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Department of Physiology, Wayne State University, School of Medicine, Detroit, Michigan
Submitted 7 July 2005 ; accepted in final form 8 August 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-adrenergic receptors that are preferentially located in the skeletal muscle vasculature. Selective activation of NTS A1 adenosine receptors evokes variable, mostly pressor effects and increases pre-ASNA, as well as lumbar sympathetic activity, which is directed to the hindlimb. These counteracting factors may have opposite effects on the hindlimb vasculature resulting in mixed vascular responses. Therefore, in chloralose-urethane-anesthetized rats, we evaluated the contribution of vasodilator versus vasoconstrictor effects of stimulation of NTS A1 receptors on the hindlimb vasculature. We compared the changes in iliac vascular conductance evoked by microinjctions into the NTS of the selective A1 receptor agonist N6-cyclopentyladenosine (330 pmol in 50 nl volume) in intact animals with the responses evoked after -adrenergic blockade, bilateral adrenalectomy, bilateral lumbar sympathectomy, and combined adrenalectomy + lumbar sympathectomy. In intact animals, stimulation of NTS A1 receptors evoked variable effects: increases and decreases in mean arterial pressure and iliac conductance with prevailing pressor and vasoconstrictor effects. Peripheral -adrenergic receptor blockade and bilateral adrenalectomy eliminated the depressor component of the responses, markedly potentiated iliac vasoconstriction, and tended to increase the pressor responses. Lumbar sympathectomy tended to decrease the pressor and vasoconstrictor responses. After bilateral adrenalectomy plus lumbar sympathectomy, a marked vasoconstriction in iliac vascular bed still persisted, suggesting that the vasoconstrictor component of the response to stimulation of NTS A1 receptors is mediated mostly via circulating factors (e.g., vasopressin, angiotensin II, or circulating catecholamines released from other sympathetic terminals). These data strongly suggest that stimulation of NTS A1 receptors exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and -adrenergic vasodilation versus vasoconstriction mediated by neural and humoral factors.
nucleus of the solitary tract; purinergic receptors; -adrenergic blockade; adrenalectomy; lumbar sympathectomy; iliac vascular conductance
The preferential activation of the sympathetic output directed to the adrenal medulla via both subtypes of NTS adenosine receptors may trigger the release of epinephrine, which would evoke vasodilation in skeletal muscles where 2-adrenergic receptors are preferentially expressed (26). Our previous study showed that selective stimulation of NTS A2a receptors elicited a large vasodilation in iliac vascular bed, whereas vasodilation in the renal and mesenteric vascular beds was much smaller (3). A subsequent study directly confirmed the hypothesis that this preferential iliac vasodilation is mediated mostly via a -adrenergic mechanism and both the adrenal glands and efferent sympathetic nerves contribute to the response (10). However, the effect of activation of NTS adenosine A1 receptors on regional hemodynamic responses have not been studied.
It is likely that stimulation of NTS A1 receptors also evokes -adrenergic vasodilation in the iliac vascular bed inasmuch as large increases in pre-ASNA occur. However, in addition to activation, pre-ASNA stimulation of NTS A1 adenosine receptors also increases LSNA (21). Therefore, the activation of NTS A1 receptors may exert reciprocal effects on the iliac vascular bed, i.e., -adrenergic vasodilation may counteract direct sympathetic vasoconstriction leading to much greater variability of the hemodynamic effects versus those observed after stimulation of NTS A2a receptors. This concept is supported by our previous observations that selective activation of NTS A1 receptors exerts more variable effects on arterial pressure compared with the uniform depressor responses observed after stimulation of NTS A2a receptors (18–21). Specifically, although the pressor responses to stimulation of NTS A1 receptors prevailed, biphasic or depressor responses were observed in 
30% of the cases (21). Therefore, in the present study, we tested the hypothesis that the variable hemodynamic responses observed following selective stimulation of NTS A1 adenosine receptors are the result of -adrenergic vasodilation opposed by simultaneous sympathetic vasoconstriction in the skeletal muscle vasculature.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Design.
The contribution of -adrenergic receptors, the adrenal glands, and the sympathetic innervation of the hindquarters to the responses evoked by selective stimulation of NTS A1 adenosine receptors was studied in 48 male Sprague-Dawley rats. In 42 animals only one microinjection was performed, and in 6 animals two microinjections separated by 90-min intervals were performed giving a total of 54 microinjections. Changes in iliac vascular conductance evoked by microinjections into the NTS of selective A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA, Tocris) were compared among five experimental groups: 1) intact (INT, n = 14), 2) after -adrenergic blockade (X, n = 11), 3) after bilateral adrenalectomy (ADX, n = 10), 4) after bilateral lumbar sympathectomy (LX, n = 10), and 5) after combined bilateral adrenalectomy plus lumbar sympathectomy (ADX+LX, n = 9).
