Anandamide content and interaction of endocannabinoid/GABA modulatory effects in the NTS on baroreflex-evoked sympathoinhibition

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure (BP). The presence of cannabinoid CB(1) receptors on fibers in the nucleus tractus solitarius (NTS) suggests that some presynaptic modulation of transmitter release could occur in this region, which receives direct afferent projections from arterial baroreceptors and cardiac mechanoreceptors. This study, therefore, was performed to determine the mechanism(s) of effects of microinjection of an endocannabinoid, arachidonylethanolamide (anandamide, AEA), into the NTS on baroreflex sympathetic nerve responses produced by phenylephrine-induced pressure changes in anesthetized rats. AEA prolonged reflex inhibition of renal sympathetic nerve activity (RSNA), suggesting an increase in baroreflex sensitivity. This effect of AEA was blocked by prior microinjection of SR-141716 to block cannabinoid CB(1) receptors. To determine whether this baroreflex enhancement by AEA involved a GABA(A) mechanism, the baroreflex response to AEA was tested after prior blockade of postsynaptic GABA(A) receptors by bicuculline, which would eliminate any effects due to modulation of GABA activity. After bicuculline, which alone prolonged the baroreflex inhibition of RSNA, AEA shortened the duration of RSNA inhibition, suggesting a possible presynaptic inhibition of glutamate release previously obscured by a more dominant GABA(A) effect. To support a possible physiological role for AEA, AEA concentration in the NTS was measured after a phenylephrine-induced increase in BP. AEA content in the NTS was increased significantly over that in normotensive animals. These results support the hypothesis that AEA content is increased by brief periods of hypertension and suggest that AEA can modulate the baroreflex through activation of CB(1) receptors within the NTS, possibly modulating effectiveness of GABA and/or glutamate neurotransmission.     

WIN-55,212-2 and SR-141716A alter nicotine-induced changes in locomotor activity, but do not alter nicotine-evoked [3H]dopamine release

Nicotine, the main psychoactive ingredient in tobacco, plays a key role in the development of cigarette smoking addiction. The endocannabinoid system has been demonstrated to have an important role in the motivational and reinforcing effects of drugs. The present study used behavioral and neurochemical techniques to study the interaction of cannabinoid receptors and nicotine pharmacology. In a locomotor activity experiment in rats, the CB(1)/CB(2) cannabinoid receptor agonist WIN-55,212-2 (0.28-2.8 mg/kg) attenuated nicotine (0.4 mg/kg)-induced hyperactivity, but did not alter nicotine (1.0 mg/kg)-induced hypoactivity. In contrast, the selective CB(1) cannabinoid receptor antagonist SR-141716A (1.0 mg/kg) diminished nicotine-induced hypoactivity, but did not alter nicotine-induced hyperactivity. In a neurochemical experiment, rat striatal slices preloaded with [(3)H]dopamine were superfused with WIN-55,212-2 or SR-141716A. A high concentration (100 microM) of WIN-55,212-2 evoked [(3)H]overflow, but this effect was not blocked by the cannabinoid receptor antagonist AM-251. SR-141716A did not evoke [(3)H]overflow, and neither WIN-55,212-2 nor SR-141716A altered nicotine-evoked [(3)H]overflow. Overall, these results indicate a behavioral interaction between cannabinoid receptors and nicotine pharmacology. Likely, WIN-55,212-2 and SR-141716A block nicotine-induced changes in behavior through an indirect mechanism, such as alteration in endocannabinoid regulation of motor circuits, rather than directly through blockade of nicotinic acetylcholine receptors.     

