the use of drugs that interfere with the mechanism of NE recapture, in an effort to study the sympathetic nerves under their most physiological conditions.
Source and Mechanisms for Electrically Elicited NO Release
Our experiments with endothelium denudation after saponin treatment support the endothelial origin of the NO surge after transmural depolarization. This experimental maneuver abolished the NO increase elicited by electrical stimulation while maintaining muscular reactivity. The finding that the total amount of NE release elicited by electrical stimulation was unchanged by saponin demonstrates that the detergent did not alter the nerve terminals or the mechanism of the NE exocytosis. On the other hand, the increase in baseline luminal NE recovery may be explained by a faster transit of the neurotransmitter into the arterial lumen in absence of the major diffusion barrier posed by the endothelium, altering the kinetics of cellular NE recapture. In addition, we demonstrated that the NO surge depends on NO synthase activation, because inhibition of this enzyme with N-nitro-L-arginine abolished NO
release evoked by exogenous NE. In the case of electrical stimulation,
an artifact signal attributable to the electrolytic formation of a
nitritelike chemical is responsible for the apparent rise in NO release.
The mechanisms underlying the basal levels of luminal NO remain only partially known. Endothelium removal and NO synthase inhibition reduced basal NO release, although not always significantly. However, neither maneuver abolished baseline NO release, possibly reflecting a contribution of nitrites and nitrosothiols not originating from NO (3). The present results do not allow us to fully discard a possible contribution of remnant functional endothelial cells, residual NO synthase activity, or the presence of resident macrophages within the mesenteric smooth muscles. We deem unlikely any significant expression of inducible NO synthase occurring in the 60-90 min lapsing between setting the preparation and the experimental maneuvers. Whatever the source of the remnant NO measured, it is most likely that the fraction sensitive to saponin treatment and/or NO synthase inhibition is the physiologically relevant fraction.
Chemical sympathectomy and -adrenoceptor blockade experiments argue
against the possibility that NO is derived from sympathetic nerves or
from the vascular smooth muscle cells. As expected, neither chemical
sympathectomy nor -adrenoceptor blockade affected basal NO release.
Guanethidine abolished the vasopressor activity and NE release evoked
by electrical stimulation, confirming previous reports in the rat
mesenteric arterial bed (14). We now demonstrate that guanethidine also
abolishes the increase in NO release after transmural depolarization.
Guanethidine blocks exocytosis of sympathetic vesicles (10), but it
would not prevent a rise in cytosolic calcium or other
depolarization-induced changes that may affect neuronal NO synthase in
the sympathetic terminals, perivascular nonadrenergic-noncholinergic
nerve fibers (22), or nitrergic nerves (35). Furthermore, it is well
established that this concentration of guanethidine does not modify
smooth muscle contractility (14, 16). The finding that guanethidine did
not affect either the motor response or the NO release induced by
exogenous NE rules out any interference of this drug on NO synthase.
Therefore, the negligible NE exocytosis and lack of vasoconstriction
explain the lack of NO release after transmural depolarization in the presence of guanethidine.
A more detailed assessment of the mechanisms involved in endothelium
release of NO can be derived from the results with -adrenoceptor blocking drugs. Prazosin, an
1-adrenoceptor antagonist,
abolished electrically evoked vasoconstriction and NO release. Prazosin does not preclude the release of NE or other sympathetic transmitters or the putative release of NO from any type of nervous terminals. Therefore, the result with prazosin supports the concept that electrically evoked NO release is secondary to vasoconstriction and
renders it unlike any possible direct effect of electrically released
NE on endothelial
2-adrenoceptors (40). This
conclusion is also supported by the finding that 1 µM yohimbine, an
2-adrenoceptor blocker, reduced
in the same proportion vasoconstriction and NO release elicited either
by electrical stimulation or exogenous NE. The efficacy of this
concentration of antagonist on presynaptic sympathetic
2-adrenoceptors was confirmed
by the increased NE released on electrical depolarization. However,
because it reduced the vasoconstriction, this concentration of
yohimbine probably also partially blocked
1-adrenoceptors present on
the rat mesenteric smooth muscle (4), as well as postjunctional
2-adrenoceptors that may
contribute to vasoconstriction in this tissue (C. Meynard and J. P. Huidobro-Toro, unpublished observations).
