Mechanisms mediating NTS P2x receptor-evoked hypotension: cardiac output vs. total peripheral resistance.

We have previously shown that P2x purinoceptor activation in the subpostremal nucleus tractus solitarius (NTS) produces dose-dependent decreases in mean arterial pressure (MAP), heart rate, efferent sympathetic nerve activity, and significant peripheral vasodilation. However, the relative roles of cardiac output (CO) and total peripheral resistance (TPR) in mediating this depressor response are unknown. Bradycardia does not necessarily result in decreased CO, because, with the greater filling time, stroke volume may increase such that CO may be unchanged. We measured changes in CO (via a chronically implanted flow probe on the ascending aorta) and MAP in alpha-chloralose- and urethane-anesthetized male Sprague-Dawley rats in response to microinjection of the selective P2x purinoceptor agonist alpha,beta-methylene ATP (25 and 100 pmol/50 nl) into the subpostremal NTS. TPR was calculated as MAP/CO. At the low dose of NTS P2x purinoceptor agonist, the reduction in MAP was primarily mediated by reductions in TPR (-31.3 +/- 3.3%), not CO (-8.7 +/- 1.7%). At the high dose, both CO (-34.4 +/- 6.6%) and TPR (-40.2 +/- 2.5%) contribute to the reduction in MAP. We conclude that the relative contribution of CO and TPR to the reduction in MAP evoked by NTS P2x purinoceptor activation is dependent on the extent of P2x purinoceptor activation.

Differential patterns of sympathetic responses to selective stimulation of nucleus tractus solitarius purinergic receptor subtypes.

1. Studies are described that indicate that stimulation of different purinergic receptor subtypes (A1, A2A and P2X) located in the sub-postremal nucleus tractus solitarius (NTS) evokes qualitatively and quantitatively different regional haemodynamic and efferent sympathetic responses. 2. Stimulation of A2A receptors evoked the most diverse pattern of regional sympathetic responses: preganglionic adrenal nerve activity (pre-ASNA) was increased, lumbar sympathetic nerve activity (LSNA) did not change, while renal (RSNA) and post-ganglionic adrenal (post-ASNA) sympathetic nerve activity was decreased. Stimulation of A1 receptors evoked qualitatively uniform, although quantitatively different, sympathoactivation: pre-ASNA > RSNA > LSNA. Stimulation of P2X receptors evoked qualitatively uniform, although quantitatively different, sympathoinhibition: RSNA=post-ASNA > LSNA = pre-ASNA. 3. These qualitatively and quantitatively different patterns of regional sympathetic responses strongly suggest that purinergic receptor subtypes may be specifically located and differentially expressed on NTS neurons/neural terminals that control different sympathetic outputs. Different NTS purinoceptors may contribute to patterned autonomic responses observed in specific physiological or pathological situations.

NTS A(2a) purinoceptor activation elicits hindlimb vasodilation primarily via a beta-adrenergic mechanism.

Previously, we have shown that activation of adenosine A(2a) receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of the selective A(2a) receptor agonist CGS-21680 elicits potent, dose-dependent decreases in mean arterial pressure and preferential, marked hindlimb vasodilation. Although A(2a) receptor activation does not change lumbar sympathetic nerve activity, it does markedly enhance the preganglionic adrenal sympathetic nerve activity, which will increase epinephrine release and could subsequently elicit hindlimb vasodilation via activation of beta(2)-adrenergic receptors. Therefore we investigated whether this hindlimb vasodilation was due to neural or humoral mechanisms. In chloralose-urethan-anesthetized male Sprague-Dawley rats, we monitored cardiovascular responses to stimulation of NTS adenosine A(2a) receptors (CGS-21680, 20 pmol/50 nl) in the intact control animals; after pretreatment with propranolol (2 mg/kg iv), a beta-adrenergic antagonist; after bilateral lumbar sympathectomy; after bilateral adrenalectomy; and after combined bilateral lumbar sympathectomy and adrenalectomy. After beta-adrenergic blockade, stimulation of NTS adenosine A(2a) receptors produced a pressor response and a hindlimb vasoconstriction. Lumbar sympathectomy reduced the vasodilation seen in the intact animals by approximately 40%, and adrenalectomy reduced it by approximately 80%. The combined sympathectomy and adrenalectomy virtually abolished the hindlimb vasodilation evoked by NTS A(2a) receptor activation. We conclude that the preferential, marked hindlimb vasodilation produced by stimulation of NTS adenosine A(2a) receptors is mediated by both the efferent sympathetic nerves directed to the hindlimb and the adrenal glands via primarily a beta-adrenergic mechanism.

