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Am J Physiol Regul Integr Comp Physiol 285: R1496-R1503, 2003. First published August 21, 2003; doi:10.1152/ajpregu.00183.2003
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NEUROHUMORAL CONTROL OF CIRCULATION AND HYPERTENSION

Cocaine- and amphetamine-regulated transcript peptide attenuates phenylephrine-induced bradycardia in anesthetized rats

Phouangmala Scruggs, Siok L. Dun, and Nae J. Dun

Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614

Submitted 7 April 2003 ; accepted in final form 11 August 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The present study was undertaken to investigate the origin of cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactive (irCART) fibers observed in the nucleus of the solitary tract (NTS) and assess the role of CART peptide on phenylephrine (PE)-induced baroreflex. Immunohistochemical and retrograde tract-tracing studies showed that some of the irCART fibers observed in the NTS may have their cell bodies in the nodose ganglia. In urethane-anesthetized rats, intracisternal or bilateral intra-NTS microinjection of the CART peptide fragment 55-102 (0.1-3 nmol), referred to herein as CARTp, consistently and dose dependently attenuated PE-induced bradycardia. CARTp, in the doses used here, caused no significant changes of resting blood pressure or heart rate. Bilateral intra-NTS injections of CART antibody (1:500) potentiated PE-induced bradycardia. Injections of saline, normal rabbit serum, or concomitant injection of CARTp and CART antiserum into the NTS caused no significant changes of PE-induced baroreflex. The result suggests that endogenously released CARTp from primary afferents or exogenously administered CARTp modulates PE-induced baroreflex.

baroreceptor reflex; nodose ganglia; nucleus tractus solitarius; phenylephrine

BAROREFLEX, which serves a crucial role in homeostasis and maintenance of arterial pressure and heart rate (HR), is the first line of defense against changes in blood pressure (BP). When baroreceptors are stretched, signals are sent to the central nervous system via the vagus (X) or glossopharyngeal (IX) nerves (2). Afferents from both nodose and petrosal ganglia enter the medulla oblongata dorsolaterally and project to second-order neurons in the nucleus tractus solitarius (NTS) where the signals are integrated and processed. Glutamate or a closely related amino acid is thought to be the principal neurotransmitter released on baroreflex stimulation (23). In addition, neuropeptides, including calcitonin gene-related peptide, cholecystokinin, enkephalin, galanin, neuropeptide Y, neurokinin A, neurokinin B, somatostatin, substance P, and vasoactive intestinal polypeptide, have been localized to neurons of the nodose and petrosal ganglia (25). More recent studies have shown that oxytocin is detectable in the nodose ganglion and may serve as a neurotransmitter to the vasomotor center (12).

Cocaine- and amphetamine-regulated transcript (CART) was first identified by PCR-differential display in the striatum after acute administration of cocaine and amphetamine to the rats (7). The CART cDNA encodes a peptide of either 129 or 116 amino acid residues in length, with a predicted leader sequence of 27 amino acids, resulting in two mature peptides of either 102 (long form) or 89 (short form) amino acid residues (7). The rat expresses both the long and short forms, whereas the human expresses only the short form (7). The preproCART contains several pairs of basic amino acids at positions 28-29, 48-49, 53-54, 60-61, and 77-78, suggesting that the peptide is processed into several smaller fragments (7). In Western blot studies, six different CART peptides varying from 4 to 14 kDa have been detected in rat tissues; these include CART55-102 and CART62-102 of the long form (16). Functional studies indicate that CART55-102 is biologically active, as it potently inhibits feeding when injected intracerebroventricularly to the rats (1) or sensitizes thermal pain-induced hindpaw withdrawal (19). Furthermore, immunohistochemical studies show that antiserum directed against the CART55-102 robustly labeled neurons in the hypothalamus, brain stem, spinal cord, pituitary, and autonomic nerves, supporting the contention that CART55-102 is present endogenously (8-10). For this reason, CART55-102, which is referred to herein as CARTp, was used in this study.