Analogs of SR-141716A (Rimonabant) alter d-amphetamine-evoked [3H] dopamine overflow from preloaded striatal slices and amphetamine-induced hyperactivity

The CB(1) cannabinoid receptor antagonist SR-141716A (Rimonabant) markedly diminishes the behavioral effects of opiates and nicotine and has been an important tool to ascertain the role of cannabinoid receptors in drug addiction. The present goal was to determine the less-explored interaction of SR-141716A and d-amphetamine in neurochemical and behavioral assays. Additionally, the effect of the substituents and substitution patterns on the phenyl ring located at the 5 position of SR-141716A (4-chlorophenyl), and of the CB(1)/CB(2) cannabinoid receptor agonist WIN-55,212-2, was determined. SR-141716A, AM-251 (4-iodophenyl) and NIDA-41020 (4-methoxyphenyl) did not alter amphetamine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine. MRI-8273-30-1 (4-fluorophenyl; 0.1-10 microM) attenuated amphetamine (3 microM)-evoked [(3)H]overflow, and MRI-8273-59 (3,4-dichlorphenyl; 0.01-10 microM) augmented amphetamine (0.3-3 microM)-evoked [(3)H]overflow. WIN-55,212-2 was without effect. In a locomotor activity experiment, SR-141716A and MRI-8273-30-1 did not alter amphetamine-induced hyperactivity. However, MRI-8273-59 (1-3 mg/kg) dose-dependently attenuated amphetamine (1 mg/kg)-induced hyperactivity. The present results suggest that SR-141716A is less efficacious to alter amphetamine effects than its reported efficacy to diminish the effects of opiates and nicotine. Modification of the 5-phenyl position of SR-141716A affords compounds that do interact with amphetamine in vitro and in vivo.     

U-46619 but not serotonin increases endocannabinoid content in middle cerebral artery: evidence for functional relevance

Cerebral vascular smooth muscle cells express the CB(1) cannabinoid receptor, and CB(1) receptor agonists produce vasodilation of cerebral arteries. The purpose of this study was to determine whether vasoconstriction of rat middle cerebral artery (MCA) results in the local formation of endocannabinoids (eCBs), which, via activation of CB(1) receptors, oppose the vasoconstriction in a feedback manner. The thromboxane A(2) (TXA(2)) mimetic U-46619 significantly increased N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol (2-AG) content of isolated MCA, whereas 5-hydroxytrypamine (5-HT) decreased AEA and 2-AG content. If eCBs play a feedback role in the regulation of MCA tone, then CB(1) receptor antagonists should enhance the constriction of MCA produced by U-46619 but not 5-HT. U-46619 caused concentration-dependent constrictions of endothelium-denuded MCA. Two CB(1) receptor antagonists SR-141716 and AM-251 decreased the EC(50) value for U-46619 to constrict endothelium-denuded MCA without affecting the maximal effect. A low concentration of CB(1) receptor agonist Win-55212-2 (30 nM) produced vasodilation of MCAs constricted with low but not saturating concentrations of U-46619. SR-141716 had no effect on the 5-HT concentration-contraction relationship. These data suggest that TXA(2) receptor activation increases MCA eCB content, which, via activation of CB(1) receptors, reduces the constriction produced by moderate concentrations of the TXA(2) agonist. Although 5-HT-induced vasoconstriction is reduced by exogenous CB(1) receptor agonist, activation of 5-HT receptors does not increase eCB content. These results suggest that MCA production of eCBs is not regulated by constriction per se but likely via a signaling pathway that is specific for TXA(2) receptors and not 5-HT receptors.     

Independent modification of baroreceptor and exercise pressor reflex function by nitric oxide in nucleus tractus solitarius