Differential role of ionotropic glutamatergic mechanisms in responses to NTS P(2x) and A(2a) receptor stimulation.

Activation of ATP P(2x) receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of alpha,beta-methylene ATP (alpha,beta-MeATP) elicits fast initial depressor and sympathoinhibitory responses that are followed by slow, long-lasting inhibitory effects. Activation of NTS adenosine A(2a) receptors via microinjection of CGS-21680 elicits slow, long-lasting decreases in arterial pressure and renal sympathetic nerve activity (RSNA) and an increase in preganglionic adrenal sympathetic nerve activity (pre-ASNA). Both P(2x) and A(2a) receptors may operate via modulation of glutamate release from central neurons. We investigated whether intact glutamatergic transmission is necessary to mediate the responses to NTS P(2x) and A(2a) receptor stimulation. The hemodynamic and neural (RSNA and pre-ASNA) responses to microinjections of alpha,beta-MeATP (25 pmol/50 nl) and CGS-21680 (20 pmol/50 nl) were compared before and after pretreatment with kynurenate sodium (KYN; 4.4 nmol/100 nl) in chloralose-urethan-anesthetized male Sprague-Dawley rats. KYN virtually abolished the fast responses to alpha,beta-MeATP and tended to enhance the slow component of the neural responses. The depressor responses to CGS-21680 were mostly preserved after pretreatment with KYN, although the increase in pre-ASNA was reduced by one-half following the glutamatergic blockade. We conclude that the fast responses to stimulation of NTS P(2x) receptors are mediated via glutamatergic ionotropic mechanisms, whereas the slow responses to stimulation of NTS P(2x) and A(2a) receptors are mediated mostly via other neuromodulatory mechanisms.

Differential control of renal vs. adrenal sympathetic nerve activity by NTS A2a and P2x purinoceptors.

Activation of adenosine A2a and ATP P2x purinoceptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of the selective agonists CGS-21680 and alpha,beta-methylene ATP (alpha, beta-MeATP), respectively, elicits large dose-dependent decreases in arterial pressure and heart rate, differential regional vasodilation, and differential inhibition of regional sympathetic outputs. With marked hypotensive hemorrhage, preganglionic adrenal sympathetic nerve activity (pre-ASNA) increases, whereas renal (RSNA) and postganglionic adrenal sympathetic nerve activity (post-ASNA) decrease. In this setting, adenosine levels in the brain stem increase. Therefore, we investigated whether stimulation of specific purinoceptors in the NTS may evoke differential sympathetic responses. RSNA was recorded simultaneously with pre-ASNA or post-ASNA in chloralose-urethan-anesthetized male Sprague-Dawley rats. CGS-21680 (2 and 20 pmol in 50 nl) inhibited RSNA and post-ASNA, whereas pre-ASNA increased markedly. alpha,beta-MeATP (25 and 100 pmol in 50 nl) inhibited all sympathetic outputs. Sinoaortic denervation plus vagotomy markedly prolonged the responses to P2x-purinoceptor stimulation. Glutamate (100 pmol in 50 nl) caused differential inhibition of all sympathetic outputs similar to that evoked by alpha,beta-MeATP. We conclude that NTS A2a-purinoceptor activation evokes differential sympathetic responses similar to those observed during hemorrhage, whereas P2x-purinoceptor and glutamate-receptor activation evokes differential inhibition of sympathetic outputs similar to arterial baroreflex responses.