Immunohistochemical and in situ hybridization studies reveal an extensive distribution of CART peptide in the central and peripheral nervous system of the rat (4, 14). A particularly dense plexus of CART-immunoreactive (irCART) fibers is noted in the NTS (9, 10, 14). The present study was undertaken to evaluate the hypothesis that CARTp, which may be released from vagal afferents, modulates baroreflex in anesthetized rats. Immunohistochemistry and tract-tracing experiments were conducted to identify the location of cell bodies that may give rise to irCART fibers observed in the NTS.


   METHODS AND MATERIALS
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 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animal protocols. A breeding colony of Sprague-Dawley rats (Harlan, Indianapolis, IN) was established at the Division of Laboratory Animal Resources, East Tennessee State University. Animals were maintained at 22 ± 1°C with an alternating 12:12-h light-dark cycle. Food and water were available ad libitum. Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats, weighing 275-350 g, were anesthetized with urethane (1.2 g/kg ip). The depth of anesthesia was monitored by observing responses to pinching the tail. Rectal temperature was maintained at 37 ± 1°C with the use of a thermoblanket. The right femoral artery was cannulated with a polyethylene tubing (Intramedic PE-50) and connected to a pressure transducer with its output to a Gould pen recorder. The BP signal was used to trigger a Biotach amplifier (Gould ECG/BiTac') for HR recording. The right femoral vein was cannulated with a polyethylene tubing for intravenous injection of phenylephrine (PE; 7-10 µg/kg). The depth of anesthesia was subsequently maintained by intravenous administration of urethane after cannulation of the femoral vein. Urethane (30-60 mg/kg) was given as needed, usually every 1.5-2 h. All intravenous injections were conducted via a 1 ml tuberculin syringe mounted on an infusion pump (KD Scientific, New Hope, PA) at a rate of 3.5 µl/s. Data were analyzed statistically using either the Kruskal-Wallis statistics followed by Dunn's post test or the Student's t-test, with P < 0.05 considered statistically significant. Results are expressed as means ± SE.

Intracisternal injections. Rats were placed in a stereotaxic frame (David Kopf, Tujunga, CA) with the bite bar 13 mm below the interaural line. An incision was made at the top of the head to expose the dorsal neck muscles. The atlanto-occipital membrane and occipital bone were exposed by using a spatula to scrape away the dorsal neck muscles. A micro-injection needle, connected to a 10-µl syringe (Hamilton, Reno, NV) via polyethylene tubing (Intramedic PE-10), was attached to the stereotaxic apparatus. The {alpha}-adrenergic agonist PE was injected intravenously at least twice, 10 min apart, to elicit a control baroreflex response before intracisternal injection of CARTp or CART antiserum was to begin. Intracisternal injections of CARTp were given at a fixed volume of 4 µl at the concentrations of 0.1, 0.3, 1, and 3 nmol over a period of 5 s. Subsequent injections of PE were administered every 10 min for 70 min to monitor the effects of CARTp on PE-induced baroreceptor reflex. Each rat received one dose of CARTp.

NTS microinjections. The atlanto-occipital membrane and occipital bone were exposed as previously indicated. A drill was used to remove part of the occipital bone. The dura and atlanto-occipital membranes were then removed with iris scissors to expose the medulla and cerebellum. Using an automatic nanoliter injector (Drummond Scientific, Broomall, PA), injections were made to the NTS at the following coordinates: 0.5 mm rostral to the calamus scriptorius, 0.5 mm lateral to the midline, and 0.5 mm deep from the dorsal medullary surface. Injections of L-glutamate (0.5 mM) in a volume of 50 nl were made to functionally identify the NTS coordinates before CARTp or CART antiserum injection. The experiment continued only if glutamate induced a depressor and bradycardic effect (6, 24). Rats were injected either with CARTp (n = 20), CART antiserum (n = 5), normal rabbit serum (NRS) (n = 5), saline (n = 5), or CARTp with CART antiserum (n = 5). All injections were made at a volume of 50 nl to each side of NTS. Before NTS injections were made, PE was administered at least twice to elicit a control baroreflex response. Subsequent injections of PE were administered every 10 min for 70 min.

