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Hypertension. 1996;27:584-590

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(Hypertension. 1996;27:584-590.)
© 1996 American Heart Association, Inc.

Articles

Platelet Activation in Carotid Sinuses Triggers Reflex Sympathoinhibition and Hypotension

Hui Z. Mao; Zhi Li; Mark W. Chapleau

From the Department of Internal Medicine, University of Iowa College of Medicine, and the Department of Veterans Affairs Medical Center, Iowa City, Iowa.

Correspondence to Mark W. Chapleau, PhD, Assistant Professor, Department of Internal Medicine, E327 General Hospital, University of Iowa College of Medicine, 200 Hawkins Dr, Iowa City, IA 52242.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
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Abstract The carotid sinuses, one of the major sites of baroreceptor innervation, are also a common site of atherosclerotic lesions and platelet aggregation. The goal of the present study was to determine whether platelet activation in carotid sinuses causes reflex-mediated changes in renal sympathetic nerve activity and arterial pressure. Rabbit platelets were isolated, resuspended in Krebs' buffer, and activated by thrombin. Injection of activated platelets (3x108 platelets/mL) into the vascularly isolated carotid sinuses of anesthetized rabbits essentially eliminated sympathetic nerve activity and acutely decreased mean arterial pressure from 126±5 to 53±4 mm Hg (n=16; P<.05). Sympathetic activity and arterial pressure returned to control levels over a period of minutes despite sustained exposure to activated platelets. Injection of U-46619, a thromboxane analogue and vasoconstrictor, into carotid sinuses did not alter sympathetic activity or arterial pressure. However, serotonin (5-hydroxytryptamine [5-HT]), which is known to be released from activated platelets, and the 5-HT3 receptor agonist phenylbiguanide mimicked the effect of platelets. Furthermore, the platelet-induced reflex inhibition of sympathetic activity and hypotension were not altered by the cyclooxygenase inhibitor indomethacin but were attenuated significantly by 5-HT receptor antagonists. Platelet activation inhibited sympathetic activity to 5±2% of control in the absence of antagonists but to only 35±11 and 76±4% of control after selective blockade of 5-HT2 and 5-HT3 receptors with ketanserin and MDL-72222, respectively. The results indicate that (1) platelet activation in carotid sinuses triggers reflex inhibition of sympathetic nerve activity and hypotension; (2) the reflex is not caused by carotid vasoconstriction and is not mediated by prostanoids; and (3) the reflex is mediated by 5-HT acting primarily on 5-HT3 and to a lesser extent on 5-HT2 receptors. We speculate that this reflex may contribute to arterial pressure lability and susceptibility to stroke in patients with carotid atherosclerotic disease.


Key Words: blood pressure • sympathetic nervous system • atherosclerosis • pressoreceptors • stroke • serotonin • carotid sinus


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
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The carotid sinuses are densely innervated by sensory nerves, including baroreceptor fibers, which have a major role in the regulation of arterial pressure and cardiovascular function.1 The carotid sinuses are also a common site of development of atherosclerotic lesions and platelet adhesion.2 3 4 5 Activation of platelets in carotid sinuses is a major cause of transient ischemic attacks and strokes.6 7 8 9 Fluctuation in arterial pressure would further increase the risk of cerebral ischemia and stroke.

We hypothesized that factors released from activated platelets alter the activity of carotid sinus sensory nerves and trigger significant reflex changes in SNA and arterial pressure. We previously demonstrated that factors released from aggregating platelets in the carotid sinus decrease the activity of type I baroreceptors with myelinated afferent fibers and increase the activity of type II baroreceptors.10 11 12 The major goal of the present study was to determine the effects of activation of rabbit platelets in isolated carotid sinuses on renal SNA, heart rate, and arterial pressure. In addition, we explored the possible roles of carotid vasoconstriction and of prostanoids and 5-HT released during platelet activation in triggering the reflex response.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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Thirty New Zealand White rabbits of either sex were anesthetized with sodium pentobarbital (30 to 35 mg/kg) administered slowly through an ear vein. The rabbits were ventilated with room air supplemented with oxygen through a tracheotomy. The arterial blood gases and pH were kept within normal ranges by adjustment of the ventilation and administration of NaHCO3 when needed. Catheters were placed in the femoral artery and vein to enable measurement of arterial pressure and administration of anesthetic, respectively. Body temperature was maintained between 36°C and 38°C by external warming. Supplemental doses of anesthetic were administered as needed. All procedures were in accordance with institutional guidelines.