Impaired arterial baroreflex regulation of heart rate after blockade of P2-purinoceptors in the nucleus tractus solitarius.

Activation of P2x-purinoceptors in the nucleus tractus solitarius (NTS) via microinjection of ATP mimics baroreflex responses (bradycardia, hypotension); however, the physiological role of these receptors in cardiovascular control remains unclear. We tested whether blockade of these receptors attenuates arterial baroreflex control of heart rate (HR). Baroreflex-induced changes in HR (via graded i.v. infusion of phenylephrine and nitroprusside) were observed in seven alpha-chloralose/urethane anesthetized male Sprague-Dawley rats before and after microinjection of the purinergic P2 receptor antagonist suramin (0.5 nmol in 50 nL) into the subpostremal NTS. Before suramin, typical baroreflex changes in HR were observed (maximum gain, Gmax = 2.94 +/- 0.54 bpm/mmHg). Suramin markedly impaired baroreflex-induced changes in HR (gain = 0.02 +/- 0.08 and 0.18 +/- 0.09 bpm/mmHg for increases and decreases in mean arterial blood pressure, respectively); however, after 90-130 min, HR and baroreflex reactivity returned to control levels. Microinjections of vehicle into the same area did not alter baroreflex function. In addition, suramin did not alter the depressor responses to microinjections of glutamate into the same site of the NTS. We conclude that normal P2x-purinoceptor function in subpostremal NTS may be necessary for baroreflex regulation of HR.

Differential arterial baroreflex regulation of renal, lumbar, and adrenal sympathetic nerve activity in the rat.

Lumbar (LSNA), renal (RSNA), or adrenal sympathetic nerve activity (ASNA) is most commonly used as an index of sympathetic nerve activity in investigations of arterial baroreflex control in the rat. Although differential regulation of sympathetic outputs to different organs has been extensively studied, no direct and simultaneous comparisons of the full range of baroreflex reactivity have been described for these sympathetic outputs. Therefore, we compared steady-state sigmoidal baroreflex stimulus-response curves (via phenylephrine-nitroprusside infusion) for RSNA recorded simultaneously with LSNA or ASNA in urethan-chloralose-anesthetized male Sprague-Dawley rats. Characteristics of the baroreflex curves differed significantly between all three sympathetic outputs. ASNA exhibited the greatest range of baroreflex regulation, the highest upper level of activity, and the widest distribution of the gain over a broad range of mean arterial pressure (MAP). RSNA exhibited greater gain than LSNA. LSNA showed the smallest range and maximal inhibition in comparison to other sympathetic outputs. However, all three nerves responded similarly to baroreflex stimulation and unloading in the range in MAP close to the operating point. We conclude that baroreflex regulation of sympathetic activity shows wide regional variability in gain, range, and maximal inhibition. Therefore, the entire stimulus-response relationship should be considered in comparing regional sympathetic responses.

Activation of A2a adenosine receptors in the nucleus tractus solitarius inhibits renal but not lumbar sympathetic nerve activity.

