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Am J Physiol Heart Circ Physiol 290: H398-H405, 2006. First published September 2, 2005; doi:10.1152/ajpheart.00712.2005
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Enhanced neurally evoked responses and inhibition of norepinephrine reuptake in rat mesenteric arteries after spinal transection

James A. Brock, Melanie Yeoh, and Elspeth M. McLachlan

Prince of Wales Medical Research Institute, University of New South Wales, Randwick, Sydney, New South Wales, Australia

Submitted 1 July 2005 ; accepted in final form 29 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In patients with high thoracic spinal lesions that remove most of the central drive to splanchnic preganglionic neurons, visceral or nociceptive stimuli below the lesion can provoke large increases in blood pressure (autonomic dysreflexia). We have examined the effects of T4 spinal transection on isometric contractions of mesenteric arteries isolated from spinalized rats. Nerve-evoked contractions involved synergistic roles for norepinephrine and ATP. At 7 wk after spinal transection, responses to perivascular stimulation at 1–5 Hz were enhanced fivefold, whereas the {alpha}1-adrenoceptor antagonist prazosin (10 nM) produced a twofold larger reduction in contraction (to 20 pulses at 10 Hz) than in unoperated controls. In contrast, the reduction in nerve-evoked contractions by the P2-purinoceptor antagonist suramin (0.1 mM) and the responses to the P2-purinoceptor agonist {alpha},{beta}-methylene ATP or to high K+ concentration did not greatly differ between groups, indicating that arteries from spinalized rats were not generally hyperreactive. Sensitivity to the {alpha}1-adrenoceptor agonist phenylephrine was enhanced in arteries from spinalized rats, and the difference from controls was abolished by the norepinephrine uptake blocker desmethylimipramine. Sensitivity to the {alpha}1-adrenoceptor agonist methoxamine, which is not a substrate for the neuronal norepinephrine transporter, was similar among the groups. Thus the increased neurally evoked response after spinal transection appeared to be due to a reduction in neuronal uptake of released norepinephrine, a mechanism that did not explain the enhanced response of tail arteries after spinal transection that we previously reported. The findings provide further support for potentiated neurovascular responses contributing to the genesis of autonomic dysreflexia.

neurovascular transmission; vascular reactivity; spinal cord injury; sympathetic nerve; autonomic dysreflexia

IN PATIENTS WITH SEVERE HIGH spinal cord lesions, resting arterial blood pressures are usually low immediately after the injury, presumably because of a marked decrease in ongoing sympathetic activity below the lesion (28, 30, 33). Partial recovery of arterial blood pressure during the first few weeks after the injury is thought to be due to adaptation of the renin-angiotensin system (21). However, patients with lesions above T6 may experience periods of excessively high blood pressure initiated by bladder or colon distension or unheeded nociceptor input from below the lesion; this symptom is known as autonomic dysreflexia. On the basis of experimentation in animal models, inflammation and plasticity of connections within the spinal cord are thought to contribute to autonomic dysreflexia by generating exaggerated reflex activity (34). However, microneurography studies of sympathetic cutaneous vasoconstrictor axons in patients with high spinal lesions showed only brief bursts of nerve activity in response to bladder compression or electrical stimulation of abdominal skin (33).

There is evidence suggesting that peripheral vascular changes might be sufficient to account for autonomic dysreflexia. Spinal cord transection at T8, which removes all bulbospinal inputs to the preganglionic neurons supplying the rat tail artery, markedly enhanced the amplitude and duration of contractile responses of isolated segments of this cutaneous artery to sympathetic nerve stimulation in vitro (35). When the postganglionic neurons that project to the rat tail artery were silenced (decentralized) by transection of their preganglionic inputs, neurovascular transmission was similarly augmented (36). These findings show that a decrease in sympathetic nerve activity after spinal cord injury leads to hyperreactivity of the cutaneous vasculature to neural activation. The brief reflex sympathetic bursts initiated in spinal patients (33) were associated with ≥30-s decreases in cutaneous blood flow. In normal subjects, similar bursts of activity decrease skin blood flow for ~10 s, indicating that reflex vasoconstriction is prolonged in the skin of patients with spinal lesions. This change would play a major role in the genesis of autonomic dysreflexia, if it applies in many vascular beds below the lesion.

In patients, autonomic dysreflexia is normally not expressed unless the lesion is above T6, i.e., when the loss of baroreflex control includes most of the splanchnic vascular bed. Rats with spinal transection at or above T5 also develop autonomic dysreflexia (15). In the present study, we have investigated the effects of spinal transection at T4 on neuroeffector responses of isolated second-order mesenteric arteries. In the rat, spinal transection at T4 removes virtually all bulbospinal inputs to the sympathetic preganglionic neurons supplying the splanchnic vascular bed while damaging <5% of these neurons directly (2, 29).

Mesenteric arteries in the rat are innervated by axons that originate from sympathetic postganglionic neurons and peptidergic primary afferent neurons with cell bodies in dorsal root ganglia (14, 19). Electrical activation of sympathetic terminals produces contraction of second-order mesenteric arteries through the combined action of norepinephrine (NE) at {alpha}1-adrenoceptors and ATP at P2X1 purinoceptors (3, 12, 27). In contrast to the rat tail artery (35), {alpha}2-adrenoceptors do not contribute to the postjunctional actions of released NE (3, 27). Calcitonin gene-related peptide released from the activated primary afferent axons opposes the effects of sympathetic axon activation (1). When investigating the effects of spinal transection on the mechanical responses to sympathetic nerve activation, we therefore pretreated the arteries with capsaicin to avoid the effects of activating the afferent axons. We determined the effects of the {alpha}1-adrenoceptor antagonist prazosin and the P2-purinoceptor antagonist suramin on neurally evoked contractions. In addition, we evaluated the effects of spinal transection on responses of the mesenteric arteries to the {alpha}1-adrenoceptor agonists phenylephrine (PE) and methoxamine, to the P2X-purinoceptor agonist {alpha},{beta}-methylene ATP ({alpha},{beta}-mATP), and to depolarization evoked by raised extracellular K+ concentration ([K+]o).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All experimental procedures conformed to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the University of New South Wales Animal Care and Ethics Committee.