Physiological Implications
We interpret the NO release as an endothelial response secondary to vasoconstriction. In chronological sequence, electrical stimulation elicits NE release that activates adrenergic receptors, leading to smooth muscle contraction and increased shear stress at the endothelial surface. Under constant flow, the elevation in shear stress may serve as the efficacious stimulus for endothelial NO synthase activation and NO release (12). Alternatively, smooth muscle activation after adrenergic stimulation may result in a rise of cytosolic calcium in endothelial cells through gap junction intercellular communication between both cell types (17). Our experiments support the notion that arteriolar NO release is sensitive to changes rather than to sustained levels of shear stress and/or vascular tone. With 1-min electrical stimulation, a clear, graded, frequency-dependent NO release proportional to the rise in perfusion pressure was observed. However, when the duration of the stimulus was changed, an apparent all-or-none NO release was evidenced after a 0.5-min threshold.Whatever the direct stimulus for endothelial NO synthase activation, the subsequently produced NO induces an increase in cGMP. The physiological response is a vasodilatory signal that counteracts the vasoconstriction elicited by NE. Evidence in favor of this counteracting mechanism is provided by the results observed in NO synthase-inhibited mesenteries, which showed prolonged vasoconstriction during 4-min electrical stimulation and a notable enhancement of the vasoconstriction induced by exogenous NE. These findings confirm what has been repeatedly shown in endothelium-denuded, isolated vascular rings that respond more vigorously to exogenous NE (13, 20, 26, 29). In intact mesenteries, the reduction in perfusion pressure and shear stress may contribute to the lack of further increase in NO release observed during 2- and 4-min stimulation.
We should also consider that ATP and NPY are coreleased with NE. ATP is
well known as an endothelium-dependent vasodilator, which putatively
acts through release of NO (9, 24, 25, 37). Therefore, it is possible
that in vivo, in addition to the increased shear stress, NE and its
cotransmitters may participate as a driving mechanism for endothelial
NO production during sympathetic stimulation. For instance, we have
observed a NO synthase-dependent hyperemia after NE-induced
vasoconstriction in the hamster microcirculation, which is enhanced by
NPY (8). Nevertheless, our results with -adrenoceptor blockers rule
out any significant vasoconstriction-independent participation of these
sympathetic cotransmitters on electrically elicited NO release in the
isolated, perfused mesentery.
In summary, our measurements of NE, NO, and cGMP release in the mesenteric arterial bed give a quantitative assessment of how sympathetically induced vasoconstriction elicits a secondary endothelial response that buffers the contractile efficacy of the neurotransmitters.
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ACKNOWLEDGEMENTS |
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The authors are grateful to J. A. Bravo for expertise in performing the cGMP RIA and to Dr. W. N. Durán (UMDNJ-New Jersey Medical School) for critical help in the interpretation and discussion of the data.
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FOOTNOTES |
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This work was funded in part by FONDECYT grants 1960502, 1971222, and 1980966 and financial aid from AGA-Chile, and Fundación Andes. In the course of this investigation, J. P. Huidobro-Toro was a recipient of a Cátedra Presidencial 1995 award.
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: M. P. Boric, Departamento de Ciencias Fisiológicas, FCB, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile (E-mail: mboric{at}genes.bio.puc.cl).
Received 6 July 1998; accepted in final form 26 March 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Aisaka, K.,
S. S. Gross,
O. W. Griffith,
and
R. Levi.
N-methyl arginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in-vivo?
Biochem. Biophys. Res. Commun.
163:
710-717,
1989[Medline].
2.
Anton, H.,
and
D. Sayre.
A study of the factors affecting the aluminum oxide trihydroxyindol procedure for the analysis of catecholamines.
J. Pharmacol. Exp. Ther.
138:
360-375,
1962
3.
Archer, S.
Measurement of nitric oxide in biological models.
FASEB J.
7:
349-360,
1993[Abstract].
4.
Barbieri, A.,
M. G. Santagostino-Barbone,
F. Zonta,
and
A. Lucchelli.
Pharmacological characterization of alpha-adrenoceptors that mediate contraction in splenic artery strips from the pig.
Naunyn Schmiedebergs Arch. Pharmacol.
375:
654-661,
1998.
5.
Beavo, J. A.
Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms.
Physiol. Rev.
75:
725-748,
1995
6.
Bitran, M.,
and
W. Tapia.
Does the release of tritiated noradrenaline accurately reflect the release of endogenous noradrenaline from rat vas deferens nerve terminals?
Biol. Res.
30:
105-115,
1997[Medline].
7.
Boric, M. P.,
and
H. R. Croxatto.
Inhibition of atrial natriuretic peptide excretory action by bradykinin.