The activation of adenosine A2a receptors in the nucleus tractus solitarius (NTS) via microinjection of the selective agonist CGS 21680 elicits long-lasting, dose-dependent decreases in mean arterial pressure (MAP) and heart rate (HR) and preferential dilation of the iliac vascular bed in comparison to the renal and mesenteric vascular beds. We investigated whether differential changes in regional sympathetic output occur with A2a receptor activation. In 24 chloralose/urethane anesthetized male Sprague-Dawley rats MAP, HR, renal (RSNA) and lumbar sympathetic nerve activity (LSNA) were recorded simultaneously. Data were analyzed as both the maximum decrease and the integral of the decrease over the duration of the depressor response. Microinjection of CGS 21680 (2 and 20 pmol in 50 nl volume) into the subpostremal NTS caused significant and dose-dependent decreases in MAP, HR and RSNA, however, did not significantly decrease LSNA in comparison to the effect of vehicle. Maximum responses of RSNA vs. LSNA in delta% of control values were: -32 +/- 4 vs. -9 +/- 2, and -59 +/- 4 vs. -19 +/- 5 for low (n = 9) and high (n = 8) doses of CGS 21680 respectively; integral responses of RSNA vs. LSNA in delta% x min were: -487 +/- 112 vs. -19 +/- 35 and -1258 +/- 164 vs. -175 +/- 126 for low and high doses of CGS 21680 respectively. Microinjections of vehicle (n = 7) did not alter integral hemodynamic or neural parameters. We conclude that activation of A2a adenosine receptors in the NTS evokes differential changes in visceral vs. somatic sympathetic nerve activity which cannot explain differential vascular responses in terms of simple sympathetic withdrawal. Lack of significant inhibition of LSNA combined with preferential vasodilation in hindquarter vascular bed suggests that active vasodilation may be triggered by activation of A2a adenosine receptors in the subpostremal NTS.

Purines and the nucleus tractus solitarius: effects on cardiovascular and respiratory function.

1. The roles of adenosine and adenosine 5'-triphosphate in cardiorespiratory regulation by the nucleus tractus solitarius (NTS) have been evaluated in a range of experiments, using micro-injections of selective agonists and antagonists of purinoceptors. 2. Adenosine injected into the caudal NTS decreases heart rate (HR), arterial blood pressure (BP) and respiratory frequency by an action at A2a receptors on glutamatergic nerve terminals. Microinjections of the A2a agonist CGS21680 caused falls in arterial BP and HR which were selectively antagonized by the A2a antagonist CGS15943. Injection of the A1 agonist CPA in the caudal NTS elevated BP and HR and its actions were selectively antagonized by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine. Adenosine enhanced glutamate release in the NTS. 3. In the rostral NTS, adenosine administration resulted in an elevation of BP. 4. ATP microinjection into the subpostremal NTS depressed HR and BP by an action on P2x purinoceptors, which are blocked by saramin. ATP microinjections differentially affected vascular conductance in regional peripheral vascular beds, with the most marked increases in conductance in the iliac bed and lesser effects in the superior mesenteric and renal vascular beds. 5. Recordings from renal and lumbar sympathetic nerves have demonstrated marked dose-dependent decreases in efferent activity following alpha, beta-methylene ATP injections into the NTS; however, only renal but not lumbar sympathetic nerve activity was inhibited following CGS21680 injections into the same site of the NTS. 6. ATP may function as a fast-acting neurotransmitter in the baroreceptor afferent pathway or in interneurons in the NTS. Adenosine is likely to play a role as a modulator of activity in baroreceptor and chemoreceptor pathways, fine tuning the functional output of both systems.

Activation of P2x-purinoceptors in the nucleus tractus solitarius elicits differential inhibition of lumbar and renal sympathetic nerve activity.

Activation of P2x-purinoceptors in the nucleus tractus solitarius (NTS) via microinjection of alpha,beta-methylene ATP (alpha,beta-MeATP) elicits large dose-dependent decreases in mean arterial pressure (MAP) and heart rate (HR) and preferential dilation of the iliac vascular bed in comparison to renal and mesenteric vascular beds. We investigated whether sympathoinhibition contributes to the depressor responses and whether differential changes in regional sympathetic output occur. In 43 chloralose/urethane anesthetized male Sprague-Dawley rats, MAP, HR, renal (RSNA) and lumbar sympathetic nerve activity (LSNA) were recorded. Data were analyzed as both the maximum decrease and the integral of the decrease over the duration of the depressor response. Microinjection of alpha,beta-MeATP (25 and 100 pmol in 50 nl volume) into the subpostremal NTS caused significant and dose-dependent decreases in MAP, HR, RSNA and LSNA. However, the changes in RSNA were significantly greater than those observed in LSNA for both doses and both methods of analysis of data (maximum responses in delta %: 84 +/- 3 vs 62 +/- 4, and 93 +/- 3 vs 74 +/- 4 for low and high dose of alpha,beta-MeATP, respectively; integral responses in delta % x min: 32 +/- 4 vs 18 +/- 3 and 179 +/- 7 vs 134 +/- 14 for low and high dose of alpha,beta-MeATP, respectively). Blockade of P2-purinoceptors in the NTS by the specific P2-receptor antagonist suramin abolished responses to 100 pmol alpha,beta-MeATP and microinjections of vehicle did not alter neural nor hemodynamic parameters. We conclude that activation of P2x-purinoceptors in the NTS inhibits sympathetic nerve activity and evokes differential regional sympathetic responses. However, differential sympathoinhibition does not explain differential vascular responses to the activation of P2x-purinoceptors in the NTS.