The spinal cord was transected in female inbred Wistar rats (~7 wk of age) under anesthesia with intraperitoneally injected ketamine (60 mg/kg) and xylazine (10 mg/kg). After infiltration of the back muscles with 0.3 ml of the long-acting local anesthetic bupivacaine hydrochloride (5 mg/ml; Marcain, AstraZeneca), the spinal cord was exposed by a laminectomy over the T4 segment, and the cord was cut with fine scissors. A piece of gelatin foam was placed between the cut surfaces and another over the laminectomy site before oxytetracycline powder was applied and the incision was closed. Warm saline (≥2 ml, 0.9% NaCl) was injected intraperitoneally, and oxytetracycline (100 mg/kg) and benzylpenicillin (90 mg/kg) were injected subcutaneously. The urinary bladder was emptied before and at frequent intervals after spinal transection. The animals were operated on a heating blanket; respiration and rectal temperature were monitored frequently until the animals recovered consciousness. After surgery, the animals recovered quickly, moving about their cages within 2 h and eating and drinking well onto the next day. Urinary bladders were emptied manually at least four times a day for the first 1–2 wk until they emptied automatically, and the animals were then monitored at least twice a day. The animals (n = 25) were caged in groups of two to four and maintained for 7.5 wk (SD 0.6). During this period, the animals appeared healthy; they recovered body weight over the 1st wk and gained weight thereafter. In sham-operated controls, the laminectomy was performed to expose the spinal cord. The sham-operated animals (n = 19) were maintained for 7.0 wk (SD 0.6). In addition, unoperated animals (n = 25) age matched to the spinalized and sham-operated animals were also used as controls.

The animals were exsanguinated under deep anesthesia with pentobarbitone (80–100 mg/kg ip), and the ileum and attached mesentery were removed and maintained in physiological saline of the following composition (in mM): 150.6 Na+, 4.7 K+, 2 Ca2+, 1.2 Mg2+, 144.1 Cl–, 1.3 H2PO4–, 16.3 HCO3–, and 7.8 glucose. The solution was gassed with 95% O2-5% CO2 (to pH 7.2) and warmed to 35–36°C. Second-order arteries supplying the distal 20 cm of ileum (measured from the point of entry into the cecum) were isolated.

Mechanical responses. Segments of second-order mesenteric artery 1.15–2.00 mm long [1.55 mm (SD 0.17)] were mounted isometrically between stainless steel wires (50 µm diameter) in a four-chamber myograph (Multi Myograph model 610M, Danish Myo Technology). Each chamber of the myograph contained 6 ml of physiological saline that was exchanged at intervals of 6–15 min throughout the recording period. To normalize the basal conditions and the isometric contractions, Laplace's equation was used to convert the measured force to the effective pressure exerted on the luminal surface of the artery (24). Initially, the inner circumference at a distending pressure of 13.3 x 103 N/m2 (100 mmHg) was determined, and the circumference was adjusted to 90% of that determined at 13.3 x 103 N/m2. At this setting, mesenteric arteries are at the peak of their length-force relation (24). Output from the myograph was recorded by using a PowerLab recording system (ADInstruments).

After they were mounted, the vessels were allowed to equilibrate for 30 min; during this period all arteries were treated with 1 µM capsaicin for 10 min to remove the effects of the peptidergic primary afferent axons (10). The vessels were then exposed to four 6-min applications of 3 µM PE, each separated by an 8-min washout interval. During this period, the amplitude of the contractions to each successive application of PE increased to reach a plateau level.

Electrical stimulation. Electrical stimuli (0.2-ms pulse width, 30 V) supplied by a Grass SD9 stimulator were applied through platinum plate electrodes mounted on either side of the artery along its length. In preliminary experiments, these stimuli produced supramaximal responses that were abolished by 0.5 µM tetrodotoxin, indicating that the electrically evoked contractions were due entirely to release of neurotransmitter from the perivascular nerves.

The arteries were stimulated with single trains of 100 stimuli at 1, 2, 3, and 5 Hz, with each train separated by a 5-min interval. These long trains of stimuli were chosen to mimic the effects of ongoing nerve activity in vivo. In arteries from unoperated animals, contractions to stimulation frequencies <1 Hz were too small to measure accurately.

In the experiments investigating the effects of blocking {alpha}1-adrenoceptors and P2 purinoceptors, the tissues mounted in two chambers were stimulated at 4-min intervals with trains of 20 stimuli at 10 Hz, with the averaged response to the third and fourth trains being used for control data. These trains of relatively high-frequency stimuli were used because they produced fast transient contractions of sufficient amplitude to enable accurate assessment of the effects of antagonists. After the fourth train of stimuli, the {alpha}1-adrenoceptor antagonist prazosin (10 nM) was added to one chamber and the P2X purinoceptor antagonist suramin (0.1 mM) was added to the other. Each drug was left in contact with the tissues for four trains of stimuli, and the response to the fourth train was used to determine its effect. Both tissues were then exposed to a combination of prazosin and suramin at these concentrations for another four trains of stimuli, with the response to the last train used to determine the combined effect of both agents.