Bilateral Isolation of Carotid Sinuses
Both carotid sinuses were vascularly isolated, as described previously.10 11 12 13 14 Catheters were placed in the common carotid and lingual arteries. The carotid sinuses were filled with Krebs-Henseleit buffer of the following composition (in mmol/L): NaCl 118.0, KCl 4.7, NaHCO3 24.0, MgSO4 1.2, CaCl2 1.1, KH2PO4 1.1, and glucose 10.0. Krebs' solution was bubbled before the experiment with a 95% O2/5% CO2 gas mixture, resulting in pH 7.3 to 7.4, PO2 of >200 mm Hg, and PCO2 of 30 to 40 mm Hg, and was kept in a sealed glass container placed in a temperature-controlled water bath (37°C). The carotid sinuses were periodically refilled with fresh Krebs' buffer.

The common carotid catheters were connected to a pressure bottle partially filled with Krebs' buffer. Pressure in the carotid sinuses was controlled by altering the inflow of air into the bottle from a pressurized air source and was measured with a transducer (model P23ID; Statham) connected to one of the lingual artery catheters. Vascular isolation of the carotid sinuses was confirmed by the ability to maintain a constant carotid sinus pressure over time after pressurization via the pressure bottle and occlusion of the common carotid catheter and by the absence of blood leaking into the carotid sinuses at a pressure of 0 mm Hg. The cervical sympathetic and aortic depressor nerves were sectioned to eliminate sympathetic influences on baroreceptor sensitivity and buffering of reflex responses by the aortic baroreceptors. In the majority of experiments, the vagus nerves were also sectioned. Decamethonium bromide (0.3 mg/kg IV) was administered before beginning the protocol to eliminate skeletal muscle contraction and associated interference with nerve recordings.

Measurement of Renal SNA
The left kidney was exposed through a flank incision, and a renal nerve was isolated and carefully placed across a platinum electrode. The nerve and electrode were encased in silicone gel. Nerve activity was recorded with a high-impedance probe (30- to 100-Hz to 3-kHz band width, model HIP511J; Grass Instrument Co). The filtered neurogram was displayed on a Tektronix dual-beam storage oscilloscope (model 5113), and we listened to the neural activity through a loudspeaker. Renal SNA was quantified by counting the frequency of action potentials that exceeded a selected voltage level set just above the electrical noise with a nerve traffic analyzer (model 706C; Department of Bioengineering, University of Iowa [Iowa City]). In a few experiments, SNA was measured by both spike counting and integrating the voltage of the neurogram with an integrator amplifier (model 13-4615-70; Gould Inc). There were no differences in the sympathetic nerve responses as measured by the two methods. Measurements of carotid sinus, pulsatile, and mean systemic arterial pressures and mean and integrated renal SNAs were recorded continuously with a pen recorder (model R611, Beckman Co, or model 11-1202-25, Gould Inc) and were analyzed manually from the chart recordings. In some experiments, heart rate was measured with a cardiotachometer triggered by the arterial pressure pulse.

Isolation of Platelets
Blood was withdrawn from anesthetized rabbits and platelets were isolated as described previously.10 11 15 16 In brief, the blood was anticoagulated with acid citrate dextrose, and platelets were isolated by multiple centrifugation and washing. The platelets were initially prepared in modified Tyrode's solution and then either resuspended or diluted into oxygenated Krebs' buffer, creating a final concentration of 3x108 platelets/mL. Platelets were maintained at 37°C in a water bath before use and often were stored at 4°C for use in experiments on subsequent days. The reactivity of platelets was confirmed by measurement of thrombin (0.1 U/mL)-induced aggregation in a dual-chamber aggregometer on the day of each experiment.