Activation of purinergic receptor subtypes in the nucleus tractus solitarius elicits specific regional vascular response patterns.

The nucleus tractus solitarius (NTS) is a major integrative site in the brain stem involved in central autonomic control. Several lines of evidence indicate that ATP, acting at P2x purinoceptors, and adenosine, acting at A2a adenosine (P1) purinoceptors, play synchronous roles as transmitter substances in NTS-mediated mechanisms of cardiovascular control. The purpose of this study was to examine regional vascular response patterns elicited by selective activation of purinergic receptor subtypes in the NTS. Adult male rats were anesthetized with a mixture of alpha-chloralose and urethane. Pulsed-Doppler flow probes were placed on the iliac, renal and superior mesenteric arteries via a midline laparotomy for measurement of regional blood flow velocities. The animal was then mounted prone in a stereotaxic unit and the dorsal surface of the medulla was surgically exposed in the region of the obex. Microinjections of alpha, beta-methylene ATP (alpha,beta-MeATP), a selective P2x purinergic receptor agonist, or 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680), a selective A2a adenosine (P1) receptor agonist, were made into the subpostremal region of the NTS via multibarrel glass micropipettes. Both alpha,beta-MeATP (25 and 100 pmoles/rat) and CGS 21680 (2 and 20 pmoles/rat) produced significant dose-related reductions in blood pressure and heart rate. These agonist-elicited depressor response patterns were associated with a pronounced and preferential dilation of the iliac vascular bed. However, alpha, beta-MeATP, but not CGS 21680, also caused significant dilation of the renal and superior mesenteric vascular beds, although lesser in magnitude compared to the iliac bed, whereas the hypotensive actions of CGS 21680 were considerably more prolonged compared to the very rapid and transient effects of alpha,beta-MeATP. These results support the view that extracellular ATP and adenosine via synchronous actions at specific purinergic receptor subtypes in the NTS may be functionally linked as neural signalling substances to selectively coordinate the regulation of regional vasomotor tone.

Daily spontaneous running attenuated the central gain of the arterial baroreflex.

Exercise training attenuates arterial baroreflex function. Mechanisms responsible may include an attenuated aortic baroreceptor reactivity (afferent mechanisms) and/or an attenuated central baroreflex gain. We tested the hypothesis that the aortic baroreceptor reactivity and/or central gain is attenuated by daily spontaneous running (DSR). Eighteen anesthetized Sprague-Dawley rats (11 control and 7 DSR) were tracheotomized and instrumented with femoral venous and right carotid arterial catheters. Electrodes were placed around the left aortic depressor nerve and the lumbar sympathetic trunk. Eight to thirteen weeks of DSR were associated with a 20% increase in heart weight-to-body weight ratio (2.83 +/- 0.04 vs. 3.39 +/- 0.10 g/kg; P < 0.001) and resting bradycardia (413 +/- 6 vs. 384 +/- 10 beats/min; P = 0.01). DSR reduced the central gain of the baroreflex regulation of heart rate (0.210 +/- 0.046 vs. 0.005 +/- 0.021 beats.min-1.%-1; P = 0.004) during decreases in arterial pressure. However, the reactivity of aortic baroreceptor afferents and the central gain of the baroreflex control of lumbar sympathetic nerve activity were not different in control and DSR rats. Thus DSR reduced the central gain of the arterial baroreflex regulation of heart rate without changing the reactivity of aortic baroreceptor afferents. We conclude that afferent mechanisms are not responsible for the training-induced reduction in arterial baroreflex function.

