© 1996 American Heart Association, Inc.
Articles |
Depression of Endothelial Nitric Oxide Synthase but Increased Expression of Endothelin-1 Immunoreactivity in Rat Thoracic Aortic Endothelium Associated With Long-term, but Not Short-term, Sympathectomy
the Department of Anatomy and Developmental Biology and Center for Neuroscience, University College London (UK).
Correspondence to Prof G. Burnstock, Department of Anatomy and Developmental Biology and Center for Neuroscience, University College London, Gower St, London WC1E 6BT, UK.
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Abstract |
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Recent pharmacological studies have shown that perivascular nerves can influence the development and function of vascular endothelial cells (ECs). However, morphological studies have not yet been carried out to investigate whether these functional changes are associated with changes in vasoactive substances in ECs. We used postembedding electron microscopy (EM) triple gold–labeling immunocytochemistry to study the effects of short-term sympathectomy (3 days after 6-hydroxydopamine [6-OHDA] treatment) and long-term sympathectomy (guanethidine and 8 days after 6-OHDA) on the distribution of vasoactive substances in ECs of the rat thoracic aorta. The postembedding immunocytochemistry, which can detect levels of label in individual cells, showed that there was a significant decrease in endothelial NO synthase (NOS3)–labeled, serotonin (5-HT)–labeled, and substance P (SP)–labeled, but a significant increase in endothelin-1 (ET-1)–labeled, gold particles in ECs after long-term, but not after short-term (3-day), sympathectomy. In conclusion, our results show that long-term sympathectomy causes an increase in ET-1 and decrease in NOS3, 5-HT, and SP immunoreactivity in ECs of the thoracic aorta. Our data also indicate that postembedding EM triple gold–labeling immunocytochemistry is a valuable technique for quantitative studies of the content of vasoactive substances in ECs.
Key Words: endothelium • sympathectomy • endothelial nitric oxide synthase • endothelin-1 • serotonin
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Introduction |
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Endothelial cells play a crucial role in the control of blood vessel tone, particularly as mediators of vasodilation and vasoconstriction to a number of vasoactive substances.1 2 EC damage or dysfunction is widely regarded as a critical initiating factor in the development of atherosclerosis and many other vascular diseases (for reviews, see References 3 and 4).
The trophic influence of perivascular nerves on blood vessel structure and function in normal development and in disease states has been previously examined (for review, see Reference 2). Since perivascular nerve fibers release many substances, it is possible that they may influence the ability of the target tissue (including ECs and SMCs) to respond to vasoactive substances. Aortic EC death was increased in a model of sympathetic nerve activation.5 Chronic stimulation of the rabbit ear artery resulted in an increase in the endothelial content of neuropeptide Y–like and calcitonin gene-related peptide–like immunoreactivity.6 Surgical sympathectomy or long-term adrenoceptor blockade by propranolol prevents or reduces the inhibition of atherosclerosis by diet, with or without exacerbation by stress.5 7 8 Sympathetic denervation before the development of hypertension results in an increased permeability of the blood-brain barrier.9
Pharmacological studies have shown that 2 to 8 weeks after sympathetic and sensory denervation of the rabbit ear artery, endothelium-dependent relaxation to methacholine is significantly depressed; endothelium-independent relaxation (to sodium nitroprusside) is unchanged.10 Chronic sensory denervation has been shown to result in impaired endothelium-mediated vasodilatation of the mesenteric arterial bed of the rat.11 12 Recently, an increase in flow-evoked ET-1 release has been shown in the isolated mesenteric arterial bed after long-term, but not short-term, sympathectomy.13 Long-term sympathetic denervation in rabbits causes an increase in the sensitivity of cerebral arteries to hypercapnia, hypoxia, and 5-HT.14 However, ultrastructural studies showed that visible morphological changes in the endothelium were not detected under these conditions.15
In the present study, our objective has been to determine whether the endothelial distribution of EDRF, namely NO,16 17 18 and one of the most important EDCFs, namely, ET-1,19 is altered by selective sympathetic denervation. NOS is the enzyme that converts L-arginine and molecular oxygen to L-citrulline and NO.20 21 Different isoforms of NOS (neuronal, NOS1; inducible, NOS2; and endothelial, NOS3) have been purified, characterized, cloned, and immunocytochemically shown in different tissues, including the endothelium of various vessels (see Reference 22). Alterations in NOS activity may play a critical role in atherogenesis.23 Several other vasoactive substances, including 5-HT and SP, are localized and synthesized in ECs in situ.2 Changes in the endothelial content of vasoactive substances could form part of a disease process before visible ultrastructural or functional damage to the endothelium can be detected. However, it is unclear whether there are changes in endothelial immunoreactivity for vasoactive substances after chronic sympathectomy. Thus, we have used short-term (3 days after 6-OHDA) and long-term (guanethidine and 8 days after 6-OHDA) sympathectomy to investigate the influence of perivascular sympathetic nerves on the immunoreactivity of endothelium-specific NOS (NOS3), ET-1, 5-HT, and SP in the thoracic aortic endothelium by using postembedding EM immunocytochemical techniques.