Experimental Protocols
Reflex responses to activated platelets and various pharmacological agents were measured while maintaining carotid sinus pressure constant at {approx}80 mm Hg. Bovine thrombin (0.4 U/mL) was added to the platelet suspension (3x108 platelets/mL) in a syringe to activate platelets. The suspension was then rapidly injected into the isolated carotid sinuses, with care taken to restore carotid sinus pressure to the same level as before the injection. The injection of platelets was completed within {approx}20 seconds. The response to activated platelets was measured at least until the maximum response began to wane (n=16), and in some experiments the platelet suspension was left in the carotid sinuses for 15 to 20 minutes (n=8). At this time, the suspension was washed out of the sinuses, and the carotid sinuses were refilled with fresh Krebs' buffer in an attempt to reverse the effects of platelets. In some experiments, arterial pressure and renal SNA were measured before and after the injection of thrombin alone (0.4 U/mL) into the carotid sinuses (n=7).

We previously demonstrated that injection of rabbit platelets into the isolated carotid sinus causes significant vasoconstriction.11 To determine whether vasoconstriction of carotid sinuses triggers reflex changes in arterial pressure and SNA, we measured responses to injection of the thromboxane analogue U-46619 (100 nmol/L) into the isolated carotid sinuses (n=6). We previously showed that this concentration of U-46619 causes a magnitude of carotid vasoconstriction similar to rabbit platelets.11

To further explore the mechanism of the platelet-induced reflex, several additional protocols were performed. In one group, the carotid sinuses and platelets were pretreated with indomethacin (40 µmol/L) for 5 to 15 minutes before injection of platelets into carotid sinuses to test for a possible role of prostanoids in mediating the reflex response (n=5). The reflex response to platelets was also measured before and after exposure of carotid sinuses and platelets to 5-HT receptor antagonists. Ketanserin (n=5) and MDL-72222 (n=8) (100 nmol/L) were used to selectively block 5-HT2 and 5-HT3 receptors, respectively.17 18 Neither indomethacin, ketanserin, nor MDL-72222 altered the magnitude of thrombin-induced platelet aggregation measured in the aggregometer. The reflex responses to injections of 5-HT (10 µmol/L) and the selective 5-HT3 receptor agonist phenylbiguanide (10 µmol/L) into carotid sinuses were also determined before and after ketanserin or MDL-72222. All of these pharmacological agents were confined to the isolated carotid sinuses and therefore could influence SNA and arterial pressure only by altering the activity of carotid sinus sensory nerves.

Intact carotid sinus innervation and central neurotransmission were confirmed at the beginning of and periodically throughout each experiment through measurement of the reflex response to a ramp increase in carotid sinus pressure from 0 to 175 mm Hg in the absence of platelets or drugs in the isolated sinuses. Failure of baroreceptor stimulation to effectively inhibit SNA was cause to exclude an experiment from data analysis.

Drugs
Bovine thrombin, indomethacin, and 5-HT were obtained from Sigma Chemical Co. Ketanserin tartrate, MDL-72222, and 1-phenylbiguanide were purchased from Research Biochemicals International. U-46619 was obtained from Cayman Chemical Co.

Data Analysis
The absolute amount of nerve activity recorded from whole nerve preparations depends on the recording conditions and varies among preparations. Therefore, platelet- and drug-induced changes in renal SNA are expressed as a percentage of the baseline level measured just before the injection of activated platelets or a particular pharmacological agent into the isolated carotid sinuses.