Gender difference in cardiopulmonary reflex inhibition of sympathetic nerve activity.

We tested the hypothesis that reflex responses to mechanical [increase in left atrial pressure (LAP) 0-25 mmHg] and chemical stimulation [left atrial injection of phenylbiguanide (PBG), 0.5-10 mg/kg] of cardiopulmonary receptors are greater in female (n = 9; 335 +/- 9 g) than in male (n = 10; 558 +/- 23 g) age-matched rats. Anesthetized (500 mg/kg urethan and 80 mg/kg alpha-chloralose), tracheotomized, and artificially ventilated (100% oxygen), sinoaortic-denervated animals were instrumented with left atrial, femoral venous, and arterial catheters and a Tygon occluder around the ascending aorta. Reflex inhibition of lumbar sympathetic nerve activity (LSNA) vs. LAP and dose PBG was higher in female rats. A two-way analysis of variance revealed a significant gender effect, males vs. females (P = 0.023), and a significant gender x dose interaction (P < 0.001) for LSNA vs. LAP. There was also a significant gender x dose interaction (P < 0.001) for LSNA vs. PBG. However, there was no influence of gender on the reflex inhibition of mean arterial pressure (P = 0.751) or heart rate (P = 0.561). These responses were associated with a higher left ventricular weight-to-body weight ratio in females (2.14 +/- 0.06 vs. 1.95 +/- 0.07 g/kg, P = 0.039).

Daily exercise improved blood pressure homeostasis of rats subjected to surgical stress.

The effect of daily spontaneous running on blood pressure homeostasis (BPH) was evaluated in 19 male and 13 female control rats and 7 male and 13 female daily spontaneous running rats subjected to surgery and subsequent repetitive hemodynamic disturbances. BPH was operationally defined as the ability to maintain mean arterial pressure above 60 mmHg during the experimental protocol. The length of time the rats maintained BPH was compared across males and females and trained and control groups. Significant sex (P = 0.01) and training (P = 0.05) effects were found. Females maintained homeostasis longer than males and trained longer than controls. Sex effects were not due to differences in the body mass. The mechanisms responsible for the higher resistance to deterioration of homeostasis merit further investigation.

Daily spontaneous running did not alter vagal afferent reactivity.

Exercise training alters the cardiopulmonary baroreflex regulation of the circulation; however, the mechanisms responsible are unknown. One possibility is an enhanced afferent response to cardiopulmonary stimulation. We therefore tested the hypothesis that daily spontaneous running (DSR) would enhance cardiopulmonary vagal afferent responses to mechanical (increase in left atrial pressure, LAP) and chemical (phenyl biguanide, PBG) stimulation. Reactivity of single-fiber cardiopulmonary vagal afferents was evaluated in 16 control and 12 DSR anesthetized Sprague-Dawley rats. Rats were weaned at 3 wk of age and randomly assigned to a control or DSR group. Eight to twelve weeks of DSR was associated with a 27% increase in heart weight-to-body weight ratio (3.27 +/- 0.08 vs. 2.56 +/- 0.05 g/kg, P < 0.001) and resting bradycardia (394 +/- 10 vs. 421 +/- 8 beats/min, P = 0.036). However, DSR did not alter the stimulus-response curves to increases in LAP (frequency of discharge vs. LAP) for either the high-frequency (maximum response, sedentary 59.6 +/- 3.2, DSR 60.1 +/- 5.0 spikes/s) or low-frequency (maximum response, sedentary 20.0 +/- 2.9 DSR 20.6 +/- 3.9 spikes/s) receptors. Dose-response curves to chemical stimulation (spikes/s vs. PBG dose) were also not altered by DSR. Thus DSR did not change vagal afferent reactivity to mechanical or chemical stimulation.