Chemical stimulation. Noncumulative concentration-response curves for the {alpha}1-adrenoceptor agonists PE (0.01–30 µM) and methoxamine (0.01–30 µM) were determined by applying concentrations that increased by half-log increments, with the tissue exposed to each concentration for 5 min and then washed for 7 min before the addition of the next concentration. During the wash, the tissue relaxed completely to basal tension. In one set of experiments, concentration-response curves to PE were determined in the presence and absence of desmethylimipramine (DMI) to determine the effects of blocking the neuronal NE transporter. In another set of experiments, after the highest concentration of PE, the artery was exposed to 3 µM {alpha},{beta}-mATP for 3 min. For groups of arteries from unoperated and spinalized rats, concentration-response curves to 0.03–10 µM {alpha},{beta}-mATP were determined by applying concentrations that increased by half-log increments, with the tissue exposed to each concentration for 3 min and then washed for 30 min before the addition of the next concentration. These experiments showed that 3 µM {alpha},{beta}-mATP produces a near-maximal contraction.

Responses to 60 mM extracellular K+ were recorded in the presence of 10 nM prazosin to prevent the excitatory actions of NE released from the sympathetic nerves. The tissues were exposed to three applications of 60 mM K+-physiological saline solution (equimolar substitution of KCl for NaCl) for 3 min, each separated by 5–10 min washes. The average responses to the second and third applications of K+ were analyzed.

In a separate series of experiments, concentration-response curves for K+ were determined for arteries from sham-operated and spinalized animals. These arteries were treated with 10 nM prazosin and exposed for 5 min to physiological saline solutions containing 0, 20, 40, 60, 80, and 100 mM K+ (equimolar substitution of KCl for NaCl), with 10-min washout periods between application of each concentration.

Data analysis. Peak amplitudes of the contractions to trains of electrical stimuli, to applications of PE, methoxamine, or {alpha},{beta}-mATP, or to changes in [K+]o were measured. For the contractions evoked by 100 stimuli at 5 Hz, the rise time was the interval between 10 and 90% of the peak contraction and 50% decay was the interval for the contraction at the end of the stimulus train to decline by 50%. The 50% decay time of high-K+-induced contraction was measured as the time from removal of the high-K+ solution until the amplitude of the contraction had fallen to 50% of its value at the start of washout. When data were obtained from more than one arterial segment from one animal, the mean value was used for statistical comparisons (i.e., n = number of animals studied).

SPSS 11 for Mac OS X (SPSS, Chicago, IL) was used for all statistical comparisons. For all nerve stimulation data and 50% decay time data for the K+ contractions, Mann-Whitney U tests were used for pairwise comparisons and Kruskal-Wallis tests for multiple comparisons because of unequal variance between the groups of data (determined by F tests). When multiple pairwise comparisons were made, P for the Mann-Whitney U-tests was adjusted using the Dunn-Sidak method. For all other multiple comparisons, one-way ANOVA and Tukey-Kramer post hoc tests were used. Other pairwise comparisons were made with two-tailed paired or unpaired Student's t-tests. EC50 and slope of the concentration-response curves for PE and methoxamine were estimated by fitting the data to the Hill equation using Igor Pro (Wavemetrics, Lake Oswego, OR). The degree of association between the response of arteries to electrical activation and their sensitivity to PE was assessed using Spearman's coefficient of rank correlation. P < 0.05 was taken as a significant difference. The nerve stimulation data and the 50% decay time data for the K+ contractions are presented as medians and interquartile range (IQR), and all other data are presented as means (SD).

Drugs. L-Phenylephrine HCl (PE), prazosin HCl, methoxamine HCl, desmethylimipramine HCl (DMI), {alpha},{beta}-mATP, and suramin were supplied by Sigma Chemical. Prazosin was prepared as a 1 mM stock solution in 10% (vol/vol) dimethyl sulfoxide in water. PE, methoxamine, DMI, and {alpha},{beta}-mATP were prepared as stock solutions in water, and suramin was made up directly in the physiological saline.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Basal conditions. The luminal diameter of the vessels and basal distending pressures measured after the equilibration period did not differ significantly between unoperated [286 µm (SD 27) diameter, 6.7 x 103 N/m2 (SD 0.7 x 103) pressure, n = 25], sham-operated [277 µm (SD 28) diameter, 7.3 x 103 N/m2 (SD 0.8 x 103) pressure, n = 19], and spinalized [276 µm (SD 32) diameter, 6.9 x 103 N/m2 (SD 1.0 x 103) pressure, n = 25] arteries (P = 0.14 by ANOVA for diameter, P = 0.10 by ANOVA for basal distending pressure).

Responses to neural stimulation. Figure 1A shows the contractile response of a sham-operated and a spinalized artery to trains of 100 stimuli at 1, 3, and 5 Hz. Arteries from spinalized animals generated much larger responses to all frequencies of stimulation than arteries from unoperated and sham-operated animals (Fig. 1B). The magnitude of this effect was similar over the range of frequencies of stimulation, the response of the spinalized arteries being increased approximately five- to sixfold compared with the sham-operated arteries. However, the peak contractile response of sham-operated arteries to stimulation at 3 and 5 Hz was smaller than that of unoperated arteries (Fig. 1B).



