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Regulatory Peptides 127 (2005) 79 – 85 www.elsevier.com/locate/regpep

Cocaine- and amphetamine-regulated transcript peptide potentiates spinal glutamatergic sympathoexcitation in anesthetized rats Phouangmala Scruggsa, Chih. C. Laib, Jesse E. Scruggsa, Nae J. Duna,* a

Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, P.O. Box 70577, Johnson City, TN 37614, United States b Department of Pharmacology, Tzu Chi University, Hualien, Taiwan Received 1 June 2004; received in revised form 22 October 2004; accepted 22 October 2004 Available online 2 December 2004

Abstract Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the rat central nervous system, notably in areas involved in control of autonomic and neuroendocrine functions. The aim of this study was to evaluate the effects of CART peptide fragment 55–102, referred to herein as CARTp, by intrathecal injection on blood pressure (BP) and heart rate (HR) before and after intrathecal glutamate in urethane-anesthetized male Sprague–Dawley rats. CARTp (0.1–10 nmol) administered intrathecally caused no or a small, statistically insignificant increase of blood pressure and heart rate, except at the concentration of 10 nmol, which caused a significant increase of blood pressure and heart rate. Intrathecal glutamate (0.1–10 nmol) produced a dose-dependent increase in arterial pressure and heart rate. Pretreatment with CARTp dose-dependently potentiated the pressor effects of glutamate (1 nmol), which by itself elicited a moderate increase of blood pressure and heart rate. Further, CARTp significantly potentiated the tachycardic effect of glutamate at 1 and 5 nmol, but attenuated the response at 10 nmol. The effect of CARTp was long-lasting, as it enhanced glutamatergic responses up to 90 min after administration. Prior injection of CARTp antiserum (1:500) but not normal rabbit serum nullified the potentiating effect of CARTp on glutamatergic responses. The result suggests that CARTp, whose immunoreactivity is detectable in sympathetic preganglionic neurons as well as in fibers projecting into the intermediolateral cell column, augments spinal sympathetic outflow elicited by glutamate at lower concentrations and may directly excite neurons in the intermediolateral cell column at higher concentrations. D 2004 Elsevier B.V. All rights reserved. Keywords: Intermediolateral cell column; Intrathecal; Sympathetic preganglionic neurons

1. Introduction Sympathetic preganglionic neurons (SPNs) correspond to the final station where neural activity related to cardiovascular, pulmonary and visceral function is integrated within the central nervous system [7,12,25]. In addition to intraspinal inputs, SPNs receive projections from supraspinal areas such as the A5 noradrenergic cell group, caudal raphe, hypothalamic paraventricular nucleus, rostral ventrolateral

* Corresponding author. Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, United States. Tel.: +1 215 707 3237; fax: +1 215 707 7068. E-mail address: [email protected] (N.J. Dun). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.10.017

medulla and ventromedial medulla [7,12,25]. The rostral ventrolateral medulla is recognized as one of the pressor areas where some of the neurons project directly to the IML region of the spinal cord. Cocaine- and amphetamine-regulated transcript (CART) is up regulated by cocaine or amphetamine [9]. Immunohistochemical and in situ hybridization studies reveal an extensive distribution of CART or protein in the central and peripheral nervous system of the rat [3,4,6,9,10,16]. In the present context, CART-like immunoreactivity (CART-LI) is expressed in a population of C1 adrenergic neurons of the RVLM [11]. Electron microscopic studies reveal synapticlike contacts between CART-immunoreactive terminals and SPNs, raising the possibility that the peptide may modulate the activity of SPNs [11]. The peptide has been shown to be

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biologically active in several systems. For example, CART peptide (CARTp) when injected intracerebroventricularly increases arterial pressure and renal sympathetic nerve activity in conscious rabbits [20]. Further, we have previously shown that CARTp administered into the nucleus of the solitary tract attenuated baroreflex in anesthetized rats [22]. l-Glutamate is thought to be the primary excitatory neurotransmitter acting on SPNs to generate phasic and/or tonic vasomotor tone [7,13–15,18,19,26]. Several peptides and catecholamines have also been shown to affect spinal autonomic functions [7]. To better understand how various transmitters are engaged in central regulation of cardiovascular functions, we explore the action of glutamate and neuropeptides individually or cooperatively. The present study was undertaken to evaluate the cardiovascular effects of intrathecally injected CARTp alone and in combination of glutamate in anesthetized rats.

pinching the tail. Rectal temperature was maintained at 37F1 8C with the use of a thermo-blanket. 2.2. Blood pressure measurements 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 blood pressure (BP) signal was used to trigger a Biotach amplifier (Gould ECG/BiTac’) for heart rate (HR) recording. Mean arterial pressure (MAP) was measured directly from the pre-calibrated chart recorder or calculated using the equation [S D/3+D], where S is systolic pressure and D is diastolic pressure. To maintain the depth of anesthesia, the right femoral vein was cannulated for subsequent intravenous administrations of urethane (30–60 mg/kg), which 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 Al/s.