Values of SNA and arterial pressure measured 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 15 to 20 minutes after the injection of activated platelets into the isolated carotid sinuses were compared with preinjection control values using one-factor ANOVA.19 When the ANOVA value was significant, differences in response at specific time points were determined with Fisher's protected least-significant differences test.19 The values of SNA, heart rate, and arterial pressure measured at the time of the maximum reflex response were compared with the corresponding baseline values by means of a paired t test.19 The magnitudes of the platelet- and drug-induced changes in SNA and arterial pressure before versus after blockade of 5-HT receptors were compared with the use of a paired t test. An unpaired t test was used to compare the level of SNA during platelet activation in the presence of the 5-HT2 receptor antagonist ketanserin with that of SNA during platelet activation in the presence of the 5-HT3 receptor antagonist MDL-72222. Data are presented as mean±SEM. Differences were considered significant at P<.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
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Reflex Response to Platelet Activation in Carotid Sinuses
Injection of thrombin-activated platelets into the carotid sinuses essentially eliminated SNA (5±2% of control) and decreased arterial pressure significantly from 126±5 to 53±4 mm Hg (n=16, Fig 1Down). Heart rate was also decreased significantly from 238±11 to 205±9 bpm (-33±4 bpm, n=10). The maximum inhibition of SNA usually occurred within 30 seconds and the maximum fall in pressure usually occurred within the first minute after injection of platelets (Figs 1Down and 2Down). SNA and arterial pressure returned toward control levels over a period of minutes despite sustained exposure of carotid sinuses to platelets, with recovery of SNA occurring more rapidly than that of arterial pressure (n=8, Figs 1Down and 2Down). Reflex responses to intracarotid injection of activated platelets were abolished after the sectioning of carotid sinus nerves (data not shown).



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  		Figure 1. Original recordings showing changes in renal SNA and arterial pressure in response to injection of thrombin-activated platelets into the isolated carotid sinuses. Carotid sinus pressure was maintained constant at 80 mm Hg throughout the periods shown. Platelet activation abolished SNA and decreased arterial pressure. Interrupted traces show gradual return of SNA and arterial pressure toward control measured 3, 10, and 20 minutes after injection of the platelets. Platelet suspension remained in the carotid sinuses throughout the 20-minute period.



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  		Figure 2. Time course of the reflex responses to injection of thrombin-activated platelets into the isolated carotid sinuses (n=8). Carotid sinus pressure was maintained constant at {approx}80 mm Hg. First data point represents baseline levels of renal SNA and arterial pressure measured just before injection of activated platelets. Data are expressed as mean±SEM. *P<.05, significant difference compared with baseline value (ANOVA and Fisher's test).

Injection of thrombin alone into the isolated carotid sinuses, in the absence of platelets, failed to trigger reflex responses. Arterial pressure averaged 113±10 and 117±10 mm Hg before and after 1 minute of exposure to thrombin, respectively (n=7). SNA averaged 102±3% of control during exposure to thrombin (n=7).

Effect of Carotid Vasoconstriction on SNA and Arterial Pressure
The purpose of this experimental group was to determine whether carotid vasoconstriction, which we have shown to occur after injection of rabbit platelets,11 mechanically triggers baroreceptor reflex–mediated inhibition of SNA. Injection of the thromboxane analogue U-46619, a powerful vasoconstrictor in this preparation,11 into the isolated carotid sinuses failed to influence SNA or arterial pressure. Arterial pressure averaged 113±6 and 116±7 mm Hg before and 1 minute after injection of U-46619 (n=6), respectively. SNA averaged 101±4% of control during exposure to U-46619 (n=6).

Role of Prostanoids in Platelet-Induced Reflex
A possible role of prostanoids in mediating the reflex response to platelet activation in carotid sinuses was examined by testing responses to platelets after inhibition of prostanoid formation with indomethacin. The maximum reflex changes in SNA and arterial pressure in response to platelets are shown in Fig 3Down for control experiments (n=16) and for experiments in which carotid sinuses and platelets were treated with indomethacin (n=5). Platelet activation in carotid sinuses triggered equivalent and reversible inhibition of SNA and decreases in arterial pressure in both groups (Fig 3Down). Baseline levels of SNA and arterial pressure were not significantly different in the control and indomethacin-treated groups (Fig 3Down).