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  		Fig. 1. Enhanced contractions to perivascular nerve stimulation after spinal transection. A: contractions of arteries from sham-operated and spinalized animals to stimulation of perivascular nerves with 100 stimuli at 1, 3, and 5 Hz. B: peak increase in pressure produced by 100 stimuli at 1, 3, and 5 Hz in arteries from unoperated (open bars, n = 14), sham-operated (hatched bars, n = 13), and spinalized (solid bars, n = 14) animals. Values are median and interquartile range. Statistical differences (Mann-Whitney U-tests) between responses to each frequency of stimulation: **P < 0.01; ***P < 0.001.

 
Time course of neurally evoked responses. The effects of spinal transection on the time course of contractions to 100 stimuli at 5 Hz were assessed. The 10–90% rise time of the contractions did not differ between unoperated, sham-operated, and spinalized arteries: 12.9 s (IQR 10.6–14.2), 13.9 s (IQR 13.2–16.3), and 13.0 s (IQR 10.5–14.2), respectively (P = 0.20 by Kruskal-Wallis test). There was no significant difference between the 50% decay times for unoperated [2.6 s (IQR 2.2–3.0)] and spinalized [3.0 s (IQR 2.5–3.7)] arteries (P = 0.15 by Mann-Whitney U-test). However, the 50% decay time for sham-operated arteries [2.3 s (IQR 2.0–2.6)] was reduced compared with that for spinalized arteries (Mann-Whitney U-test, P < 0.01).

Effects of {alpha}1-adrenoceptor and P2-purinoceptor blockade on neurally evoked contractions. The effects of the {alpha}1-adrenoceptor antagonist prazosin (10 nM) and the P2-purinoceptor antagonist suramin (0.1 mM) were investigated on contractions evoked by 20 stimuli at 10 Hz. The increase in pressure produced by this train of stimuli did not differ between unoperated [7.2 x 103 N/m2 (IQR 5.3–9.4), n = 14] and sham-operated [5.6 x 103 N/m2 (IQR 4.0–6.6), n = 13, P = 0.13 by Mann-Whitney U-test] arteries, but the response of the spinalized arteries was increased compared with both of these groups of arteries [14.5 x 103 N/m2 (IQR 12.6–17.0), n = 12, P < 0.001 for both comparisons by Mann-Whitney U-test].

Figure 2, A and B, shows the reduction in pressure of the neurally evoked responses produced by suramin and prazosin, respectively. Despite the marked difference in the peak amplitude of the neurally evoked responses, the net reduction in the pressure produced by suramin was similar for unoperated and spinalized arteries (Fig. 2A). The reduction in pressure produced by suramin was smaller for sham-operated than for spinalized arteries but did not differ from that for unoperated arteries (Fig. 2A). In contrast, the decrease in pressure produced by prazosin was much greater in spinalized than in unoperated and sham-operated arteries (Fig. 2B). These findings indicate that the increase in the neurally evoked response of spinalized arteries is due primarily to an increase in the prazosin-sensitive (noradrenergic) component of contraction.



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  		Fig. 2. Effects of 0.1 mM suramin and 10 nM prazosin on contractions of arteries from unoperated (Un-op, n = 13), sham-operated (n = 13), and spinalized (n = 12) animals evoked by 20 stimuli at 10 Hz. A and B: reduction in pressure of nerve-evoked responses produced by suramin and prazosin. C and D: percent block of nerve-evoked responses produced by suramin and prazosin. Values are median and interquartile range. Statistical differences (Mann-Whitney U-tests) between groups of arteries: *P < 0.05; **P < 0.01.

 
Figure 2, C and D, shows the percent block of the neurally evoked contractions produced by suramin and prazosin, respectively. The percent block produced by suramin varied greatly between the groups of arteries, with the contraction of sham-operated arteries being most affected by this agent and that of spinalized arteries being least affected (Fig. 2C). In contrast, the percent block produced by prazosin was similar (80–85%) for all groups of arteries (Fig. 2D), although there was a small but significant difference between unoperated and sham-operated arteries. In all groups of arteries, the combined application of suramin and prazosin reduced the peak force of contraction by ~95%: 96% (IQR 96–98) for unoperated, 95% (IQR 86–96) for sham-operated, and 97% (IQR 95–99) for spinalized arteries.

Responses to PE. Figure 3A shows the concentration-response curves for PE in unoperated, sham-operated, and spinalized arteries. Compared with unoperated and sham-operated arteries, spinalized arteries showed a leftward shift in their concentration-response curve for PE (Fig. 3A; P < 0.01 for both comparisons by Tukey-Kramer tests). In addition, compared with unoperated arteries, the PE concentration-response curve for sham-operated arteries was significantly shifted to the right (Fig. 3A; P < 0.01 by Tukey-Kramer test). The EC50 for PE also differed between the three groups of arteries, but there was no difference between the slopes of the Hill equation fits (Table 1). There was no difference in the maximal increase in pressure produced by PE between unoperated, sham-operated, and spinalized arteries (Table 1).



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  		Fig. 3. Increased sensitivity of arteries to phenylephrine, but not methoxamine, after spinal transection. A: concentration-response curves for phenylephrine in arteries from unoperated ({bullet}, n = 19), sham-operated ({blacktriangleup}, n = 13), and spinalized ({blacksquare}, n = 19) animals. B: concentration-response curves for methoxamine in arteries from unoperated ({bullet}, n = 12), sham-operated ({blacktriangleup}, n = 6), and spinalized ({blacksquare}, n = 12) animals. Curves are best fits to Hill equation. Values are means (SD).

 

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  		Table 1. Properties of concentration-response relations for PE and methoxamine

 
A significant negative correlation was found between the response of arteries to neural activation (100 stimuli at 5 Hz) and their EC50 for PE (r = –0.82, P < 0.001).