It has been suggested that nitric oxide (NO) is a key modulator of both baroreceptor and exercise pressor reflex afferent signals processed within the nucleus tractus solitarius (NTS). However, studies investigating the independent effects of NO within the NTS on the function of each reflex have produced inconsistent results. To address these concerns, the effects of microdialyzing 10 mM L-arginine, an NO precursor, and 20 mM N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, into the NTS on baroreceptor and exercise pressor reflex function were examined in 17 anesthetized cats. Arterial baroreflex regulation of heart rate was quantified using vasoactive drugs to induce acute changes in mean arterial pressure (MAP). To activate the exercise pressor reflex, static hindlimb contractions were induced by electrical stimulation of spinal ventral roots. To isolate the exercise pressor reflex, contractions were repeated after barodenervation. The gain coefficient of the arterial cardiac baroreflex was significantly different from control (-0.24 +/- 0.04 beats.min(-1).mmHg(-1)) after the dialysis of L-arginine (-0.18 +/- 0.02 beats.min(-1).mmHg(-1)) and L-NAME (-0.29 +/- 0.02 beats.min(-1).mmHg(-1)). In barodenervated animals, the peak MAP response to activation of the exercise pressor reflex (change in MAP from baseline, 39 +/- 7 mmHg) was significantly attenuated by the dialysis of L-arginine (change in MAP from baseline, 29 +/- 6 mmHg). The results demonstrate that NO within the NTS can independently modulate both the arterial cardiac baroreflex and the exercise pressor reflex. Collectively, these findings provide a neuroanatomical and chemical basis for the regulation of baroreflex and exercise pressor reflex function within the central nervous system.     

Medial prefrontal cortex endocannabinoid system modulates baroreflex activity through CB(1) receptors

Neural reflex mechanisms, such as the baroreflex, are involved in the regulation of cardiovascular system activity. Previous results from our group (Resstel LB, Correa FM. Medial prefrontal cortex NMDA receptors and nitric oxide modulate the parasympathetic component of the baroreflex. Eur J Neurosci 23: 481-488, 2006) have shown that glutamatergic synapses in the ventral portion of the medial prefrontal cortex (vMPFC) modulate baroreflex activity. Moreover, glutamatergic neurotransmission in the vMPFC can be modulated by the endocannabinoids system (eCBs), particularly the endocannabinoid anandamide, through presynaptic CB(1) receptor activation. Therefore, in the present study, we investigated eCBs receptors that are present in the vMPFC, and more specifically whether CB(1) receptors modulate baroreflex activity. We found that bilateral microinjection of the CB(1) receptor antagonist AM251 (100 or 300 pmol/200 nl) into the vMPFC increased baroreflex activity in unanesthetized rats. Moreover, bilateral microinjection of either the anandamide transporter inhibitor AM404 (100 pmol/200 nl) or the inhibitor of the enzyme fatty acid amide hydrolase that degrades anandamide, URB597 (100 pmol/200 nl), into the MPFC decreased baroreflex activity. Finally, pretreatment of the vMPFC with an ineffective dose of AM251 (10 pmol/200 nl) was able to block baroreflex effects of both AM404 and URB597. Taken together, our results support the view that the eCBs in the vMPFC is involved in the modulation of baroreflex activity through the activation of CB(1) receptors, which modulate local glutamate release.     

Differential baroreflex control of sympathetic drive by angiotensin II in the nucleus tractus solitarii

Microinjection of angiotensin II into the nucleus tractus solitarii attenuates the baroreceptor reflex-mediated bradycardia by inhibiting both vagal and cardiac sympathetic components. However, it is not known whether the baroreflex modulation of other sympathetic outputs (i.e., noncardiac) also are inhibited by exogenous angiotensin II (ANG II) in nucleus tractus solitarii (NTS). In this study, we determined whether there was a difference in the baroreflex sensitivity of sympathetic outflows at the thoracic and lumbar levels of the sympathetic chain following exogenous delivery of ANG II into the NTS. Experiments were performed in two types of in situ arterially perfused decerebrate rat preparations. Sympathetic nerve activity was recorded from the inferior cardiac nerve, the midthoracic sympathetic chain, or the lower thoracic-lumbar sympathetic chain. Increases in perfusion pressure produced a reflex bradycardia and sympathoinhibition. Microinjection of ANG II (500 fmol) into the NTS attenuated the reflex bradycardia (57% attenuation, P < 0.01) and sympathoinhibition of both the inferior cardiac nerve (26% attenuation, P < 0.05) and midthoracic sympathetic chain (37% attenuation, P < 0.05) but not the lower thoracic-lumbar chain (P = 0.56). We conclude that ANG II in the nucleus tractus solitarii selectively inhibits baroreflex responses in specific sympathetic outflows, possibly dependent on the target organ innervated.     