Injection sites were marked by injecting diluted India ink (50 nl) at the end of each experiment. Brains were removed, fixed in 4% paraformaldehyde, and immersed in 30% sucrose overnight. Brain stems were cut into 50-µm sections, mounted on glass slides, and stained with Cresyl violet. Injection sites were then viewed under a microscope and verified with a standard atlas (21).

Immunohistochemical studies. Rats were anesthetized with urethane (1.7 g/kg ip) and intracardially perfused with PBS followed by 4% paraformaldehyde in PBS. Brain stems and nodose ganglia were removed and postfixed in the same fixative for 2 h followed by immersion into 30% sucrose in PBS overnight. Brain stems were embedded in agar and then cut into 50-µm coronal sections using a vibratome. Nodose ganglia were embedded and frozen in Tissue Tek OCT compound (Pella, Redding, CA) and cut into 50-µm sections with a cryostat (International Equipment, Needham, MA). Tissue, which was collected as free floating sections, was thoroughly washed with PBS, blocked with normal goat serum (1:10) for 2 h, and followed by incubation with CART antiserum (1:1,500) for 2 days. Normal goat serum and CART antiserum were diluted with 0.4% Triton X-100 and 0.5% BSA in PBS. Tissues were washed with PBS before incubation with biotinylated anti-rabbit IgG (1:50) for 2 h and washed again before incubation with avidin fluorescein isothiocyanate (FITC) (1:50) for 3 h. Tissues were mounted on gelatin-coated glass slides, and Citifluor mountant media (Pella) was applied before coverslipped.

Retrograde tracing studies. Rats were anesthetized with intraperitoneal injection of urethane (0.6 g/kg), ketamine (50 mg/kg), and xylazine (12 mg/kg). Ketamine was injected first, followed by xylazine and then urethane. Rats were then placed in a stereotaxic apparatus where the dorsal medulla was exposed as previously described. A glass micropipette (tip diameter = 20-40 µm) connected to the automatic nanoliter injector was used for unilateral injection of 1% Fluorogold in a volume of 50 nl. The micropipette was introduced into the dorsal medulla at least 5 min before injection and left in the medulla at least 5 min after injection. Site of injection was 0.5 mm rostral to calamus scriptorius, 0.5 mm lateral to midline, and 0.5 mm deep from the dorsal surface of the medulla. The incision was closed, and animals were allowed to recover from anesthesia. All procedures were performed in aseptic conditions. Animals were given a subcutaneous injection of buprenorphine (0.2 mg/kg) before recovery to reduce pain and discomfort.

After 72 h, rats were reanesthetized with urethane and intracardially perfused with PBS followed by 4% paraformaldehyde. Brains were removed, fixed, and sectioned as previously described. Brain stem sections were viewed as free-floating sections under a microscope for verification of injection site. Injections sites were taken as a tract in the tissue made by the micropipette. The damaged sites were easily identified due to the presence of residual blood even after the animal was flushed with PBS and paraformaldehyde. Tissues were then processed for immunohistochemistry and double-stained for CART and Fluorogold immunoreactivity using FITC and Texas red, respectively. Double-labeling procedures are as follows. Tissues were processed as previously described. Following the completion of CART immunostaining, tissues were thoroughly washed with PBS and incubated with the second primary antiserum, i.e., Fluorogold antiserum, for 2 days. Tissues were washed with PBS, incubated with biotinylated anti-rabbit IgG (1:50), washed with PBS, incubated with avidin Texas red (1:50) for 3 h, and mounted. Fluorogold antiserum was used at a dilution of 1:3,000 with 0.4% Triton X-100 and 0.5% BSA in PBS. Sections were examined under a laser confocal microscope (Leica TCS SP2). The excitation and emission wavelengths were set to 488/543 nm for FITC and 520/620 nm for Texas red.

Chemicals and reagents. CART peptide (CART55-102) was from American Peptide (Sunnyvale, CA). CART antiserum was a gift from Dr. J. K. Chang, Phoenix Pharmaceuticals (Belmont, CA), and normal rabbit serum albumin was from Vector Laboratories (Burlingame, CA). All other chemicals were from Sigma (St. Louis, MO).