Changes in effects of the neuronal NE transporter. The leftward shift in the concentration-response curve to PE could result from a postjunctional increase in reactivity of the vascular smooth muscle to {alpha}1-adrenoceptor activation and/or a reduction in its uptake by the nerve terminals. To determine the role of PE uptake into nerve terminals, the effects of blocking the neuronal NE transporter on the reactivity to PE were evaluated in pairs of arteries from the same animal, one being treated with DMI. In a second series of experiments, concentration-response curves to the {alpha}1-adrenoceptor agonist methoxamine, which is not a substrate for the neuronal NE transporter (31), were constructed.

The maximum increase in pressure produced by PE did not differ between unoperated and spinalized arteries in the absence or presence of 30 nM DMI (Table 2). In unoperated and spinalized arteries, DMI decreased the EC50 for PE without changing the slopes of the Hill equation fits (Table 2). Importantly, although in the absence of DMI the EC50 values for PE differed significantly between unoperated and spinalized arteries, in the presence of this agent the EC50 values were similar (Table 2).


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  		Table 2. Properties of concentration-response relations for PE with and without DMI

 
The concentration-response curves for methoxamine did not differ between unoperated, sham-operated, and spinalized arteries (Fig. 3B; P = 0.59 by ANOVA). There was also no difference between the EC50 values, the slope of the Hill equation fits, or the maximum increase in pressure produced by methoxamine for these groups of arteries (Table 1).

Responses to {alpha},{beta}-mATP. Application of 3 µM {alpha},{beta}-mATP produced a contraction that peaked shortly after its addition and then waned. The peak increase in pressure produced by {alpha},{beta}-mATP did not differ significantly between unoperated [23.8 x 103 N/m2 (SD 3.2 x 103), n = 8] and spinalized [25.4 x 103 N/m2 (SD 2.8 x 103), n = 7] arteries, but the response of the sham-operated arteries [12.2 x 103 N/m2 (SD 5.9 x 103), n = 7] was ~50% smaller (P < 0.01 by Tukey-Kramer test for comparisons with unoperated and spinalized arteries). For unoperated (n = 5) and spinalized (n = 5) arteries, there was no difference between the concentration-response curves (P = 0.37 by ANOVA) or the EC50 values [0.5 µM (SD 0.2) for unoperated and 0.5 µM (SD 0.1) for spinalized, P = 0.79 by unpaired t-test] for {alpha},{beta}-mATP.

Responses to high [K+]o. The peak increase in pressure produced in 60 mM extracellular K+ did not differ between unoperated [32.0 x 103 N/m2 (SD 4.0 x 103) n = 8], sham-operated [32.0 x 103 N/m2 (SD 4.8 x 103), n = 7], and spinalized [33.1 x 103 N/m2 (SD 2.3 x 103), n = 7] arteries (P = 0.84 by ANOVA). On return to normal bathing solution, the 50% decay of the K+-evoked contraction was slower in spinalized [9.3 s (IQR 8.6–12.4)] than in unoperated [7.4 s (IQR 7.1–7.8), P < 0.05 by Mann-Whitney U-test] and sham-operated [6.8 s (IQR 6.0–7.8) P < 0.05 by Mann-Whitney U-test] arteries.

In a separate series of experiments, there was no difference in the K+ concentration-response curves between sham-operated and spinalized arteries (Fig. 4; P = 0.21 by ANOVA).



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  		Fig. 4. Absence of change in reactivity to raised K+ concentration ([K+]) after spinal transection, shown as concentration-response curves for K+ in arteries from sham-operated and spinalized animals. Values are means (SD); n = 6.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Spinal transection at T4 in the rat produced a marked increase in the contractile response of second-order mesenteric arteries to electrical stimulation of the perivascular sympathetic nerves. This change was attributable to increased {alpha}1-adrenoceptor activation and was correlated with an increased sensitivity to the {alpha}1-adrenoceptor agonist PE. The latter appeared to be due to a decrease in PE removal by the neuronal NE transporter. The responses of spinalized arteries to the P2X-purinoceptor agonist {alpha},{beta}-mATP and to raised [K+]o were unchanged compared with unoperated arteries. There was also no change in the neurally evoked increase in pressure that was due to released ATP. Together, these findings suggest that the noradrenergic component of the nerve-evoked response in the mesenteric artery is selectively increased after spinal cord transection.

NE and ATP are known to play a role in nerve-evoked contractions of rat mesenteric arteries. In the present study, prazosin reduced the contractions of the unoperated and sham-operated arteries evoked by 20 stimuli at 10 Hz by ~80%, whereas suramin reduced these contractions by 40–70%. Together, prazosin and suramin virtually abolished the responses to nerve stimulation. The nonadditive nature of the blockade produced by prazosin and suramin suggests that NE and ATP released from the sympathetic nerves act synergistically to produce activation of the vascular smooth muscle. This suggestion is consistent with the demonstration, in rat mesenteric arteries, that low concentrations of {alpha},{beta}-mATP produce a leftward shift in the concentration-response curve for NE without increasing the maximum response to this agent (26). It appears that the synergism between neurally released NE and ATP in control mesenteric arteries has not previously been recognized. However, ATP and NE have been proposed to act synergistically in neurally evoked contractions of the rat tail artery (6). Importantly, although the relative contribution of ATP to nerve-evoked contractions was reduced in spinalized arteries, the net increase in pressure attributable to purinoceptor activation was similar to that of unoperated arteries (Fig. 2A). This finding indicates that there was no change in the contribution of purinoceptors to the neurally evoked response of spinalized arteries.