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Materials and Methods |
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Guanethidine Sympathectomy
Matched litters of male Sprague-Dawley rats were given subcutaneous injections of either 50 mg/kg guanethidine sulfate (Ismelin) or saline from day 8 after birth for 3 weeks, 5 days per week.13 24 25 Rats were killed at 12 to 14 weeks of age by asphyxiation with ether.
6-OHDA Sympathectomy
Short-term sympathectomy was induced with 6-OHDA (since guanethidine treatment requires an extended injection regimen). Sprague-Dawley rats that were age-matched to those used for experiments with guanethidine sympathectomy were intraperitoneally injected with 6-OHDA dissolved in sterile saline with 1 mg/mL (0.1%) ascorbic acid to retard oxidation of the drug or with ascorbic acid alone. The injection regimen was 100 mg/kg on day 1 of treatment and 250 mg/kg on day 2; the rats were killed on day 3 or 813 25 by asphyxiation with ether.
Immunocytochemistry
Rat thoracic aortas were dissected out, immersion-fixed for 3 hours at 4°C with fixative containing 4% paraformaldehyde and 0.25% glutaraldehyde (in 0.1 mol/L cacodylate buffer at pH 7.4), and then transferred to cacodylate buffer and stored overnight at 4°C. Aortas were cut to produce 6- to 8-mm-long strips, which were then processed for postembedding EM immunocytochemistry.
Postembedding Triple Gold–Labeling Immunocytochemistry
Strips of aorta (osmicated or nonosmicated) were dehydrated in ethanol and propylene oxide and embedded in Unicryl. Localization of NOS3 (10 nm), ET-1 (5 nm), 5-HT (20 nm), and SP (20 nm) in ECs was examined in ultrathin sections of the specimens (on uncoated nickel grids) using the immunogold labeling technique (adapted from References 26 through 28). The procedure was as follows: Sections were (1) exposed to 5% H2O2 solution at room temperature for 15 minutes, (2) washed five times in bidistilled water at 5-minute intervals, (3) incubated with hot inactivated normal goat serum (1:10) in Tris buffer containing 0.5% BSA, 0.1% Tween-20, 0.9% NaCl, and 0.1% sodium azide (pH 8), (4) washed five times with buffer solution every 20 minutes, (5) incubated with primary antibody (NOS3/monoclonal H-32 purified from bovine aortic ECs) diluted 1:300 to 1:400 overnight at 4°C in Tris buffer (the same buffer used for dilution of goat serum), (6) washed with Tris buffer containing Tween-20 five times every 20 minutes, (7) incubated with goat anti-mouse IgG serum–coated colloidal gold probe coupled to 10-nm gold particles at a dilution of 1:75 to 1:90 in Tris buffer (the same buffer used for dilution of the normal goat serum) for 3 to 4 hours at room temperature, (8) washed five times with Tris buffer containing Tween-20 every 20 minutes, and (9) washed five times with bidistilled water every 20 minutes. The same procedure was used for double and triple labeling with polyclonal antibody against ET-1, SP, or 5-HT. Polyclonal antibody specimens were incubated with hot inactivated normal goat serum diluted in the same Tris buffer. Dilutions of primary antibody for second and third labeling were 1:200 and 1:100 to 1:120, respectively. The concentrations of goat anti-rabbit IgG serum–coated colloidal gold particles were 1:50 to 1:60 and 1:30 to 1:40 for second and third labeling, respectively. Labeling of ET-1, 5-HT, or SP was with 5- or 20-nm goat anti-rabbit IgG serum–coated gold particles. Other steps of the immunogold labeling procedure were the same as above. After several more washes with Tris buffer and bidistilled water, specimens were stained with uranyl acetate and lead citrate and examined using a JEOL TEM-1010 microscope.