   RESULTS
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
CART immunoreactivity in the medulla oblongata and nodose ganglia. As reported earlier (9, 14), CART immunoreactivity (irCART) was observed in a dense plexus of cell processes throughout the NTS and dorsal motor nucleus of the vagus; fibers were also noted in the area postrema (Fig. 1, A and B). Few irCART somata were detected in these areas. With respect to nodose ganglia, numerous irCART neurons and varicose fibers were noted throughout the ganglion (Fig. 1, C and D). Labeled ganglion cells were predominantly spherical and measured 10-30 µm in diameter (Fig. 1, C and D).



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  		Fig. 1. Confocal imaging of sections through the rat medulla oblongata and nodose ganglion labeled with cocaine- and amphetamine-regulated transcript (CART) antiserum. A: CART-immunoreactive (irCART) fibers are detected in the nucleus of the solitary tract (sol), dorsal motor nucleus of the vagus (10), and area postrema (AP); irCART fibers are generally not seen in the hypoglossal nucleus (12). CC, central canal. B: enlarged area outlined in A, where numerous irCART fibers are seen throughout the nucleus of the solitary tract. C: section of nodose ganglion containing numerous irCART perikarya and cell processes. D: enlarged area outlined in C, where immunoreactive ganglion cells with their cell processes are clearly seen. The anterior-posterior coordinate of the medullary section shown in A is approximately -14.08 mm caudal to bregma.

 

irCART nodose ganglion cells project to the NTS. To determine whether irCART fibers observed in the NTS originate from neurons in the nodose ganglion, Fluorogold was unilaterally injected into the NTS 3 days before the terminal experiment. Nodose ganglion sections double-labeled with CART and Fluorogold antisera showed numerous nodose ganglion cells expressing CART and Fluorogold immunoreactivity (Fig. 2). These neurons were noticeably smaller than those neurons that were labeled only with CART or Fluorogold (Fig. 2). The number of cells containing both irCART and Fluorogold was visually counted. Approximately 80% of neurons in the nodose ganglion expressed Fluorogold. Of those that contained Fluorogold, 64% were irCART, indicating that ~54% of the cells in the nodose ganglion are irCART and may project to the NTS.



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  		Fig. 2. Confocal images of a section of nodose ganglion and medulla labeled with CART antiserum and Fluorogold antiserum. The retrograde tracer 1% Fluorogold (50 nl) was injected unilaterally to the nucleus of the solitary tract (NTS) of a urethane-anesthetized rat 3 days before terminal experimentation. A and B: a section of nodose ganglion double-labeled with Fluorogold antiserum (A) and CART antiserum (B). C: a merged image of A and B where cells expressing both irCART and Fluorogold appear orange yellow. Note that cells expressing both irCART and Fluorogold generally had smaller diameter compared with CART-negative and/or Fluorogold-negative cells.

 

Intracisternal administration of CARTp attenuates PE-induced bradycardia. The mean MAP and HR in urethane-anesthetized rats were 103 ± 2 mmHg and 368 ± 6 beats/min (n = 20). CARTp was administered in four doses: 0.1, 0.3, 1, and 3 nmol, in a volume of 4 µl by intracisternal injection. Each rat received one dose of CARTp (0.1-3 nmol). Administration of CARTp did not cause a significant change in either the basal HR or MAP (Fig. 3). However, CARTp consistently and dose dependently attenuated the PE-induced bradycardia, as assessed by the decrease in baroreflex gain (Fig. 4A). The effect of CARTp reached its peak ~10 min after injection and dissipated in the ensuing 10-20 min.



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  		Fig. 3. Representative tracings of blood pressure and heart rate (HR) in response to phenylephrine (PE) before and after intracisternal administration of CART peptide fragment 55-102 (CARTp) in a urethane-anesthetized rat. The first 2 injections of PE were controls. CARTp injected intracisternally did not cause a significant change in basal pulsatile arterial pressure (PAP), mean arterial pressure (MAP), or HR. Subsequent PE-induced bradycardia, but not blood pressure, were attenuated by CARTp in a dose-dependent manner. A: effect of CARTp at the lowest (0.1 nmol) dose. B: effect of CARTp at the highest (3 nmol) dose. Arrows indicate time of injection. bpm, Beats/min.