Serotonin2 Receptors in the Nucleus Tractus Solitarius: Characterization and Role in the Baroreceptor Reflex Arc

1. There is a general agreement concerning the key role of the baroreceptor reflex in blood pressure homeostasis. It is also well accepted that baroreceptor afferent messages are first integrated within the nucleus tractus solitarius (NTS) and that an excitatory amino acid, probably glutamate, is the principal neurotransmitter of corresponding afferents fibers. However, important points concerning the processing of baroreceptor messages within the NTS remain to be clarified, in particular the possible modulatory role of other neuroactive substances at this particular level in the medulla oblongata.2. In this context, the present review focuses on serotonin, and the possible facilitatory influence of NTS serotonergic afferents and receptors on the baroreceptor reflex arc. Relevant pharmacological, electrophysiological, immunohistochemical, and biochemical data, are presented and discussed. They can be summarized as follows.3. The selective destruction of the nodose ganglion-NTS serotonergic pathway produces a long-term increase in blood pressure variability, similar to that caused by baroreceptor denervation.4. Microinjection of picomolar doses of 5-HT into the NTS elicits the typical responses of baroreceptor activation.5. The cardiovascular effects elicited by local microinjections of specific agonists and antagonists into the NTS of intact rats and of animals that underwent nodose ganglionectomy indicate that the baroreceptor-like effects of locally administered 5-HT are mediated by the activation of postsynaptic 5-HT₂ receptors.6. The medullary pathways which mediate NTS 5-HT₂ receptor-evoked responses are similar to those involved in the baroreceptor reflex arc.7. Pharmacological and electrophysiological studies suggest that the cardiovascular effects of intra-NTS 5-HT involve the 5-HT_2A receptor subtype expressed by NTS barosensitive neurons that receive polysynaptic vagal afferents.8. Intra-NTS microinjection of a subthreshold dose of DOI, a 5-HT₂ receptor agonist, which, on its own, does not produce any cardiovascular changes, significantly enhances the bradycardiac component of the baroreflex.9. Altogether, the data summarized above show that, in the NTS, 5-HT acting at 5-HT_2A receptors exerts a facilitatory influence on the baroreceptor reflex, especially on the cardiac component of this reflex.10. Convergent pharmacological and electrophysiological data indicate that, in the NTS, functional interactions between NMDA- and 5-HT_2A-receptors coexpressed by the same neurons probably underlie the facilitatory influence of 5-HT upon the baroreceptor reflex.11. Under physiological conditions, the 5-HT_2A receptor-mediated facilitatory modulation of the cardiovagal component of the baroreflex might be triggered by 5-HT released from nodose ganglion-NTS serotoninergic afferent neurons and/or for serotoninergic projections originating in raphe nuclei. The latter possibility might notably occur during recovery after physical exercise and/or during the freezing reaction in stressed animals.     

Stimulation of NTS A1 adenosine receptors differentially resets baroreflex control of regional sympathetic outputs