Instrumentation and measurements.
All the procedures were described in detail previously (3, 16–21). Briefly, male Sprague-Dawley rats (350–400 g) (Charles River) were anesthetized with a mixture of 
-chloralose (80 mg/kg) and urethane (500 mg/kg ip), tracheotomized, connected to a small animal respirator (SAR-830, CWE, Ardmore, PA), and artificially ventilated with 40% oxygen-60% nitrogen mixture. Arterial blood gases were tested occasionally (Radiometer, ABL500, OSM3), and animals were maintained slightly hyperventilated to prevent spontaneous respiratory movements. Average values measured at the end of each experiment were the following: PO2 = 143.3 ± 5.5 mmHg, PCO2 = 31.1 ± 0.8 mmHg, and pH = 7.416 ± 0.006. The same level of artificial ventilation was applied in all experimental groups; therefore, this factor most likely did not affect the differences between the groups. The right femoral artery and vein were catheterized to monitor arterial blood pressure and infuse drugs.
From a midabdominal incision, the left iliac artery was exposed. A pulsed Doppler blood flow velocity transducer (Baylor Electronics) was placed around the artery and connected to the flowmeter. From the same incision in some animals, bilateral adrenalectomy or bilateral lumbar sympathectomy L1–L6 or combined adrenalectomy plus lumbar sympathectomy were performed. The intermesenteric nerves were also severed in sympathectomized animals. Blockade of -adrenergic receptors was performed similarly as in our previous study via injection of propranolol (2 mg/kg iv) 10 min before the NTS microinjections (10).
The arterial pressure and iliac flow signals were digitized and recorded with an analog-digital converter (Modular Instruments) interfaced to a laboratory computer. The signals were recorded continuously using Biowindows software (Modular Instruments), averaged over 5-s intervals, and stored on a hard disk for subsequent analysis.
Microinjections into the NTS.
After the exposure of the brain stem via dissection of the atlantoocipital membrane, the animals were allowed to stabilize for at least 30 min before microinjections. Unilateral microinjections of CPA (330 pmol in 50 nl of artificial cerebrospinal fluid, ACF) were made with multibarrel, glass micropipettes into the medial region of the caudal subpostremal NTS as described previously (3, 18–21). This dose of CPA produced the most consistent, predominantly pressor responses in our previous study (21). In several previous studies, we have shown that microinjections of the same amount of vehicle (ACF) into the same site of the NTS did not markedly affect mean arterial pressure (MAP), heart rate (HR), RSNA, LSNA, and pre-ASNA and blood flow in iliac, renal, and mesenteric arteries. The changes in all these variables were either not different from zero or smaller than natural, random fluctuations of these variables over the time of the measurements (3, 18–21). To avoid the effect of desensitization of A1 adenosine receptors, in all experiments, only one dose of the agonist was microinjected into the left and/or right side of the NTS. If the agonist was injected bilaterally, at least a 90-min interval between the injections was allowed. No simultaneous bilateral microinjections were performed. CPA was dissolved in ACF, and the pH was adjusted to 7.2. All microinjection sites were verified histologically as described previously (3, 18–21) and are presented in Fig. 1.
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Iliac vascular conductance (IVC) was calculated by dividing iliac blood flow (IBF), expressed as a Doppler shift (in Hz) by MAP (in mmHg). Hemodynamic responses were analyzed over a 20-min period following the microinjections and quantified in two ways as described previously (3, 18–21): 1) the maximal changes from a 60-s baseline control period taken immediately before the microinjection and 2) integration of the changes from the control values. The integral reflects a predominant trend of the response despite transient, sometime large, bidirectional fluctuations in each variable. Because hemodynamic effects evoked by stimulation of NTS A1 receptors were variable, often biphasic, or even polyphasic, as we reported previously (21), we used the integral values for the comparisons between the experimental groups. Both absolute and percent changes of hemodynamic variables evoked by stimulation of NTS A1 receptors were compared between the groups. Because the absolute values of blood flow depend to some extent on positioning of the probe around the iliac artery, the comparisons between the percent changes in MAP, IBF, and IVC seem more reliable. The HR responses, calculated from pulse intervals, were expressed in absolute values (beats/min). To characterize the time course of the responses, the total time (in minutes) and the time to maximum of the responses were calculated for MAP and IVC and averaged separately for the increases and decreases in these variables in each experimental group. One-way ANOVA for independent measures was used to compare hemodynamic responses versus experimental conditions. Differences observed were further evaluated by t-test with Bonferroni adjustment for independent measures. The changes in all recorded variables were also compared with zero by means of SYSTAT univariate F test. An level of P < 0.05 was used to determine statistical significance.