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  		Figure 3. Effect of inhibiting prostanoid formation on response to platelets. Maximum changes in SNA and arterial pressure in response to injection of activated platelets into isolated carotid sinuses. Results are from experiments in which carotid sinuses and platelets were not treated with indomethacin (Activated Platelets, n=16) and experiments in which carotid sinuses and platelets were treated with indomethacin (40 µmol/L) (Plts after Indomethacin, n=5). C indicates control; Plts, thrombin-activated platelets; and R, recovery. Data are expressed as mean±SEM. *P<.05, significant difference compared with control baseline value (paired t test).

Role of 5-HT in Platelet-Induced Reflex
Selective blockade of either 5-HT2 or 5-HT3 receptors with ketanserin (n=5) or MDL-72222 (n=8), respectively, significantly attenuated the reflex response to platelet activation in carotid sinuses (Figs 4Down and 5Down). MDL-72222 was considerably more effective than ketanserin in inhibiting the platelet-induced reflex (Fig 5Down). SNA was inhibited by platelet activation to a significantly greater extent in the presence of ketanserin than in the presence of MDL-72222 (SNA, 35±11% and 76±4% of baseline, respectively). Although the reflex was markedly attenuated by MDL-72222, there was still significant reflex inhibition of SNA and a decrease in arterial pressure in response to platelets (Figs 4Down and 5Down). Neither ketanserin nor MDL-72222 significantly influenced baseline SNA or arterial pressure (Fig 5Down). The attenuation of the platelet-induced reflex by 5-HT receptor antagonists was not caused by desensitization of the response because the reflex was reproducible with repeated injections of activated platelets in the absence of antagonists (data not shown).



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  		Figure 4. Original recordings showing reflex responses to injection of activated platelets and serotonin into isolated carotid sinuses before (control) and after blockade of 5-HT3 receptors with MDL-72222 (100 nmol/L). Platelets and serotonin each inhibited SNA and decreased arterial pressure (AP), and these responses were attenuated after MDL-72222.



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  		Figure 5. Reflex responses to activated platelets before and after 5-HT receptor antagonists. Maximum changes in SNA and arterial pressure in response to injection of activated platelets into isolated sinuses. Results are shown before and after blockade of 5-HT2 receptors with ketanserin (100 nmol/L, n=5) and before and after blockade of 5-HT3 receptors with MDL-72222 (100 nmol/L, n=8). C indicates control; Plts, thrombin-activated platelets; and R, recovery. Data are expressed as mean±SEM. *Significant difference compared with control baseline value. {dagger}Magnitude of response to platelets was significantly less after compared with before 5-HT receptor antagonist (paired t test).

Injection of exogenous 5-HT into the isolated carotid sinus mimicked the effects of activated platelets on SNA and arterial pressure (Figs 4Up and 6Down). 5-HT–induced inhibition of SNA was significantly attenuated by ketanserin (n=5; Fig 6Down, left). Similar to that observed with platelets, MDL-72222 was more effective than ketanserin in inhibiting both inhibition of SNA and the hypotension caused by 5-HT (n=5; Fig 6Down, right). Injection of the 5-HT3 receptor agonist phenylbiguanide into the sinuses significantly inhibited SNA to 57±12% of control and decreased arterial pressure from 119±7 to 79±11 mm Hg (n=7).