Control for Postembedding Immunocytochemistry
The NOS3 antisera (H-32) used in the present study was monoclonal and was from Abbott Laboratories. The specificity and characteristics of this antibody have been reported previously.22 Rabbit polyclonal antibody to human ET-1 and rabbit antiserum to 5-HT and SP were from Cambridge Research Biochemicals, and the specificities of these antibodies have been reported previously.29
In preliminary experiments, we first tested the specificity of consecutive triple gold–labeling techniques. The single and double labeling for different antibodies was also tested using different sizes of gold particles (5, 10, and 20 nm). Specificity of faces A and B of the sections was also tested. Moreover, some grids were incubated with mixed antibody (NOS3 in combination with other polyclonal antibodies: NOS3 and ET-1, NOS3 and 5-HT, and NOS3 and SP), and then specimens were labeled for the third antibody. The density of gold particles for different vasoactive substances was the same as for triple labeling (data not shown). After stabilization of triple labeling, postembedding triple gold immunocytochemistry procedures were generally used. Control specimens were exposed to the same procedure, after the omission of primary antibody or with incubation with synthetic antibody.
Statistical Analysis
The specific volume (number of gold particles per µm2) of gold particles for antibody against NOS3, ET-1, 5-HT, and SP was evaluated by counting gold granules and tissue points of randomly selected photographic fields (80 observations from each of five animals per experimental group), as reported previously.28 30 31 All morphometric measurements were carried out at each of the time points studied. All values are expressed as mean±SEM of n observations. A two-way ANOVA was used to analyze results from repeated measurements. End-point experiments were tested by ANOVA. This was followed by a least significant difference procedure to determine differences of response (SPSS Inc). Values of P<.05 were considered statistically significant. Tables of correlation analyses between each of the pairs of markers (NOS1, ET-1, 5-HT, and SP) measured at each of the time points studied were provided by using the program Excel 5.0 for Windows on an IBM 386 DX-40.
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Postembedding EM Triple Gold–Labeling Cytochemistry
ECs from control aortas showed large numbers of NOS3-positive gold particles (10 nm) in the cell cytoplasmic matrix (Fig 1A
). NOS3-containing gold particles were rarely found in the cell nucleus. There were generally few gold particles in relation to the cytoplasmic organelles. The NOS3-containing gold particles were usually localized in the cytoplasm and particularly in the external membrane of the granular endoplasmic reticulum and the external membranes of mitochondria. At 3 days after 6-OHDA treatment, the number of NOS3-containing gold particles appeared to decrease, particularly in the abluminal parts of the cytoplasmic matrix, but this was not statistically significant (Figs 1B, 2A, and 3). A considerable decrease in NOS3-containing gold particles was seen 8 days after 6-OHDA and after long-term guanethidine sympathectomy. Sometimes there were only a few NOS3-positive gold particles in the cytoplasmic matrix of ECs (Fig 4A).
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), and compared with 8-day 6-OHDA and guanethidine treatment (
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The distribution of gold particles against ET-1 (5 nm) from control aortic ECs showed a pattern similar to that of NOS3-containing gold particles. Aortas from sympathectomized animals showed an increasing number of ET-1–containing gold particles in the cytoplasmic matrix. A feature of ET-1–containing gold particles was the appearance of cluster-type localization (Fig 2B
). In particular, abluminal and basal parts of the cytoplasmic matrix of ECs showed a large number of ET-1–positive gold particles. The presence of ET-1–positive gold particles in EC organelles was sometimes also observed (see Figs 2B, 4B).
The antibody against 5-HT was coupled to a large-sized gold particle (20 nm), which showed a lower density within ECs than did NOS3 and ET-1. In control experiments, 5-HT–containing gold particles were randomly distributed throughout the cytoplasm (Fig 1A). After 3 days of 6-OHDA treatment, the density of 5-HT–containing gold particles appeared to increase, but this was not statistically significant (Fig 3). The increase was only seen near the thoracic aortic ostia, mainly in regions where SMCs were present in the subendothelium; cytoplasmic parts of ECs that sharply protruded into the vessel lumen displayed clusters of 5-HT–containing gold particles (Fig 1B). At 8 days after 6-OHDA treatment, a decrease in the density of 5-HT–containing gold particles, compared with control and after 3 days of 6-OHDA treatment, was seen (Figs 2 and 3). After long-term guanethidine sympathectomy, the density of 5-HT–containing gold particles was considerably decreased (Fig 3).