 


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  		Fig. 4. Dose-response relationships of CARTp on PE-induced blood pressure and HR changes in anesthetized rats. The baroreflex gain (beats/mmHg) is determined by plotting the value of the corresponding bradycardia for a given increase in MAP (10 mmHg). A: baroreflex gain before intracisternal (IC) CARTp administration was -0.97 ± 0.03. After treatment with CARTp, baroreflex gain was -0.93 ± 0.04 (0.1 nmol), -0.78 ± 0.02 (0.3 nmol), -0.71 ± 0.05 (1 nmol), and -0.69 ± 0.09 (3 nmol). B: baroreflex gain before intra-NTS administration of CARTp was -1.11 ± 0.04. After treatment with CARTp, baroreflex gain was -0.99 ± 0.05 (0.1 nmol), -0.85 ± 0.01 (0.3 nmol), -0.68 ± 0.08 (1 nmol), and -0.61 ± 0.01 (3 nmol). The maximal gain was greater before CARTp than after CARTp was administered. Kruskal-Wallis statistics followed by Dunn's post test were used to calculate statistical significance. Vertical bars are means ± SE, and asterisks indicate statistical significance of P < 0.05.

 

Intra-NTS administration of CARTp attenuates PE-induced bradycardia. To assess whether the attenuation of PE-induced bradycardia occurred within the NTS, CARTp was administered bilaterally into the NTS. Figure 5 shows the schematic diagram of a transverse section of medulla oblongata, as adapted from the atlas of Paxinos and Watson (21), where the injection sites were marked by India ink. The mean MAP and HR in urethane-anesthetized rats in this set of experiments were 104 ± 2 mmHg and 350 ± 5 beats/min (n = 20). CARTp was given at doses of 0.1, 0.3, 1, and 3 nmol in 50 nl unilaterally (or 100 nl bilaterally). CARTp consistently and dose dependently attenuated the PE-induced bradycardia (Fig. 4B).



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  		Fig. 5. A schematic diagram of a section of medulla oblongata where CARTp injection sites are marked by solid dots. Cu, cuneate nucleus; Gr, gracile nucleus; IO, inferior olive; LRt, lateral reticular nucleus; py, pyramidal tract; RPa, raphe pallidus nucleus; sp5, spinal trigeminal tract; Sp5C, spinal trigeminal nucleus; Sol, solitary tract.

 

CART antiserum potentiates PE-induced bradycardia. Because specific CART receptor antagonists are currently not available, we used CART antiserum to evaluate the specificity of the effects observed after CARTp administration. CART antiserum is a rabbit polyclonal directed against the active fragment CART55-102 (8-10). The antiserum exhibits 100% cross-reactivity with the rat CART55-102 (8). CART antiserum (1:500) was injected bilaterally into the NTS. Administration of CART antiserum to the NTS produced no significant changes in basal MAP or HR (Fig. 6A). The antiserum potentiated the bradycardia induced by PE but did not significantly alter the PE-induced MAP changes (Fig. 6A). PE produced an average increase of 64 ± 4 mmHg in MAP and a decrease of -77 ± 3 beats/min in HR (n = 5) before and 62 ± 3 mmHg and -96 ± 12 beats/min after CART antiserum.



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  		Fig. 6. Representative tracings of effects of bilateral intra-NTS injections of CART antiserum (1:500; A), saline (B), concomitant injection of CARTp (0.3 nmol) and CART antiserum (1:500) (C), and normal rabbit serum (NRS; D) on PE-induced baroreflex.

 

Administration of CARTp with CART antiserum, normal rabbit serum, and saline. To assess the specificity of CART antiserum, CARTp (0.3 nmol) and CART antiserum (1:500) were coadministered to the NTS. Administration of CARTp and CART antiserum did not produce any significant changes in basal MAP or HR nor did it produce any significant changes in PE-induced baroreflex response (Fig. 6C). Similarly, injection of physiological saline or normal rabbit serum (1:500) to the NTS did not cause any significant changes in resting MAP or HR or in the PE-induced baroreflex (Fig. 6, B and D, respectively). Results from five rats assigned to each series of experiments are shown in Fig. 7.