In unoperated arteries, the peak force of contractions to 20 stimuli at 10 Hz (mainly due to NE) was only ~20% of the maximum increase in force produced by PE. Therefore, the selective increase in the noradrenergic component of neurally evoked contraction in spinalized arteries might be simply explained by the leftward shift in the agonist concentration-response curve for PE, which is a substrate for the neuronal NE transporter (31). This interpretation supposes that neurally released NE acts in the same manner as exogenously applied PE. In second-order mesenteric arteries, the majority of sympathetic varicosities do not make close neuroeffector junctions (20). If some of the neurally released NE originates from noncontacting varicosities, then NE diffusing from the sites of transmitter release might act similarly to exogenously applied NE. In support of this idea, the increase in NE concentration detected at the adventitial surface of second-order mesenteric arteries in response to trains of 10 stimuli at 10 Hz peaked at >0.1 µM (9) and the EC50 for bath-applied NE at {alpha}1-adrenoceptors is ~0.8 µM (8). This indicates that the concentration of endogenous NE in the adventitia is adequate to activate the same receptors that are activated by exogenous NE.

It is possible that NE release is enhanced after spinalization, in accord with disuse, increasing neurotransmitter release at other synapses (25). There is no direct evidence for this possibility, but an increase in NE release is likely to contribute to the augmented responses of tail arteries from spinalized animals (35). If it is assumed that NE and ATP are coreleased (7), the fact that there was no change in the neurally evoked increase in pressure attributable to ATP in mesenteric arteries argues against an increase in transmitter release in this vessel.

The above-mentioned arguments do not address the mechanisms that underlie the increased sensitivity of the spinalized arteries to PE. In general, supersensitivity after decentralization of sympathetic postganglionic neurons is nonspecific, with tissues displaying increased sensitivity to a wide range of agents (11). Because of this lack of specificity, postjunctional hyperreactivity is believed to result from an alteration in smooth muscle function, rather than from a change in receptor expression. However, because the sensitivity of the spinalized arteries to {alpha},{beta}-mATP and high [K+]o was unchanged compared with unoperated arteries, their increased sensitivity to PE was not due to a generalized increase in the reactivity of the smooth muscle. Two findings indicate that increased reactivity to PE is accounted for by a reduction in its removal by the neuronal NE uptake transporter: 1) Blockade of neuronal uptake with DMI produced a leftward shift in the concentration-response curves for PE in unoperated and spinalized arteries and abolished the supersensitivity to PE. 2) The sensitivity of spinalized arteries to the {alpha}1-adrenoceptor agonist methoxamine was the same as in controls; methoxamine, in contrast to PE, is not a substrate for the neuronal NE uptake transporter (31). This suggests that the efficacy of uptake by the transporter is modified by the levels of ongoing nerve activity.

Sympathetic neuroeffector transmission in the rat tail artery was augmented after spinalization by quite different mechanisms. Nerve-evoked contractions of the tail artery involve the synergistic interactions of {alpha}1- and {alpha}2-adrenoceptor activation (5, 35). After spinalization, the increased responses to nerve stimulation were correlated with an increased sensitivity to the {alpha}2-adrenoceptor agonist clonidine and raised [K+]o. This suggests that the {alpha}2-adrenoceptor-mediated component of contraction and the overall reactivity of the tail artery were enhanced (35). Tail arteries displayed an increased reactivity to PE at 2 wk, but not at 8 wk, after spinal transection, when neurally evoked contractions were still markedly potentiated. For all measured parameters, there were no differences between tail arteries from unoperated and sham-operated animals. Thus the mechanisms underlying the augmentation of the neurally evoked contractions after spinal transection differ markedly between the cutaneous and splanchnic vascular beds. In the cutaneous artery the changes are mostly postjunctional, whereas in the mesenteric artery the changes are prejunctional.

Surprisingly, compared with the unoperated mesenteric arteries, responses to nerve stimulation were reduced in the sham-operated arteries. This change was associated with an increased EC50 for PE. The decreased reactivity of sham-operated arteries to PE, relative to unoperated arteries, was most probably due to increased neuronal uptake of this substance, because the sensitivities of these arteries to methoxamine were similar. It should be noted that the 50% decay times of the neurally evoked contractions of sham-operated arteries were faster than those of spinalized arteries, which may reflect the more rapid removal of released NE. The sham-operated arteries also were less reactive to {alpha},{beta}-mATP but not to raised [K+]o. These findings suggest that neurally evoked responses of the sham-operated arteries are selectively reduced. Indeed, the changes in responses to nerve stimulation and PE in sham-operated arteries were the opposite of those observed for the spinalized arteries. This might indicate that the sympathetic outflow to the mesenteric arteries increased after the sham operation. Increased sympathetic nerve activity and/or catecholamine release from the adrenal medulla might be produced by operative stress, but this is unlikely to have lasted for 7 wk. In all operated animals, reflex changes in nerve activity could result from inflammation of dorsal root ganglia near the lesion (23), but again this is unlikely to persist. Regardless of the reason for the changes in the arteries of sham-operated animals, the fact that their responsiveness was reduced relative to unoperated animals indicates that the effects of spinal transection are even more marked than indicated by the differences from unoperated controls.