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  		Figure 6. Reflex responses to 5-HT (10 µmol/L) before and after 5-HT receptor antagonists. Maximum changes in SNA and arterial pressure in response to injection of 5-HT into isolated carotid sinuses. Results are shown before and after blockade of 5-HT2 receptors with ketanserin (100 nmol/L, n=5) and before and after blockade of 5-HT3 receptors with MDL-72222 (100 nmol/L, n=5). C indicates control; R, recovery. Data are expressed as mean±SEM. *Significant difference compared with control baseline value. {dagger}Magnitude of response to 5-HT was significantly less after compared with before 5-HT receptor antagonist (paired t test).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
The major new findings of the present study are that (1) injection of thrombin-activated rabbit platelets, but not thrombin alone, into carotid sinuses triggers reflex-mediated inhibition of SNA and hypotension; (2) the thromboxane analogue U-46619 injected into the isolated carotid sinus does not influence SNA or arterial pressure, whereas 5-HT and the 5-HT3 receptor agonist phenylbiguanide mimic the effect of platelets; and (3) the platelet-induced reflex is not altered after inhibition of prostanoid formation with indomethacin but rather is attenuated significantly by either the 5-HT3 receptor antagonist MDL-72222 or the 5-HT2 receptor antagonist ketanserin. MDL-72222 is more effective than ketanserin in attenuating the reflex.

These results suggest that 5-HT released from activated platelets acts directly on carotid sinus sensory nerve endings, most likely baroreceptors, via both 5-HT3 and 5-HT2 receptors to trigger reflex inhibition of SNA and hypotension. We discuss the results of the present study in relation to previous studies that have investigated the effects of aggregating platelets and 5-HT on sensory nerves and the possible pathophysiological implications of our findings.

Effects of Activated Platelets on Sensory Nerves
We previously demonstrated that sustained exposure of the isolated carotid sinus of rabbits to thrombin-activated platelets for 10 to 20 minutes suppresses baroreceptor activity recorded from either the whole carotid sinus nerve or from single baroreceptor fibers classified as type I baroreceptors10 11 14 based on criteria described by Seagard et al.20 The suppression of baroreceptor activity was mediated by a stable, diffusible factor14 that was not thromboxane, 5-HT, or adenosine diphosphate.10 11 Suppression of baroreceptor activity would predict that platelet activation in carotid sinuses should increase instead of decrease SNA and arterial pressure. Therefore, we were surprised at first by the pronounced inhibition of SNA that occurred when activated platelets were injected into the carotid sinuses.

Preliminary results from recent experiments provide a possible explanation for these findings.12 In contrast to that observed with type I baroreceptors, platelet activation in the carotid sinus increased the activity of type II baroreceptors.12 Furthermore, the increase in activity was evident soon after injection of platelets before the delayed inhibition of activity of type I baroreceptors. The results suggest that type II baroreceptors may have a predominant role in mediating the rapid inhibition of SNA during platelet activation.

We noticed that the increased activity of type II baroreceptors persisted throughout the period of exposure to activated platelets.12 Therefore, the question remains why SNA and arterial pressure returned to control levels despite the sustained exposure of carotid sinuses to the platelets (see Figs 1Up and 2Up). We speculate that the recovery of SNA and arterial pressure may reflect the opposing influence of the delayed decrease in activity of type I baroreceptors or adaptation within the central nervous system to the increased type II baroreceptor activity.

It is also possible that activation of unmyelinated afferent C-fibers contributes to the platelet-induced reflex inhibition of SNA and hypotension. Activated human platelets have been shown to excite nociceptor C-fiber afferents innervating skin.21

The mechanisms responsible for causing reflex inhibition of SNA during localized activation of platelets in carotid sinuses may also be applicable during systemic thrombotic disorders associated with hypotension. Wiggins et al22 found that intravenous administration of dextran sulfate to rabbits triggered hypotension and bradycardia associated with release of 5-HT from degranulating platelets before but not after depletion of circulating platelets. The hypotension in response to either dextran or 5-HT was markedly attenuated by sectioning the vagus and aortic depressor nerves and was nearly abolished after additional carotid artery ligation, indicating the reflex nature of the response.22

Role of Serotonin in Mediating Reflex Sympathoinhibition
The conclusion that 5-HT is responsible for triggering the platelet-induced inhibition of SNA is based on the findings that the reflex was attenuated by 5-HT receptor antagonists and mimicked by 5-HT and phenylbiguanide. Furthermore, the facts that MDL-72222 did not totally block the reflex and that ketanserin attenuated the reflex suggest that both 5-HT2 and 5-HT3 receptors contribute to the response to platelets. These conclusions rely heavily on the effectiveness and selectivity of the antagonists.