Hypertension
26:
1167-1172,
1995
8.
Boric, M. P.,
M. V. Donoso,
A. Martínez,
and
J. P. Huidobro-Toro.
Neuropeptide Y is a vasoconstrictor and adrenergic modulator in the hamster microcirculation by acting on neuropeptide Y1 and Y2 receptors.
Eur. J. Pharmacol.
294:
391-401,
1995[Medline].
9.
Brake, A. J.,
and
D. Julius.
Signaling by extracellular nucleotides.
Annu Rev. Cell Dev. Biol.
12:
519-541,
1996[Medline].
10.
Brock, J. A.,
and
T. C. Cunane.
Studies on the mode of action of bretylium and guanethidine in postganglionic sympathetic nerves.
Naunyn Schmiedebergs Arch. Pharmacol.
338:
504-509,
1988[Medline].
11.
Conrad, K. P.,
and
S. L. Whittemore.
N-monomethyl-L-arginine and nitroarginine potentiate pressor responsiveness of vasoconstrictors in conscious rats.
Am. J. Physiol.
262 (Regulatory Integrative Comp. Physiol. 31):
R1137-R1144,
1992
12.
Corson, M. A.,
N. L. James,
S. E. Latta,
R. M. Nerem,
B. C. Berk,
and
D. G. Harrison.
Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress.
Circ. Res.
79:
984-991,
1996
13.
Cortes, V.,
M. V. Donoso,
N. Brown,
R. Fanjul,
C. López,
A. Fournier,
and
J. P. Huidobro-Toro.
Synergism between neuropeptide Y and norepinephrine highlights sympathetic cotransmission: studies in rat arterial mesenteric bed with neuropeptide Y, analogs, and BIBP 3226.
J. Pharmacol. Exp. Ther.
289:
1313-1322,
1999
14.
Donoso, M. V.,
N. Brown,
C. Carrasco,
V. Cortes,
A. Fournier,
and
J. P. Huidobro-Toro.
Stimulation of the sympathetic perimesenteric arterial nerves releases neuropeptide Y potentiating the vasomotor activity of noradrenaline: involvement of neuropeptide Y Y1 receptors.
J. Neurochem.
69:
1048-1059,
1997[Medline].
15.
Donoso, M. V.,
H. Faundez,
G. Rosa,
A. Fournier,
L. Edvinsson,
and
J. P. Huidobro-Toro.
Pharmacological characterization of the ETA receptor in the vascular smooth muscle comparing its analogous distribution in the rat mesenteric artery and in the arterial mesenteric bed.
Peptides
17:
1145-1153,
1996[Medline].
16.
Donoso, M. V.,
M. Steiner,
and
J. P. Huidobro-Toro.
BIBP 3226, suramin and prazosin identify neuropeptide Y, adenosine 5'-triphosphate and noradrenaline as sympathetic co-transmitters in the rat arterial mesenteric bed.
J. Pharmacol. Exp. Ther.
282:
691-698,
1997
17.
Dora, K. A.,
M. P. Doyle,
and
B. R. Duling.
Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.
Proc. Natl. Acad. Sci. USA
94:
6529-6534,
1997
18.
Figueroa, X., M. P. Boric, and J. P. Huidobro-Toro. Bradykinin-stimulated nitric oxide (NO) release in
mesenteric arterial bed is blunted in Goldblatt hypertensive rats
(Abstract). In Proceedings of Fifteenth International
Conference on Kinins, Kinin 98 edited by M. Katori. Nara, Japan,
1998, p. 95. (Publ. P-111-4).
19.
Furchgott, R.
The role of the endothelium in the responses of vascular smooth muscle to drugs.
Annu. Rev. Pharmacol. Toxicol.
24:
175-198,
1984[Medline].
20.
Furchgott, R.,
and
J. V. Zawadski.
The obligatory role of endothelial cells in the relaxation of the arterial smooth muscle by acetylcholine.
Nature
299:
373-376,
1980.
21.
Griffith, O. W.,
and
D. J. Stuehr.
Nitric oxide synthases: properties and catalytic mechanism.
Annu. Rev. Physiol.
57:
707-736,
1995[Medline].
22.
Han, S.-P.,
L. Naes,
and
T. C. Westfall.
Calcitonin gene-related peptide is the endogenous mediator of nonadrenergic-noncholinergic vasodilation in rat mesentery.
J. Pharmacol. Exp. Ther.
255:
423-428,
1990
23.