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  		Fig. 7. Histograms of percent changes in PE-induced HR and MAP after administration of various agents to anesthetized rats. A: percent changes of MAP and HR induced by PE before and after CART antiserum (1:500) (n = 5). B: percent changes of MAP and HR induced by PE before and after physiological saline (n = 5). C: percent changes of MAP and HR induced by PE before and after CART antiserum plus CARTp (0.3 nmol) (n = 5). D: percent change of MAP and HR induced by PE before and after NRS (n = 5). The Student's t-test was used for statistical analysis. Vertical bars are means ± SE, and asterisks indicate statistical significance of P < 0.05.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The major observation made in this study is that a subset of neurons in the nodose ganglion may give rise to a dense plexus of irCART fibers in the NTS and that CARTp administered intracisternally or directly into the NTS attenuated PE-induced bradycardia, whereas CART antiserum injected into the NTS potentiated PE-induced bradycardia.

It is well established that vagal afferents project to the NTS and that the NTS is the predominant site of autonomic cardiovascular control. Lesions or other perturbations to the NTS result in increased arterial pressure (11, 22). Previous immunohistochemical and in situ hybridization studies show that irCART is present in fibers of the NTS and in neurons of the vagal nerves, respectively (3, 9, 14). Our immunohistochemical results confirm the presence of a dense plexus of irCART fibers throughout the rostrocaudal axis of the NTS. Although the possibility that irCART fibers observed in the NTS may be derived from other sources cannot be excluded, retrograde tract-tracing studies demonstrate that some of the irCART fibers in the NTS arise from neurons in the nodose ganglion. In our study, ~50% of nodose ganglion cells that are Fluorogold positive are also irCART, which is consistent with a previous report that ~50% of vagal afferents are irCART (3). More interestingly, irCART nodose ganglion cells are generally of smaller diameter compared with Fluorogold and/or CART-negative nodose neurons. Because of their small size, irCART nodose neurons that project to the NTS may belong to the slowly conducting type C neurons. It is pertinent to mention that not all nodose ganglion cells projecting to the NTS are baroreceptor afferents. Hence, not all irCART nodose ganglion cells projecting to the NTS are baroreceptor related.

Intracisternal injection of CARTp consistently attenuated PE-induced bradycardia in a dose-dependent manner. CARTp injected intracisternally may reach a number of sites in the dorsal medulla that could potentially alter the cardiovascular response. To assess the role of NTS in relation to the action of CARTp, the peptide was injected directly into an area of NTS that is thought to be the predominant site of baroreceptor afferent termination (24). CARTp injected into the NTS attenuated the PE-induced bradycardia in a dose-dependent manner as well, suggesting that CARTp acts at the level of NTS. Our finding does not necessarily exclude the possibility that CARTp may act on other sites in the dorsal medulla to alter cardiovascular responses. Matsumura et al. (18) reported that CARTp by intracerebroventricular injection increased MAP, HR, and renal sympathetic nerve activity (RSNA) in conscious rabbits. Intracisternal or intra-NTS administration of CARTp in the concentrations used in our study did not cause a significant change in basal arterial pressure or HR in anesthetized rats. Differences in experimental species and sites of administration as well as the observed time frame may have accounted for this discrepancy. Elevation of MAP and RSNA was observed 30-40 min and increase of HR was observed ~90 min after CARTp administration in rabbits, whereas in this study, the effect of CARTp reached its peak ~10 min after injection in rats. Furthermore, CARTp was injected intracerebroventricularly, which may activate supramedullary neurons, leading to a different profile of cardiovascular responses in the case of rabbits.