The density of SP-containing gold particles (20 nm) in control aortic ECs was similar to that of 5-HT–containing gold particles. The cytoplasmic matrix of ECs showed a diffuse localization of SP-containing gold particles. After long-term sympathectomy, there was a decrease in the density of SP-positive gold particles (Fig 4B). After guanethidine treatment, an absence of SP-containing gold particles was frequently observed.
Control specimens, which were processed according to the same procedure but with the omission of primary antibody or with synthetic antibodies, showed an absence of any gold particles in the cytoplasmic matrix or EC nucleus (Fig 5A). Rarely, one or two gold particles were found when rabbit polyclonal antibodies were used (Fig 5B).
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Morphometric Measurement
The specific volumes of gold particles containing eNOS, ET-1, 5-HT, and SP after short- and long-term sympathectomy are summarized in Fig 3.
The specific volume of eNOS-containing gold particles 3 days after 6-OHDA treatment was slightly, but not significantly, decreased. However, 8 days after 6-OHDA treatment, a significant decrease in the specific volume of gold particles was seen compared with the control aortic endothelium (P<.001). After guanethidine treatment, the specific volume of eNOS-containing gold particles was significantly less compared with control and 3-day 6-OHDA–treated groups (P<.001 and P<.05, respectively). There was no significant difference at 8 days between 6-OHDA–treated and guanethidine-treated groups (Fig 3).
The specific volume of ET-1–containing gold particles in control groups was similar to eNOS-containing gold particles (Fig 3). At 3 days after 6-OHDA treatment, the specific volume of ET-1–containing gold particles was significantly increased (P<.05). At 8 days after 6-OHDA treatment, this difference was even greater (P<.001). The guanethidine-treated group showed a significant increase in the specific volume of ET-1–containing gold particles compared with control and 3- and 8-day 6-OHDA–treated groups (P<.001, P<.01, and P<.05, respectively) (Fig 3).
The specific volume of 5-HT–containing gold particles was not significantly different in control and 3-day 6-OHDA–treated groups (0.26±0.008 and 0.28±0.02 particles per µm2, respectively). However, 8 days after 6-OHDA treatment, there was a significant decrease in the specific volume of 5-HT–containing gold particles compared with the control (P<.05). In aortic ECs from long-term sympathectomized animals, there was a significant decrease in the volume of 5-HT–containing gold particles compared with control and 3-day 6-OHDA–treated groups (P<.01 and P<.05, respectively).
The specific volume of SP-containing gold particles decreased progressively with increasing duration of sympathectomy. However, this only reached statistical significance after long-term guanethidine treatment (P<.05 compared with controls). Correlation analyses between each of the pairs of markers measured at each of the time points studied are presented in the Table in control, 3- and 8-day 6-OHDA–treated, and guanethidine-treated groups. In controls, correlation analysis between NOS1 and ET-1 showed a strong positive correlation between these two vasoactive substances. There was a slight negative correlation between NOS1 and 5-HT and between ET-1 and 5-HT. There was no statistically significant correlation between other pairs of vasoactive substances. At 3 days and 8 days after 6-OHDA treatment, there was a strong positive correlation between NOS1 and ET-1, although less than in the controls. Between other vasoactive substances, there was no significant correlation. In the guanethidine-treated group, there was a positive correlation between NOS1 and ET-1, although this was significantly less strong than in the other groups. In contrast to the other groups, a slight positive correlation between 5-HT and SP was observed. There was no correlation between other pairs of vasoactive substances.
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Discussion |
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The results of the present study show that there is a significant decrease in NOS3, 5-HT, and SP and an increase in ET-1 immunoreactivity in rat thoracic aortic endothelium after long-term, but not after short-term, sympathectomy. These findings identify a selective influence of sympathetic nerves on the vasoactive substances in the rat thoracic aortic endothelium and lend support to existing pharmacological evidence that perivascular nerves have a trophic influence on ECs.
The application of postembedding EM single and double gold–labeling cytochemistry for different vasoactive substances in ECs (ET-1, vasopressin, SP, and 5-HT) by using polyclonal antibodies in primary cultures of ECs has been reported previously.29 In the present study, we have extended this technique to show for the first time application of a triple label for different vasoactive substances in the same cells. The physiological relevance for the localization of endothelial vasoactive substances within ECs comes from demonstration of their release in response to various stimuli (for review, see Reference 2), which is selective, since not all substances are synthesized or released under all conditions.32 33 34 The influence of perivascular nerves on the endothelial content of vasoactive substances was examined by using guanethidine or 6-OHDA treatment to cause selective denervation of sympathetic nerves.23 25 35 36 37 The aortic endothelium can modulate vascular relaxation or contraction by the synthesis and release of several factors including EDRF (NO) and EDCF (ET-1). An implication of our results showing a significant decrease in the number of eNOS-containing gold particles after long-term, but not after short-term, sympathectomy is that perivascular sympathetic nerves may be modulators of endothelium-specific NOS3 activity in the thoracic aortic endothelium. Conversely, under conditions of intact innervation, NO may have a physiological role to counterbalance constrictor effects mediated by sympathetic nerves.