We have assumed that the changes observed in the spinalized mesenteric arteries, as for the tail arteries, result from silencing of ongoing sympathetic nerve activity after spinal transection (35). If this is the case, the changes should apply equally well to arterial vessels in patients with lesions at any level that disrupt the descending drive to the vasoconstrictor outflow. Sublingual nitrovasodilators or Ca2+ antagonists and removal of the stimulus are the most commonly used acute treatments for autonomic dysreflexia. However, the foot veins of quadriplegics have raised sensitivity to local infusions of NE that did not produce a significant pressor response (4). Pressor responses to intravenously infused NE (22) and PE (17) are enhanced in patients with cervical spinal lesions; however, there is no evidence of a reduced rate of catecholamine clearance that could explain these changes in vascular reactivity (13, 16, 22). In men with spinal injury, prophylactic treatment with {alpha}-adrenoceptor antagonists (phenoxybenamine and the selective {alpha}1-adrenoceptor antagonists prazosin and terazosin) has proved effective in improving bladder function and reducing autonomic dysreflexia (7, 18). However, prophylactic treatment with terazosin was also effective in treating recurrent episodes of autonomic dysreflexia where the precipitating cause could not be identified as bladder related (32), supporting a reduction in vascular {alpha}-adrenoceptor-mediated responsiveness as the primary mechanism of action of this agent.