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RESULTS |
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-adrenergic blockade was not significantly different compared with the intact group, it was significantly lower compared with resting HR measured just before the blockade in the X group (325.1 ± 7.7 vs. 338.9 ± 8.7 beats/min, P < 0.05). IBF and IVC were significantly increased after bilateral lumbar sympathectomy, as expected. Surprisingly, IBF and IVC were also significantly greater following -adrenergic blockade compared with the intact group. However, IBF and IVC measured in the same animals before and after the blockade were not different (IBF, 1148.3 ± 134.8 vs. 1089.6 ± 123.5 Hz and IVC, 12.4 ± 1.5 vs. 12.0 ± 1.3 Hz/mmHg, respectively). Therefore, the differences between the intact versus -adrenergic blockade groups reflect the differences between animals rather than the experimental conditions.
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X, ADX, LX, and ADX+LX). In addition to prevailing pressor and vasoconstrictor responses to stimulation of NTS adenosine A1 receptors presented in Fig. 2, biphasic or depressor and vasodilatory responses were also occasionally observed. The patterns of the responses observed in intact animals were similar to those that were observed in our previous study (21). Table 2 presents the numbers of responses in which increases or decreases in MAP, HR, IBF, and IVC prevailed in each experimental group as shown by the integral responses. In the intact group, the pressor and vasoconstrictor responses were observed in 70% and 65% of the cases, respectively. After -adrenergic blockade, the variability disappeared, and only pressor and vasoconstrictor responses were observed, indicating that -adrenergic vasodilation was responsible for the variability of the effects observed in intact animals. Also, bilateral adrenalectomy eliminated the variability in vascular responses leading to consistent vasoconstriction. In both X and ADX groups, the pressor and iliac vasoconstrictor responses were markedly enhanced compared with those observed in intact animals (Fig. 2, left three panels). This indicated that the adrenal medulla contributed to the initial variability of the vascular effects probably via release of epinephrine and -adrenergic vasodilation, which competed with simultaneous, marked vasoconstriction. Surprisingly, bilateral lumbar sympathectomy did not eliminate the vasoconstrictor component of the response but only tended to decrease it (Fig. 2). Instead of expected uniform depressor and vasodilatory responses after LX, the variability of the responses actually increased in this group; pressor and iliac vasoconstrictor responses prevailed in 50% and 40% of cases, respectively (Table 2). After combined adrenalectomy and lumbar sympathectomy, marked iliac vasoconstriction persisted in all the responses (Fig. 2 and Table 2), indicating that stimulation of NTS A1 receptors triggers powerful nonsympathetic vasoconstriction in the iliac vascular bed. There were no significant differences in the time course of hemodynamic responses between the experimental groups (Table 3). Time to maximum and total time of MAP and IVC responses measured in the intact group were not different versus all other groups (P > 0.05). There were also no significant differences between time to maximum as well as total time of increases versus decreases in MAP and IVC in those experimental groups where bidirectional responses were observed (INT, LX, and ADX+LX) (Table 3).
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-adrenergic blockade and adrenalectomy a large, severalfold decrease in the IBF and IVC responses was observed compared with those measured in the intact group. Also, pressor responses tended to increase in these groups compared with the intact control group, although the differences did not reach statistical significance. Lumbar sympathectomy virtually abolished the responses (the averaged responses after LX were not different from zero, P > 0.05). However, the responses in the LX group were also not different from those observed in the intact control animals, indicating that lumbar sympathetic nerves contribute somewhat to the overall responses evoked by stimulation of NTS A1 receptors. After combined ADX+LX, marked iliac vasoconstriction persisted. The decreases in IBF and IVC observed in this group were severalfold greater than those observed in the intact control group and not different from those observed following -adrenergic blockade and bilateral adrenalectomy. This additionally confirmed the small contribution of sympathetic nerves to the responses and strongly suggested that nonsympathetic, perhaps humoral, vasoconstriction of the iliac vascular bed is triggered by stimulation of NTS A1 receptors. The analysis of absolute values of the responses, shown in Table 4, leads to the same conclusions as those described above based on percentage changes in hemodynamic parameters presented in Fig. 3. The changes in HR in response to stimulation of NTS A1 receptors were irregular with prevailing cardiac slowing, as reported previously (1, 21). No significant differences in HR responses were observed between the experimental groups (Table 4).