Previously we showed that pressor and differential regional sympathoexcitatory responses (adrenal > renal >/= lumbar) evoked by stimulation of A(1) adenosine receptors located in the nucleus of the solitary tract (NTS) were attenuated/abolished by baroreceptor denervation or blockade of glutamatergic transmission in the NTS, suggesting A(1) receptor-elicited inhibition of glutamatergic transmission in baroreflex pathways. Therefore we tested the hypothesis that stimulation of NTS A(1) adenosine receptors differentially inhibits/resets baroreflex responses of preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activity. In urethane-chloralose-anesthetized male Sprague-Dawley rats (n = 65) we compared baroreflex-response curves (iv nitroprusside and phenylephrine) evoked before and after bilateral microinjections into the NTS of A(1) adenosine receptor agonist (N(6)-cyclopentyladenosine, CPA; 0.033-330 pmol/50 nl). CPA evoked typical dose-dependent pressor and differential sympathoexcitatory responses and similarly shifted baroreflex curves for pre-ASNA, RSNA, and LSNA toward higher mean arterial pressure (MAP) in a dose-dependent manner; the maximal shifts were 52.6 +/- 2.8, 48.0 +/- 3.6, and 56.8 +/- 6.7 mmHg for pre-ASNA, RSNA, and LSNA, respectively. These shifts were not a result of simple baroreceptor resetting because they were two to three times greater than respective increases in baseline MAP evoked by CPA. Baroreflex curves for pre-ASNA were additionally shifted upward: the maximal increases of upper and lower plateaus were 41.8 +/- 16.4% and 45.3 +/- 8.7%, respectively. Maximal gain (%/mmHg) measured before vs. after CPA increased for pre-ASNA (3.0 +/- 0.6 vs. 4.9 +/- 1.3), decreased for RSNA (4.1 +/- 0.6 vs. 2.3 +/- 0.3), and remained unaltered for LSNA (2.1 +/- 0.2 vs. 2.0 +/- 0.1). Vehicle control did not alter the baroreflex curves. We conclude that the activation of NTS A(1) adenosine receptors differentially inhibits/resets baroreflex control of regional sympathetic outputs.     

Cannabinoid antagonist SR-141716 inhibits endotoxic hypotension by a cardiac mechanism not involving CB1 or CB2 receptors

Endocannabinoids and CB1 receptors have been implicated in endotoxin (LPS)-induced hypotension: LPS stimulates the synthesis of anandamide in macrophages, and the CB1 antagonist SR-141716 inhibits the hypotension induced by treatment of rats with LPS or LPS-treated macrophages. Recent evidence indicates the existence of cannabinoid receptors distinct from CB1 or CB2 that are inhibited by SR-141716 but not by other CB1 antagonists such as AM251. In pentobarbital-anesthetized rats, intravenous injection of 10 mg/kg LPS elicited hypotension associated with profound decreases in cardiac contractility, moderate tachycardia, and an increase in lower body vascular resistance. Pretreatment with 3 mg/kg SR-141716 prevented the hypotension and decrease in cardiac contractility, slightly attenuated the increase in peripheral resistance, and had no effect on the tachycardia caused by LPS, whereas pretreatment with 3 mg/kg AM251 did not affect any of these responses. SR-141716 also elicited an acute reversal of the hypotension and decreased contractility when administered after the response to LPS had fully developed. The LPS-induced hypotension and its inhibition by SR-141716 were similar in pentobarbital-anesthetized wild-type, CB1(-/-), and CB1(-/-)/CB2(-/-) mice. We conclude that SR-141716 inhibits the acute hemodynamic effects of LPS by interacting with a cardiac receptor distinct from CB1 or CB2 that mediates negative inotropy and may be activated by anandamide or a related endocannabinoid released during endotoxemia.     

Delta9-tetrahydrocannabinol selectively acts on CB1 receptors in specific regions of dorsal vagal complex to inhibit emesis in ferrets

The aim of this study was to investigate the efficacy, receptor specificity, and site of action of Delta9-tetrahydrocannabinol (THC) as an antiemetic in the ferret. THC (0.05-1 mg/kg ip) dose-dependently inhibited the emetic actions of cisplatin. The ED50 for retching was approximately 0.1 mg/kg and for vomiting was 0.05 mg/kg. A specific cannabinoid (CB)1 receptor antagonist SR-141716A (5 mg/kg ip) reversed the effect of THC, whereas the CB2 receptor antagonist SR-144528 (5 mg/kg ip) was ineffective. THC applied to the surface of the brain stem was sufficient to inhibit emesis induced by intragastric hypertonic saline. The site of action of THC in the brain stem was further assessed using Fos immunohistochemistry. Fos expression induced by cisplatin in the dorsal motor nucleus of the vagus (DMNX) and the medial subnucleus of the nucleus of the solitary tract (NTS), but not other subnuclei of the NTS, was significantly reduced by THC rostral to obex. At the level of the obex, THC reduced Fos expression in the area postrema and the dorsal subnucleus of the NTS. The highest density of CB1 receptor immunoreactivity was found in the DMNX and the medial subnucleus of the NTS. Lower densities were observed in the area postrema and dorsal subnucleus of the NTS. Caudal to obex, there was moderate density of staining in the commissural subnucleus of the NTS. These results show that THC selectively acts at CB1 receptors to reduce neuronal activation in response to emetic stimuli in specific regions of the dorsal vagal complex.     