Ikeda, K.,
O. G. Gutierrez, Jr.,
and
Y. Yamori.
Dietary N-nitro-L-arginine induces sustained hypertension in normotensive Wistar-Kyoto rats.
Clin. Exp. Pharmacol. Physiol.
19:
583-586,
1992[Medline].
24.
Khakh, B. S.,
and
C. Kennedy.
Adenosine and ATP: progress in their receptors' structures and functions.
Trends Pharmacol. Sci.
19:
39-41,
1998[Medline].
25.
Lundberg, J. M.
Pharmacology of co-transmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate amino acids and nitric oxide.
Pharmacol. Rev.
48:
113-178,
1996[Medline].
26.
Luscher, T. F.,
L. Raij,
and
P. M. Vanhoutte.
Endothelium-dependent responses in normotensive and hypertensive Dahl rats.
Hypertension
9:
157-163,
1987
27.
Marín, J.,
and
M. A. Rodríguez-Martínez.
Role of vascular nitric oxide in physiological and pathological conditions.
Pharmacol. Ther.
75:
111-134,
1997[Medline].
28.
McGregor, D. D.
The effect of sympathetic nerve stimulation on vasoconstriction responses in perfused mesenteric blood vessels of the rat.
J. Physiol. (Lond.)
177:
21-43,
1965.
29.
Moncada, S.,
R. M. Palmer,
and
E. A. Higgs.
Nitric oxide: physiology, pathophysiology and pharmacology.
Pharmacol. Rev.
43:
109-142,
1991[Medline].
30.
Myers, P. R.,
R. Guerra, Jr.,
and
D. G. Harrison.
Release of NO and EDRF from cultured bovine aortic endothelial cells.
Am. J. Physiol.
256 (Heart Circ. Physiol. 25):
H1030-H1037,
1989
31.
Oehlenshlager, W. F.,
S. W. Kubolak,
and
M. G. Currie.
A rapid and ecconomical method of preparing radiodinated cyclic nucleotide derivatives for use in radioimmunoassays.
J. Immunoassay
11:
109-118,
1990[Medline].
32.
O'Neil, W. C.
Flow-mediated NO release from endothelial cells is independent of K+ channel activation or intracellular Ca2+.
Am. J. Physiol.
269 (Cell Physiol. 38):
C863-C869,
1995
33.
Peredo, H. A.,
and
M. A. Enero.
Effect of endothelium removal on basal and muscarinic cholinergic stimulated rat mesenteric vascular bed prostanoid synthesis.
Prostaglandins Leukot. Essent. Fatty Acids
48:
373-378,
1993[Medline].
34.
Racchi, H.,
A. J. Schliem,
M. V. Donoso,
A. Rahmer,
A. Zuñiga,
S. Guzmán,
K. Rudolf,
and
J. P. Huidobro-Toro.
Neuropeptide Y Y1 receptors are involved in the vasoconstriction caused by human sympathetic nerve stimulation.
Eur. J. Pharmacol.
329:
79-83,
1997[Medline].
35.
Rand, M. J.,
and
C. G. Li.
Nitric oxide as a neurotransmitter in peripheral nerves: nature of transmitter and mechanism of transmission.
Annu. Rev. Physiol.
57:
659-682,
1995[Medline].
36.
Rees, D. D.,
R. M. Palmer,
and
S. Moncada.
Role of endothelium-derived nitric oxide in the regulation of blood pressure.
Proc. Natl. Acad. Sci. USA
86:
3375-3378,
1989
37.
Sedaa, K. O.,
R. A. Bjur,
S. Kazumasa,
and
D. P. Westfall.
Nerve and drug-induce release of adenine nucleosides and nucleotides from rabbit aorta.
J. Pharmacol. Exp. Ther.
252:
1060-1067,
1990
38.
Vallance, P.,
J. Collier,
and
S. Moncada.
Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man.
Lancet
2:
997-1004,
1989[Medline].
39.
Vallance, P.,
A. Leone,
A. Calver,
J. Collier,
and
S. Moncada.
Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure.
Lancet
339:
572-575,
1992[Medline].
40.
Vanhoutte, P.,
and
V. M. Miller.
Alpha2-adrenoceptors and endothelium derived relaxing factor.
Am. J. Med.
87, Suppl. 3C:
1S-5S,
1989[Medline].
41.
Wu, K. K.,
and
P. Thiagarahan.
Role of endothelium in thrombosis and hemostasis.
Annu. Rev. Med.
47:
315-331,
1996[Medline].
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