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DISCUSSION |
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-adrenergic vasodilation, which is counteracted by neural and humoral vasoconstriction; and 3) the neural component of the iliac vasoconstriction triggered by stimulation of NTS A1 receptors is negligible compared with the humoral component.
The present study confirmed our hypothesis that preferential activation pre-ASNA after stimulation of NTS A1 adenosine receptors (19) elicits -adrenergic vasodilation in the iliac vascular bed. The overall iliac conductance tended to decrease following stimulation of NTS A1 receptors, although the decreases measured as both absolute and relative values were not significantly different from zero (Table 4 and Fig. 3, respectively). This indicates that the -adrenergic iliac vasodilation was completely buffered by slightly greater iliac vasoconstriction in intact animals.
We have previously reported that stimulation of NTS A2a adenosine receptors evokes preferential hindlimb vasodilation via activation of the adrenal medulla and -adrenergic vasodilation (10). However, the preferential hindlimb vasodilation elicited by NTS A2a receptors was a result of synergistic action of -adrenergic vasodilation, withdrawal of sympathetic vasoconstriction, and probably simultaneous active sympathetic vasodilation. All these factors led to the uniform vasodilatory responses, which were abolished by bilateral adrenalectomy plus lumbar sympathectomy (10). In contrast, NTS A1 adenosine receptors elicit -adrenergic vasodilation in the hindlimb vascular bed and simultaneously trigger neural and humoral vasoconstriction leading to marked variability of the hemodynamic responses observed in intact rats in this and previous studies (21). It is unlikely that the variability of the responses evoked by microinjections of the highly selective A1 adenosine receptor agonist CPA may be caused by nonselective activation of other adenosine receptor subtypes, which may be present in the NTS (A2a, A2b, and A3). In our previous study (21), we found that the variability of the responses decreased with increasing doses of CPA, whereas nonselective activation of other receptor subtypes should increase with increasing doses of the agonist. In addition, the variability of the hemodynamic responses was completely eliminated after -adrenergic blockade or adrenalectomy. Finally, although we observed variable, even bidirectional responses, especially in INT and LX experimental groups, there were no significant differences between time parameters of the responses across the groups (Table 3). Taken together, the above observations strongly suggest that the interactions of peripheral factors were mostly responsible for the observed variability of the iliac vascular responses, whereas the time course of activation of NTS A1 adenosine receptors was similar in all experimental groups.
Interestingly, although stimulation of NTS A1 receptors significantly increases LSNA, the contribution of sympathetic nerves to iliac vasoconstriction following stimulation of NTS A1 receptors was negligible compared with nonsympathetic, likely humoral vasoconstriction. This may be explained by simultaneous activation of both vasoconstrictor and active vasodilatory fibers directed to the hindlimb in response to selective activation of adenosine A1 receptors in the NTS. Vascular effects of two counteracting components of the increased LSNA may counteract each other. Therefore, removal of sympathetic nerves supplying the hindquarter may have little or no effect on the vascular response to stimulation of NTS A1 receptors as we found in the present study. We previously postulated a similar mechanism to explain that LSNA significantly contributes to the iliac vasodilation elicited by activation of NTS A2a receptors, although LSNA remains virtually unaltered, most likely as a result of simultaneous inhibition of sympathetic vasoconstrictor fibers and activation of sympathetic vasodilatory fibers (10). Both of the above concepts are consistent with the reports from Lewis' laboratory that in addition to withdrawal of sympathetic vasoconstrictor tone, the nitrosyl factors released from sympathetic terminals contribute to active vasodilation in hindlimb vasculature in the rat (7, 8).