AT1 receptors in the nucleus tractus solitarii mediate the interaction between the baroreflex and the cardiac sympathetic afferent reflex in anesthetized rats

The cardiac "sympathetic afferent" reflex (CSAR) has been reported to increase sympathetic outflow and depress baroreflex function via a central angiotensin II (ANG II) mechanism. In the present study, we examined the role of ANG II type 1 (AT(1)) receptors in the nucleus tractus solitarii (NTS) in mediating the interaction between the CSAR and the baroreflex in anesthetized rats. We examined the effects of bilateral microinjection of AT(1) receptor antagonist losartan (100 pmol) into the NTS on baroreflex control of renal sympathetic nerve activity (RSNA) before and after CSAR activation by epicardial application of capsaicin (0.4 microg). Using single-unit extracellular recording, we further examined the effects of CSAR activation on the barosensitivity of barosensitive NTS neurons and the effects of intravenous losartan (2 mg/kg) on CSAR-induced changes in activity of NTS barosensitive neurons. Bilateral NTS microinjection of losartan significantly attenuated the increases in arterial pressure, heart rate, and RSNA evoked by capsaicin but also markedly (P < 0.01) reversed the CSAR-induced blunted baroreflex control of RSNA (Gain(max) from 1.65 +/- 0.10 to 2.22 +/- 0.11%/mmHg). In 17 of 24 (70.8%) NTS barosensitive neurons, CSAR activation significantly (P < 0.01) inhibited the baseline neuronal activity and attenuated the neuronal barosensitivity. In 11 NTS barosensitive neurons, intravenous losartan effectively (P < 0.01) normalized the decreased neuronal barosensitivity induced by CSAR activation. In conclusion, blockade of NTS AT(1) receptors improved the blunted baroreflex during CSAR activation, suggesting that the NTS plays an important role in processing the interaction between the baroreflex and the CSAR via an AT(1) receptor-dependent mechanism.     

Modulation of the baroreceptor reflex by the dorsomedial hypothalamic nucleus and perifornical area

Neurons within the dorsomedial hypothalamic nucleus (DMH) and perifornical area (PeF), which lie within the classic hypothalamic defense area, subserve the cardiovascular response to psychological stress. Previous studies have shown that electrical stimulation of the hypothalamic defense area causes inhibition of the cardiac and (in some cases) sympathetic components of the baroreceptor reflex. In contrast, naturally evoked psychological stress does not appear to be associated with such inhibition. In this study, we tested the effect of specific activation of neurons within the DMH and PeF on the baroreflex control of renal sympathetic nerve activity and heart rate in urethane-anesthetized rats. Microinjection of bicuculline (a GABA(A) receptor antagonist) into the DMH caused dose-dependent increases in heart rate and renal sympathetic activity, shifted the baroreflex control of both variables to higher levels (i.e., increased the upper and lower plateaus of the baroreflex function curves, and increased the threshold, midpoint, and saturation levels of mean arterial pressure). The maximum gain of the sympathetic component of the baroreflex was also increased, while that of the cardiac component was not significantly changed. Increases in the midpoint were very similar in magnitude to the evoked increases in baseline mean arterial pressure. Microinjection of bicuculline into the PeF evoked very similar effects. The results indicate that disinhibition of neurons in the DMH/PeF region not only increases sympathetic vasomotor activity and heart rate but also resets the baroreceptor reflex such that it remains effective, without any decrease in sensitivity, over a higher operating range of arterial pressure.     