CARTp receptor antagonists are currently not available. CART antiserum, a rabbit polyclonal directed against the biologically active fragment CART55-102 (8-10), was employed here to evaluate the pharmacological specificity of the effects of CARTp. Intra-NTS injections of CART antiserum augmented PE-induced bradycardia but did not produce a significant change in basal MAP or HR. If CARTp were tonically released, one would expect to see changes in baseline MAP and HR after CART antiserum. Because there were no significant changes in baseline activity, the peptide may not be tonically released under current experimental conditions. Alternatively, the lack of effect may indicate that the quantity of CARTp being released under basal conditions is negligible. Intra-NTS injections of physiological saline, normal rabbit serum, and concomitant injection of CARTp and CART antiserum did not produce any significant changes in baseline MAP and HR or in PE-induced bradycardia. The ineffectiveness of CARTp and CART antiserum when injected simultaneously to the NTS further attests to the specificity of CART antiserum. More importantly, CART antiserum augmented the PE-induced bradycardia, implying CARTp is released in a quantity sufficiently large to produce a significant damping effect on medullary outflow to the heart during PE-induced baroreflex. As a corollary, CART antiserum may augment PE-induced bradycardia by disinhibition.

Perspectives

CART-containing neurons and cell processes are widely distributed in the forebrain, hindbrain, spinal cord, peripheral autonomic ganglia, and adrenal medulla (8, 14). Information relative to the pathway-specific function of this peptide is relatively sparse. CARTp injected intracerebroventricularly attenuates feeding, as assessed by food consumption in rats (1, 15, 17). Intracisternal, but not intravenous, injection of CARTp inhibits gastric emptying (20). Other studies have shown that CARTp by intravenous administration increases amylase secretion from the rat pancreas (5). CARTp when injected intrategmentally increases locomotor activity in rats (13). Because CARTp is injected either intravenously or intracerebroventricularly, it is not possible to establish a direct correlation between function and pathway in these studies. Viewed in this context, our finding provides the first evidence of a pathway-specific function of CARTp.


   DISCLOSURES
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by National Institutes of Health Grants NS-18710 and HL-51314 from the Department of Health and Human Services.