After bilateral adrenalectomy combined with bilateral lumbar sympathectomy, stimulation of NTS A1 adenosine receptors still evoked marked iliac vasoconstriction, indicating that other, likely humoral mechanisms are involved. The potential vasoconstrictor factors released to the blood stream following stimulation of NTS A1 receptors remain unknown. However, among many possibilities, the release of vasopressin, norepinephrine, and renin/angiotensin II should be considered. According to our previous studies, the pressor and sympathoactivatory responses to stimulation of NTS A1 receptors are most likely mediated via inhibition of the release of glutamate from baroreceptor afferents terminating in the NTS (27). This hypothesis was supported by our observations that the pressor and sympathoactivatory responses were abolished following bilateral sinoaortic denervation combined with bilateral vagotomy as well as following blockade of ionotropic glutamatergic mechanisms in the NTS (21). Assuming that the stimulation of A1 adenosine receptors inhibits baroreflex transmission at the level of the NTS, it is likely that this mechanism may decrease baroreflex restraint of vasopressin release and consequently increase the circulating levels of vasopressin (6). Therefore, vasopressin may be a major humoral factor responsible for the vasoconstriction that persisted following combined lumbar sympathectomy and adrenalectomy. Another humoral factor that may contribute to iliac vasoconstriction may be norepinephrine released from other sympathetic terminals and reaching the iliac vascular bed via circulating blood. This is likely, because stimulation of NTS A1 receptors evokes uniform sympathoactivation in several sympathetic outputs (21). Finally, because activation of NTS A1 receptors increases sympathetic activity directed to the kidney, it may facilitate the renin/angiotensin system, and this mechanism may also contribute to humoral vasoconstriction (9). All the above possibilities await further investigation.
The present study directly indicates that one of the most important sources of variability of the hemodynamic effects evoked by stimulation of NTS A1 receptors in the hindlimb vascular bed is simultaneously triggered vasoconstriction and epinephrine-mediated -adrenergic vasodilation. Our data also provide direct evidence that in addition to increased sympathetic vasoconstrictor activity, NTS A1 adenosine receptors trigger a powerful humoral vasoconstriction in the iliac and probably other vascular beds. A detailed assessment of neural versus humoral factors mediating NTS A1 receptor responses in regional vascular beds awaits further investigation.
In our previous study, predominantly pressor or depressor responses to microinjections of CPA were evoked from similar sites of the caudal subpostremal NTS, indicating that the type of the response was not related to anatomically specific groups of NTS neurons, for example, "depressor" (rostral and subpostremal) versus "pressor" (caudal) portion of the NTS, which respond reciprocally to microinjections of small doses of glutamate (13). In the present study, the depressor responses to activation of NTS A1 receptors seem to be more frequent in the rostral than caudal portion of the subpostremal NTS (Fig. 1). However, the pressor responses were evenly distributed across the NTS. In addition, after -adrenergic blockade, only the pressor responses were observed, and the majority of the microinjections in this setting was located in the most rostral portion of the subpostremal NTS. Therefore, we believe that the variable hemodynamic responses were rather determined by the relative ratio of vasoconstrictor versus vasodialator factors triggered by activation of NTS A1 adenosine receptors, mechanisms that overlap anatomically but are functionally different.
In summary, stimulation of adenosine A1 receptors located on NTS neurons/neural terminals triggers simultaneous -adrenergic vasodilation and humoral/sympathetic vasoconstriction. These reciprocal effects are the source of variability of the responses of the hindlimb vasculature and contribute to the variability of the general pressor/depressor responses observed in intact animals.
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FOOTNOTES |
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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.
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-adrenergic mechanism. Am J Physiol Heart Circ Physiol 278: H1775–H1782, 2000.This article has been cited by other articles:
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  J. M. McClure, N. F. Rossi, H. Chen, D. S. O'Leary, and T. J. Scislo Vasopressin is a major vasoconstrictor involved in hindlimb vascular responses to stimulation of adenosine A1 receptors in the nucleus of the solitary tract Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1661 - H1672. [Abstract] [Full Text] [PDF]
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T. J. Scislo, T. K. Ichinose, and D. S. O'Leary Stimulation of NTS A1 adenosine receptors differentially resets baroreflex control of regional sympathetic outputs Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H172 - H182. [Abstract] [Full Text] [PDF]
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 G. Burnstock Physiology and Pathophysiology of Purinergic Neurotransmission Physiol Rev, April 1, 2007; 87(2): 659 - 797. [Abstract] [Full Text] [PDF]
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 J. M McClure, T. J Scislo, and D. S O'Leary Mechanisms Mediating Regional Vascular Responses to Stimulation of Adenosine A1 Receptors in the Nucleus of the Solitary Tract (NTS) FASEB J, April 1, 2007; 21(5): A466 - A466.
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T. J. Scislo and D. S. O'Leary Adenosine receptors located in the NTS contribute to renal sympathoinhibition during hypotensive phase of severe hemorrhage in anesthetized rats Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2453 - H2461. [Abstract] [Full Text] [PDF]
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, 
), after 
, 
), and after bilateral adrenalectomy (
, 
). B: microinjections of CPA after bilateral lumbar sympathectomy (
, 
) and combined bilateral adrenalectomy and lumbar sympathectomy (
, 
). Pressor or depressor responses to CPA were evoked from similar anatomical sites of NTS.