The marked increase in ET-1 immunoreactivity in 6-OHDA–treated and, in particular, guanethidine-treated rat thoracic aortic ECs indicates a possible role for this peptide in modulating thoracic aortic blood flow. The increase in the number of ET-1–containing gold particles after sympathectomy may be a consequence of changes in the mRNA expression of ET or of changes in mechanisms of synthesis or production and, together with the simultaneous decrease in other vasoactive substances, may represent a local compensatory response of the aortic wall to maintain tone. We recently demonstrated a selective increase in flow-evoked ET release from the isolated mesenteric arterial vasculature of the rat after long-term, but not short-term, sympathectomy.13
A decrease in EDRF has been reported in various situations, some of which are also associated with changes in perivascular innervation, including hypertension and different models of atherosclerosis.10 11 12 38 39 40 41 42 43 44 One explanation for the decrease in EDRF-mediated relaxation is that there may be an increase in the production of EDCF. Such a reciprocal change is indicated in the present study, where the decrease in NOS3-containing gold particles was associated with an increase in ET-1–containing gold particles. Saijonmaa and Fyhrquist45 have shown that oxyhemoglobin, an inhibitor of NO, stimulates ET-1 production in cultured human umbilical cord vein ECs. The stimulated release of ET-1 was reduced by the NO donor (SIN-1, the active metabolite of molsidomine) acting through cGMP.46 In our experiments, the increase in the number of ET-1–containing gold particles was approximately equal to the decrease in the number of NOS3-containing gold particles, suggesting that there may be antagonism between NOS3 and ET-1 in ECs. A strong positive correlation between NOS3-containing and ET-1–containing gold particles was observed. This became less strong after sympathectomy, particularly after guanethidine treatment, suggesting breakdown of normal vascular control mechanisms.
5-HT has been shown to induce vasoconstriction via direct actions on the vascular smooth muscle and endothelium-dependent vasodilation in arteries of different species.47 48 Burnstock et al49 have shown that ECs release 5-HT into the heart perfusate during hypoxia-induced vasodilation. The physiological consequences of the decrease in EC 5-HT seen in the present study with respect to production and release and the action of 5-HT during hypoxia and other conditions remain to be investigated. The decrease in number of SP-positive gold particles after long-term sympathectomy suggests that sympathetic nerves also have trophic effects on the content of neuropeptides in ECs of the thoracic aorta.
The mechanism by which perivascular nerves modulate the endothelial content of vasoactive substances is not known but may involve nerve impulse regulation of activity, a specific involvement of the neurotransmitter itself, or an unidentified trophic factor.15 50 51 It is unlikely that changes in blood pressure are responsible for the observed changes, since previous workers have shown no changes in mean blood pressure after chronic 6-OHDA52 or guanethidine treatment.53 However, it is possible that changes in circulating substances may influence the endothelial content of vasoactive substances. Plasma noradrenaline and adrenaline levels were reported to be markedly higher in 6-OHDA–treated dogs than in control dogs.54 An increase in plasma levels of vasopressin has been reported after chronic guanethidine sympathectomy.55 It is also possible that the changes in EC immunoreactivity seen in the present study are not due to the removal of a trophic influence of sympathetic nerves, per se, but may be due to the resulting increase in the remaining populations of perivascular nerves.
In conclusion, the results from the present study show that long-term sympathectomy causes a decrease in eNOS (NOS3), 5-HT, and SP and an increase in ET-1 immunoreactivity in the thoracic aortic endothelium of the rat, although the biological significance of these changes remains to be shown. Our study also indicates that postembedding EM triple gold–labeling immunocytochemistry is a valuable technique for studying the relative changes in the content of vasoactive substances in ECs.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by the British Heart Foundation. The authors wish to thank Abbott Laboratories for providing the eNOS (NOS3) antiserum (H-32) and Dr D. Christie for editorial assistance.
Received October 31, 1995; accepted April 11, 1996.
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