Effects of CB(1) cannabinoid receptor activation on cerebellar granule cell nitric oxide synthase activity.

Cerebellar granule cells (CGCs) express the CB(1) subtype of cannabinoid receptor. CB(1) receptor agonists Win 55212-2, CP55940 and HU210 inhibit KCl-induced activation of nitric oxide synthase (NOS) in CGCs. Win 55212-2 has no effect on either basal NOS activity or on activation by N-methyl-D-aspartate and its effect is abolished by pre-treatment of the cells with pertussis toxin. The CB(1) receptor antagonist/inverse agonist SR141716A both reverses the effects of Win 55212-2 and produces an increase in NOS activity that is additive with KCl. These results support the hypothesis that activation of the CB(1) receptor in CGCs results in a decreased influx of calcium in response to membrane depolarization, resulting in a decreased activation of neuronal NOS.     

The role of enzymatic processing in the biological actions of substance P

There is considerable evidence that substance P (SP) is a neurotransmitter in the CNS. Current findings suggest that the effects of synaptically released SP are terminated by enzymatic breakdown, primarily by endopeptidase 3.4.24.11 (endo 24.11). The products of cleavage by endo 24.11 include the amino-terminal fragment SP(1-7). Evidence suggests that SP is involved in mediating baroreceptor reflex activity in the nucleus of the solitary tract (NTS). Microinjection of SP into the NTS lowered blood pressure and heart rate. Microinjection of SP(1-7) into the NTS reproduced the effects of SP on both heart rate and blood pressure. Intra-NTS injection of phosphoramidon, an inhibitor of endo 24.11 activity, completely blocked the effects of a subsequent injection of SP. This blocking effect of phosphoramidon was unaltered by pretreatment with the opiate inhibitor naloxone. In contrast, phosphoramidon failed to block the depressor and bradycardic effects of SP(1-7). The implications of these findings regarding the role of endo 24.11 in the metabolism of SP are discussed.     

Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons.

Neuronal cannabinoid receptors (CB(1)) are coupled to inhibition of voltage-sensitive Ca(2+) channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB(1) receptors and VSCCs in cerebellar granule neurons (CGN). Ca(2+) transients were evoked by KCl-induced depolarization and imaged using fura-2. The CB(1) receptor agonists CP55940, Win 55212-2 and N-arachidonylethanolamine (anandamide) produced concentration-related decreases in peak amplitude of the Ca(2+) response and total Ca(2+) influx. Pre-treatment of CGN with pertussis toxin abolished agonist-mediated inhibition. The inhibitory effect of Win 55212-2 on Ca(2+) influx was additive with inhibition produced by omega-agatoxin IVA and nifedipine but not with omega-conotoxin GVIA, indicating that N-type VSCCs are the primary effector. Paradoxically, the CB(1) receptor antagonist, SR141716, also inhibited KCl-induced Ca(2+) influx into CGN in a concentration-related manner. SR141716 inhibition was pertussis toxin-insensitive and was not additive with the inhibition produced by Win 55212-2. Confocal imaging of CGN in primary culture demonstrate a high density of CB(1) receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB(1) receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca(2+) influx through N-type VSCC.     

Postexercise hypotension in conscious SHR is attenuated by blockade of substance P receptors in NTS