   ACKNOWLEDGMENTS
 
We thank C. Brailoiu and J. Hoard for assistance with the confocal imaging.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. J. Dun, Dept. of Pharmacology, James H. Quillen College of Medicine, East Tennessee State Univ., PO Box 70577, Johnson City, TN 37614 (E-mail: dunnae{at}mail.etsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Aja S, Sahandy S, Ladenheim EE, Schwartz GJ, and Moran TH. Intracerebroventricular CART peptide reduces food intake and alters motor behavior at a hindbrain site. Am J Physiol Regul Integr Comp Physiol 281: R1862-R1867, 2001.[Abstract/Free Full Text]
  2. Andresen MC and Kunze DL. Nucleus tractus solitarius: gateway to neural circulatory control. Annu Rev Physiol 56: 93-116, 1994.[ISI][Medline]
  3. Broberger C, Holmberg K, Kuhar MJ, and Hokfelt T. Cocaine- and amphetamine-regulated transcript in the rat vagus nerve: a putative mediator of cholecystokinin-induced satiety. Proc Natl Acad Sci USA 93: 13506-13511, 1999.
  4. Couceyro PR, Koylu EO, and Kuhar MJ. Further studies on the anatomical distribution of CART by in situ hybridization. J Chem Neuroanat 12: 229-241, 1997.[ISI][Medline]
  5. Cowles RA, Segura BJ, and Mulholland MW. Stimulation of rat pancreatic exocrine secretion by cocaine- and amphetamine-regulated transcript peptide. Regul Pept 99: 61-68, 2001.[ISI][Medline]
  6. Dhruva A, Bhatnagar T, and Sapru HN. Cardiovascular responses to microinjections of glutamate into the nucleus tractus solitarii of unanesthetized supracollicular decerebrate rats. Brain Res 801: 88-100, 1998.[ISI][Medline]
  7. Douglas J, McKenzie AA, and Couceyro P. PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. J Neurosci 15: 2471-2481, 1995.[Abstract]
  8. Dun NJ, Dun SL, Kwok EH, Yang J, and Chang JK. Cocaine- and amphetamine-regulated transcript-immunoreactivity in the rat sympathoadrenal axis. Neurosci Lett 283: 97-100, 2000.[ISI][Medline]
  9. Dun SL, Castellino SJ, Yang J, Chang JK, and Dun NJ. Cocaine- and amphetamine-regulated transcript peptide-immunoreactivity in dorsal motor nucleus of the vagus neurons of immature rats. Dev Brain Res 131: 93-102, 2001.[Medline]
  10. Dun SL, Ng YK, Brailoiu GC, Ling EA, and Dun NJ. Cocaine- and amphetamine-regulated transcript peptide-immunoreactivity in adrenergic C1 neurons projecting to the intermediolateral cell column of the rat. J Chem Neuroanat 23: 123-132, 2002.[ISI][Medline]
  11. Ferrario CM, Barnes KL, and Bohonek S. Neurogenic hypertension produced by lesions of the nucleus tractus solitarii alone or with sinoaortic denervation in the dog. Hypertension 396: 112-118, 1981.
  12. Higa KT, Mori E, Viana FF, Morris M, and Michelini LC. Baroreflex control of heart rate by oxytocin in the solitary-vagal complex. Am J Physiol Regul Integr Comp Physiol 282: R537-R545, 2002.[Abstract/Free Full Text]
  13. Kimmel HL, Gong W, Vechia SD, Hunter RG, and Kuhar MJ. Intra-ventral tegmental area injection of rat cocaine and amphetamine-regulated transcript peptide 55-102 induces locomotor activity and promotes conditioned place preference. J Pharmacol Exp Ther 294: 784-792, 2000.[Abstract/Free Full Text]
  14. Koylu EO, Couceyro PR, Lambert PD, and Kuhar MJ. Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J Comp Neurol 391: 115-132, 1998.[ISI][Medline]
  15. Kristensen P, Judge ME, Thim L, Ribel U, Christjansen KN, Wulff BS, Clausen JT, Jensen PB, Madsen OD, Vrang N, Larsen PJ, and Hastrup S. Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393: 72-76, 1998.[Medline]
  16. Kuhar MJ and Yoho LL. CART peptide analysis by Western blotting. Synapse 33: 163-171, 1999.[ISI][Medline]
  17. Lambert PD, Couceyro PR, McGirr KM, Dall Vechia SE, Smith Y, and Kuhar MJ. CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 29: 293-298, 1998.[ISI][Medline]
  18. Matsumura K, Tsuchihashi T, and Abe I. Central human cocaine- and amphetamine-regulated transcript peptide 55-102 increases arterial pressure in conscious rabbits. Hypertension 38: 1096-1100, 2001.[Abstract/Free Full Text]
  19. Ohsawa M, Dun SL, Tseng LF, Chang JK, and Dun NJ. Decrease of hindpaw withdrawal latency by cocaine- and amphetamine-regulated transcript peptide to the mouse spinal cord. Eur J Pharmacol 399: 165-169, 2000.[ISI][Medline]
  20. Okumura T, Yamada H, Motomura W, and Kohgo Y. Cocaine- and amphetamine-regulated transcript (CART) acts in the central nervous system to inhibit gastric acid secretion via brain corticotropin-releasing factor system. Endocrinology 141: 2854-2860, 2000.[Abstract/Free Full Text]
  21. Paxinos G and Watson C. The Rat Brain in Stereotaxic Coordinates (4th ed.). San Diego, CA: Academic, 1998.
  22. Sved AF. Peripheral pressor systems in hypertension caused by nucleus tractus solitarius lesions. Hypertension 9: 742-747, 1986.
  23. Talman WT, Perrone MH, and Reis DJ. Evidence for L-glutamate as the neurotransmitter of primary baroreceptor afferent nerve fibers. Science 209: 813-815, 1980.[Abstract/Free Full Text]
  24. Viard E and Sapru HN. Cardiovascular responses to activation of metabotropic glutamate receptors in the nTS of the rat. Brain Res 952: 308-321, 2002.[ISI][Medline]
  25. Zhou H, Ichikawa H, and Helke CJ. Neurochemistry of the nodose ganglion. Prog Neurobiol 52: 79-107, 1997.[ISI][Medline]




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