Selectivity of Antagonists
Based on the literature, ketanserin and MDL-72222 at the concentration that we used (100 nmol/L) should effectively block 5-HT2 and 5-HT3 receptors, respectively, in a selective manner.17 18 The reflex inhibition of SNA and decrease in arterial pressure caused by the specific 5-HT3 receptor agonist phenylbiguanide were inhibited by >85% by MDL-72222 (n=2) but were not influenced by ketanserin (n=4). Phenylbiguanide decreased SNA to 59±21% and 59±19% of control before and after ketanserin, respectively. Phenylbiguanide decreased arterial pressure from 120±12 to 70±16 mm Hg (-50±24 mm Hg) before ketanserin and from 129±11 to 76±16 mm Hg (-53±21 mm Hg) after ketanserin (n=4). These data demonstrate the effectiveness of MDL-72222 in blocking 5-HT3 receptors and the selective blockade of 5-HT2 receptors by ketanserin in our preparation.

We also considered the possibility that the marked attenuation of the platelet-induced reflex by MDL-72222 may have resulted from nonspecific interference with impulse conduction in carotid sinus afferent nerves. To address this possibility, we examined whether MDL-72222 would also inhibit baroreflex-mediated inhibition of SNA. We found that a ramp increase in nonpulsatile carotid sinus pressure to 175 mm Hg inhibited SNA to a similar extent before and after MDL-72222; at 175 mm Hg of pressure, SNA averaged 10±6% and 5±4% of control before and after MDL-72222, respectively (n=6). Therefore, the attenuation of platelet-induced inhibition of SNA by MDL-72222 was not the result of nonspecific inhibition of afferent nerve activity.

Vascular Versus Neural Mechanism of Action of 5-HT
An important question is whether the reflex inhibition of SNA caused by 5-HT (or by activated platelets) is the result of a direct action of 5-HT on sensory nerve endings or the result of mechanical activation of baroreceptors secondary to vascular contraction. Local exposure of carotid sinuses to norepinephrine, epinephrine, and vasopressin may trigger significant reflex bradycardia and hypotension.23 24 This reflex has been attributed to mechanical deformation of baroreceptor nerve endings, particularly those with unmyelinated C-fiber afferents.23 24 25 It is important to note, however, that direct excitation of sensory nerves by these factors was not ruled out in these studies. We recently demonstrated that rabbit platelets and U-46619 (100 nmol/L) injected into the isolated carotid sinus of rabbits cause significant and equivalent vasoconstriction.11 The striking difference in the reflex responses to platelets and U-46619 despite similar vascular responses strongly suggests that the platelet-induced reflex inhibition of SNA was not the result of carotid vasoconstriction per se. Furthermore, if the platelet- or 5-HT–induced reflex were the result of mechanical activation of baroreceptors, one would not expect it to be blocked by the 5-HT3 receptor antagonist MDL-72222. 5-HT3 receptors are not present on vascular muscle; vascular contraction caused by 5-HT is mediated through 5-HT2 and 5-HT1 receptors26 and therefore should not be blocked by MDL-72222. In addition, MDL-72222 did not attenuate baroreflex-mediated inhibition of SNA in response to increased carotid sinus pressure in our experiments, indicating that it does not interfere with mechanoelectrical transduction and therefore should also not interfere with vascular contraction–induced mechanical activation of baroreceptors. Thus, several lines of evidence suggest that the reflex-mediated inhibition of SNA triggered by platelet activation and 5-HT involves a direct action of 5-HT on the sensory nerve endings.