In conclusion, the findings of the present study provide further evidence that increased reactivity of the vasculature to sympathetic nerve activation is likely to contribute prominently to the etiology of autonomic dysreflexia. Therefore, interventions that inhibit or prevent the development of vascular hyperactivity after spinal injury have considerable therapeutic potential. Our data indicate that decreases in sympathetic activity modify vascular reactivity by distinct mechanisms in different arterial beds and that this might be exploited to alleviate different symptoms. Whether hyperreactivity contributes to modified vascular perfusion under resting conditions in spinally injured humans is not clear. Furthermore, the findings suggest that the ongoing level of sympathetic activity in intact humans may modulate the effectiveness of neurovascular transmission.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by Christopher Reeve Paralysis Foundation Contracts BAC1–0101-1 and BAC1–0101-2 and National Health and Medical Research Council of Australia Project Grant 209632. J. A. Brock is a National Health and Medical Research Council of Australia Senior Research Fellow.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Brock, Prince of Wales Medical Research Institute, Barker St., Randwick, NSW 2031, Australia (e-mail: j.brock{at}unsw.edu.au)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Ahluwalia A and Vallance P. Interaction between sympathetic and sensory nerves in rat small arteries: involvement of nitric oxide. Am J Physiol Heart Circ Physiol 271: H969–H976, 1996.[Abstract/Free Full Text]
  2. Anderson CR, McLachlan EM, and Srb-Christie O. Distribution of sympathetic preganglionic neurons and monoaminergic nerve terminals in the spinal cord of the rat. J Comp Neurol 283: 269–284, 1989.[CrossRef][ISI][Medline]
  3. Angus JA, Broughton A, and Mulvany MJ. Role of {alpha}-adrenoceptors in constrictor responses of rat, guinea-pig and rabbit small arteries to neural activation. J Physiol 403: 495–510, 1988.[Abstract/Free Full Text]
  4. Arnold JM, Feng QP, Delaney GA, and Teasell RW. Autonomic dysreflexia in tetraplegic patients: evidence for {alpha}-adrenoceptor hyper-responsiveness. Clin Auton Res 5: 267–270, 1995.[CrossRef][ISI][Medline]
  5. Bao JX, Gonon F, and Stjärne L. Frequency- and train length-dependent variation in the roles of postjunctional {alpha}1- and {alpha}2-adrenoceptors for the field stimulation-induced neurogenic contraction of rat tail artery. Naunyn Schmiedebergs Arch Pharmacol 347: 601–616, 1993.[CrossRef][ISI][Medline]
  6. Bradley E, Law A, Bell D, and Johnson CD. Effects of varying impulse number on cotransmitter contributions to sympathetic vasoconstriction in rat tail artery. Am J Physiol Heart Circ Physiol 284: H2007–H2014, 2003.[Abstract/Free Full Text]
  7. Chancellor MB, Erhard MJ, Hirsch IH, and Stass WE. Prospective evaluation of terazosin for the treatment of autonomic dysreflexia. J Urol 151: 111–113, 1994.[ISI][Medline]
  8. Chen H, Fetscher C, Schafers RF, Wambach G, Philipp T, and Michel MC. Effects of noradrenaline and neuropeptide Y on rat mesenteric microvessel contraction. Naunyn Schmiedebergs Arch Pharmacol 353: 314–323, 1996.[CrossRef][ISI][Medline]
  9. Dunn WR, Brock JA, and Hardy TA. Electrochemical and electrophysiological characterization of neurotransmitter release from sympathetic nerves supplying rat mesenteric arteries. Br J Pharmacol 128: 174–180, 1999.[CrossRef][ISI][Medline]
  10. Dunn WR, Hardy TA, and Brock JA. Electrophysiological effects of activating the peptidergic primary afferent innervation of rat mesenteric arteries. Br J Pharmacol 140: 231–238, 2003.[CrossRef][ISI][Medline]
  11. Fleming WW and Westfall DP. Adaptive supersensitvity. In Handbook of Experimental Pharmacology: Catecholamines, edited by Trendelenburg U. Berlin: Springer-Verlag, 1988, vol. 90/1.
  12. Gitterman DP and Evans RJ. Nerve evoked P2X receptor contractions of rat mesenteric arteries; dependence on vessel size and lack of role of L-type calcium channels and calcium-induced calcium release. Br J Pharmacol 132: 1201–1208, 2001.[CrossRef][ISI][Medline]
  13. Karlsson AK, Friberg P, Lonnroth P, Sullivan L, and Elam M. Regional sympathetic function in high spinal cord injury during mental stress and autonomic dysreflexia. Brain 121: 1711–1719, 1998.[Abstract/Free Full Text]
  14. Kawasaki H, Nuki C, Saito A, and Takasaki K. Role of calcitonin gene-related peptide-containing nerves in the vascular adrenergic neurotransmission. J Pharmacol Exp Ther 252: 403–409, 1990.[Abstract/Free Full Text]
  15. Krassioukov AV and Weaver LC. Episodic hypertension due to autonomic dysreflexia in acute and chronic spinal cord-injured rats. Am J Physiol Heart Circ Physiol 268: H2077–H2083, 1995.[Abstract/Free Full Text]
  16. Krum H, Brown DJ, Rowe PR, Louis WJ, and Howes LG. Steady-state plasma [3H]-noradrenaline kinetics in quadriplegic chronic spinal cord injury patients. J Auton Pharmacol 10: 221–226, 1990.[ISI][Medline]
  17. Krum H, Louis WJ, Brown DJ, and Howes LG. Pressor dose responses and baroreflex sensitivity in quadriplegic spinal cord injury patients. J Hypertens 10: 245–250, 1992.[CrossRef][ISI][Medline]
  18. Krum H, Louis WJ, Brown DJ, and Howes LG. A study of the {alpha}1 adrenoceptor blocker prazosin in the prophylactic management of autonomic dysreflexia in high spinal cord injury patients. Clin Auton Res 2: 83–88, 1992.[CrossRef][Medline]
  19. Luff SE, Young SB, and McLachlan EM. Ultrastructure of substance P-immunoreactive terminals and their relation to vascular smooth muscle cells of rat small mesenteric arteries. J Comp Neurol 416: 277–290, 2000.[CrossRef][ISI][Medline]
  20. Luff SE, Young SB, and McLachlan EM. Hyperinnervation of mesenteric arteries in SHR rats by sympathetic but not primary afferent axons: an ultrastructural analysis. J Vasc Res 42: 348–358, 2005.[CrossRef][ISI][Medline]
  21. Mathias CJ and Frankel H. Autonomic disturbances in spinal cord lesions. In: Autonomic Failure, edited by Mathias C and Banister R. Oxford, UK: Oxford University Press, 1999.
  22. Mathias CJ, Frankel HL, Christensen NJ, and Spalding JM. Enhanced pressor response to noradrenaline in patients with cervical spinal cord transection. Brain 99: 757–770, 1976.[Free Full Text]
  23. McKay SM and McLachlan EM. Inflammation of rat dorsal root ganglia below a mid-thoracic spinal transection. Neuroreport 15: 1783–1786, 2004.[CrossRef][ISI][Medline]
  24. Mulvany MJ and Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ Res 41: 19–26, 1977.[Free Full Text]
  25. Murthy VN, Schikorski T, Stevens CF, and Zhu Y. Inactivity produces increases in neurotransmitter release and synapse size. Neuron 32: 673–682, 2001.[CrossRef][ISI][Medline]
  26. Ralevic V and Burnstock G. Postjunctional synergism of noradrenaline and adenosine 5'-triphosphate in the mesenteric arterial bed of the rat. Eur J Pharmacol 175: 291–299, 1990.[CrossRef][ISI][Medline]
  27. Stephens N, Bund SJ, Jagger C, and Heagerty AM. Arterial neuroeffector responses in early and mature spontaneously hypertensive rats. Hypertension 18: 674–682, 1991.[Abstract/Free Full Text]
  28. Stjernberg L, Blumberg H, and Wallin BG. Sympathetic activity in man after spinal cord injury. Outflow to muscle below the lesion. Brain 109: 695–715, 1986.[Abstract/Free Full Text]
  29. Taylor RB and Weaver LC. Spinal stimulation to locate preganglionic neurons controlling the kidney, spleen, or intestine. Am J Physiol Heart Circ Physiol 263: H1026–H1033, 1992.[Abstract/Free Full Text]
  30. Teasell RW, Arnold JM, Krassioukov A, and Delaney GA. Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord injury. Arch Phys Med Rehabil 81: 506–516, 2000.[CrossRef][ISI][Medline]
  31. Trendelenburg U, Maxwell RA, and Pluchino S. Methoxamine as a tool to assess the importance of intraneuronal uptake of l-norepinephrine in the cat's nictitating membrane. J Pharmacol Exp Ther 172: 91–99, 1970.[Abstract/Free Full Text]
  32. Vaidyanathan S, Soni BM, Sett P, Watt JW, Oo T, and Bingley J. Pathophysiology of autonomic dysreflexia: long-term treatment with terazosin in adult and paediatric spinal cord injury patients manifesting recurrent dysreflexic episodes. Spinal Cord 36: 761–770, 1998.[CrossRef][ISI][Medline]
  33. Wallin BG and Stjernberg L. Sympathetic activity in man after spinal cord injury. Outflow to skin below the lesion. Brain 107: 183–198, 1984.[Abstract/Free Full Text]
  34. Weaver LC, Marsh DR, Gris D, Meakin SO, and Dekaban GA. Central mechanisms for autonomic dysreflexia after spinal cord injury. Prog Brain Res 137: 83–95, 2002.[ISI][Medline]
  35. Yeoh M, McLachlan EM, and Brock JA. Tail arteries from chronically spinalized rats have potentiated responses to nerve stimulation in vitro. J Physiol 556: 545–555, 2004.[Abstract/Free Full Text]
  36. Yeoh M, McLachlan EM, and Brock JA. Chronic decentralization potentiates neurovascular transmission in the isolated rat tail artery, mimicking the effects of spinal transection. J Physiol 561: 583–596, 2004.[Abstract/Free Full Text]



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