In hypertensive subjects, a single bout of dynamic exercise results in an immediate lowering of blood pressure back toward normal. This postexercise hypotension (PEH) also occurs in the spontaneously hypertensive rat (SHR). In both humans and SHRs, PEH features a decrease in sympathetic nerve discharge, suggesting the involvement of central nervous system pathways. Given that substance P is released in the nucleus tractus solitarius (NTS) by activation of baroreceptor and skeletal muscle afferent fibers during muscle contraction, we hypothesized that substance P acting at neurokinin-1 (NK-1) receptors in the NTS might contribute to PEH. We tested the hypothesis by determining, in conscious SHRs, whether NTS microinjections of the NK-1 receptor antagonist SR-140333 before exercise attenuated PEH. The antagonist, in a dose (60 pmol) that blocked substance P- and spared D,L-homocysteic acid-induced depressor responses, significantly attenuated the PEH by 37%, whereas it had no effect on blood pressure during exercise. Vehicle microinjection had no effect. The antagonist also had no effect on heart rate responses during both exercise and the PEH period. The data suggest that a substance P (NK-1) receptor mechanism in the NTS contributes to PEH.     

Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function

Following inducible expression in HEK293 cells, the human orexin-1 receptor was targeted to the cell surface but became internalized following exposure to the peptide agonist orexin A. By contrast, constitutive expression of the human cannabinoid CB1 receptor resulted in a predominantly punctate, intracellular distribution pattern consistent with spontaneous, agonist-independent internalization. Expression of the orexin-1 receptor in the presence of the CB1 receptor resulted in both receptors displaying the spontaneous internalization phenotype. Single cell fluorescence resonance energy transfer imaging indicated the two receptors were present as heterodimers/oligomers in intracellular vesicles. Addition of the CB1 receptor antagonist SR-141716A to cells expressing only the CB1 receptor resulted in re-localization of the receptor to the cell surface. Although SR-141716A has no significant affinity for the orexin-1 receptor, in cells co-expressing the CB1 receptor, the orexin-1 receptor was also re-localized to the cell surface by treatment with SR-141716A. Treatment of cells co-expressing the orexin-1 and CB1 receptors with the orexin-1 receptor antagonist SB-674042 also resulted in re-localization of both receptors to the cell surface. Treatment with SR-141716A resulted in decreased potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. Treatment with SB-674042 also reduced the potency of a CB1 receptor agonist to phosphorylate ERK1/2 only when the two receptors were co-expressed. These studies introduce an entirely novel pharmacological paradigm, whereby ligands modulate the function of receptors for which they have no significant inherent affinity by acting as regulators of receptor heterodimers.     

Activation of NTS A1 adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex

Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 μg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes integrated in the NTS.     

5-HT-mediated inhibition of cardiovagal baroreceptor reflex response during defense reaction in the rat

Previous studies showed that the cardiac response of the baroreceptor reflex (bradycardia) is inhibited during the defense reaction evoked by direct electrical or chemical stimulation of the periaqueductal gray (dPAG) in the rat. Whether central serotonin and nucleus tractus solitarius (NTS) serotonin(3) (5-HT(3)) receptors might participate in this inhibition was investigated in urethane-anesthetized and atenolol-pretreated rats. Our results showed that both electrical and chemical stimulation of the dPAG produced a drastic reduction of the cardiovagal component of the baroreceptor reflex triggered by either intravenous administration of phenylephrine or aortic nerve stimulation. This inhibitory effect of dPAG stimulation on the baroreflex bradycardia was not observed in rats that had been pretreated with p-chlorophenylalanine (300 mg/kg ip daily for 3 days) to inhibit serotonin synthesis. Subsequent 5-hydroxytryptophan administration (60 mg/kg ip), which was used to restore serotonin synthesis, allowed the inhibitory effect of dPAG stimulation on both aortic and phenylephrine-induced cardiac reflex responses to be recovered in p-chlorophenylalanine-pretreated rats. On the other hand, in nonpretreated rats, the inhibitory effect of dPAG stimulation on the cardiac baroreflex response could be markedly reduced by prior intra-NTS microinjection of granisetron, a 5-HT(3) receptor antagonist, or bicuculline, a GABA(A) receptor antagonist. These results show that serotonin plays a key role in the dPAG stimulation-induced inhibition of the cardiovagal baroreceptor reflex response. Moreover, they support the idea that 5-HT(3) and GABA(A) receptors in the NTS contribute downstream to the inhibition of the baroreflex response caused by dPAG stimulation.