5-HT has been shown to enhance membrane excitability and increase spike discharge frequency of many types of sensory nerves, including vagal afferents innervating the heart and lungs27 28 and type II baroreceptors29 and chemoreceptors30 31 32 located in the carotid sinus region. Activation of chemoreceptors reflexly increases SNA and therefore is not responsible for our finding of reflex inhibition of SNA during exposure of carotid sinuses to platelets or 5-HT. Injection of 5-HT or phenylbiguanide into blood perfusing the nodose ganglion in cats causes reflex apnea, bradycardia, and hypotension that are thought to result from a direct action of these substances on the cell soma of nodose neurons.33 34 In our experiments, activation of carotid sinus sensory nerves by 5-HT was responsible for triggering the reflex response to activated platelets because the sectioning of carotid sinus nerves abolished the reflex.

Intravenous administration of 5-HT or phenylbiguanide evokes reflex inhibition of SNA, bradycardia, and hypotension.27 35 This reflex is essentially eliminated by vagotomy or intrapericardial procaine (for an exception see Reference 22), indicating the absence of a contribution of carotid sinus afferents in mediating the reflex.27 35 This finding is not surprising considering that the concentration of the intravenously administered agonists likely falls considerably before reaching the carotid sinuses and that these compounds activate numerous types of afferents with potentially opposing influences on the circulation. Our results indicate that 5-HT produced locally from activated platelets as well as phenylbiguanide in carotid sinuses triggers reflex sympathoinhibition and hypotension.

Two different ionic mechanisms most likely mediate the increase in sensory nerve activity caused by activation of 5-HT3 and 5-HT2 receptors. The 5-HT3 receptor is a receptor/ion channel complex.36 37 Opening this nonselective cation channel leads to depolarization of sensory nerves.28 36 Activation of 5-HT2 receptors inhibits K+ channel activity,38 39 which may depolarize and/or block spike after–hyperpolarization in visceral sensory neurons.40 41 These results along with those of the present study suggest that the ionic mechanism of platelet-induced activation of carotid sinus sensory nerves may involve both opening of the 5-HT3 cation channel and inhibition of K+ channels.

Pathophysiological Implications
The carotid sinuses are particularly prone to development of atherosclerotic lesions and platelet adhesion.2 3 4 5 The proximity of platelets aggregating in carotid sinuses to sensory nerve endings increases the likelihood that factors released from platelets in vivo may produce significant changes in sensory nerve activity.

It is established that platelets aggregating in carotid sinuses are an important cause of transient cerebral ischemia and strokes.6 7 8 9 The pronounced reflex decrease in arterial pressure resulting from inappropriate activation of carotid sinus sensory nerves would further compromise cerebral blood flow by reducing cerebral perfusion pressure. We speculate that fluctuations in SNA in response to episodic activation of platelets may also provide a neural mechanism of increased arterial pressure lability in atherosclerotic and thrombotic states.


*    Selected Abbreviations and Acronyms
 
bpm = beats per minute
5-HT = serotonin (5-hydroxytryptamine)
MDL-72222 = 3-tropanyl-3,5-dichlorobenzoate
SNA = sympathetic nerve activity
U-46619 = methanoepoxyprosta-5,13-dienoic acid


*    Acknowledgments
 
This work was supported by research funds from the Department of Veterans Affairs and grant PO1-HL-14388 from the National Institutes of Health. Dr Mao was the recipient of an Institutional National Research Service Award from the National Institutes of Health when this study was performed. The authors thank Shawn M. Roach and Debra J. Schiek for preparation of figures and Colleen Chapleau and Jackie Schneider for typing the manuscript.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

  1. Kirchheim HR. Systemic arterial baroreceptor reflexes. Physiol Rev. 1976;56:100-176. [Free Full Text]
  2. Solberg LA, Eggen DA. Localization and sequence of development of atherosclerotic lesions in the carotid and vertebral arteries. Circulation. 1971;43:711-724. [Abstract/Free Full Text]
  3. Heath D, Smith P, Harris P, Winson M. The atherosclerotic human carotid sinus. J Pathol. 1973;110:49-58. [Medline] [Order article via Infotrieve]
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