2. Materials and methods 2.3. Intrathecal injection 2.1. 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 22F1 8C with an alternating 12:12-h light–dark cycle. Food and water were available ad libitum. Animal protocols were reviewed and approved by the Institution Animal Care and Use Committee. Male Sprague–Dawley rats, weighing 250–350 g, were anesthetized with urethane (1.2 g/kg, i.p.). The depth of anesthesia was monitored by observing responses to

Rats were placed in a stereotaxic frame (David Kopf, Tujunga, CA). An incision was made at the top of the head to expose the dorsal neck muscles. The atlanto-occipital membrane was exposed by using a spatula to remove the dorsal neck muscles. A slit was made in the atlanto-occipital membrane. The spinal catheter (PE-10) was inserted down into the spinal subarachnoid space so that the tip was placed in the vicinity of T1–T2 segment; the catheter was then sutured to the top of the head. The position of the tubing was visually verified at the end of the experiment. Animals in which the intrathecal tubing was misplaced inside the

Fig. 1. Representative tracings of intrathecally administered saline and glutamate in an urethane-anesthetized rat. The first row is pulsatile arterial pressure (PAP), middle row is mean arterial pressure (MAP) and third row is heart rate (HR). Glutamate (0.1–10 nmol) produced dose-dependent increases in MAP and HR, while saline had no appreciable effects. Arrows indicate time of injection. Horizontal bar =5 min.


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spinal cord or outside the subarachnoid space were not used for statistical analyses. Rats were kept in a prone position for the duration of the experiment. 2.4. Chemicals and statistics CARTp and glutamate were dissolved in distilled, deionized water and frozen in aliquots. Before administration, peptides were thawed, dissolved in saline to the desired concentration and injected intrathecally at a volume of 5 Al, which was followed by 5 Al saline to wash in the agent. CARTp was from American Peptide Company (Sunnyvale, CA) and glutamate was from Sigma (St. Louis, MO). CARTp-antiserum (Phoenix Pharmaceuticals, Belmont, CA) or normal rabbit serum (Vector Laboratories, Burlingame, CA) was also injected in a volume of 5 Al. Results were reported as percentage of maximal change in MAP and HR relative to the control value elicited by glutamate (1 nmol) administered prior to intrathecal injection of CARTp. Data were analyzed statistically using one factor ANOVA followed by Dunnett’s multiple comparison post test, with Pb0.05 considered statistically significant. Results are expressed as meansFstandard error of the mean (S.E.M.).

3. Results 3.1. Glutamate pressor and tachycardic effects The MAP and HR were 116.1F6.4 mm Hg and 309.6F 5.8 beats per minute (bpm) in urethane-anesthetized rats (n=28). Prior to every experiment, saline was given intrathecally as a negative control. Intrathecal saline did not elicit any significant changes in blood pressure and HR in the majority of rats tested. Animals in which saline produced any cardiovascular responses were excluded for statistical analyses, since that usually indicated the catheter was positioned improperly in the spinal cord. Intrathecal glutamate (0.1–10 nmol), in agreement with previous findings [14,28], caused a rise in MAP and HR. Representative tracings of intrathecal saline and glutamate are shown in Fig. 1. Glutamate caused a dose dependent increase in MAP and HR (Fig. 2). HR responses to glutamate developed rapidly and lasted 15–25 min depending on the concentration of glutamate. Peak responses occurred approximately 1–2 min after injection. Injections of saline or glutamate were given approximately 30 min apart, permitting MAP and HR to recover to baseline. At a given dose of glutamate, the increase in HR was proportionally larger as compared to blood pressure (Fig. 2). 3.2. Potentiation of glutamate response by CARTp In these experiments, glutamate (1 nmol) was first administered intrathecally to establish a control response.

Fig. 2. Dose – response curves of intrathecally injected glutamate on mean arterial pressure and heart rate of urethane-anesthetized rats. Glutamate (0.1–10 nmol) was administrated intrathecally to the T1–T2 spinal segment. (A) Change in mean arterial pressure (MAP). (B) Changes in heart rate (HR). Each point on the curves represents the meanFS.E.M. of four responses.

Thirty minutes later, animals were pretreated with intrathecal saline or CARTp (0.1–10 nmol) 5 min prior to a subsequent administration of intrathecal glutamate (1 nmol). Representative tracings of cardiovascular responses elicited by glutamate before and after pretreatment with either saline or different concentrations of CARTp are shown in Fig. 3. At lower concentrations, i.e., V1 nmol, intrathecal CARTp did not produce any noticeable effects on the heart rate and mean arterial pressure. However, at doses of 5 and 10 nmol, slight pressor responses and tachycardia were observed 1–2 min after administration. CARTp increased MAP by 0.75F0.75 (0.1), 1.8F1.8 (0.5), 1F1 (1), 3.3F3.3 (5) and 8.5F6.4 mm Hg (10 nmol). The rises in MAP were not statistically significant as compared to saline injection. CARTp increased HR by 0.5F0.5 (0.1), 2.4F1.4 (0.5), 6.3F3.8 (1), 7.5F2.5 (5) and 19F8.3 bpm (10 nmol). Increases in HR were not statistically significant except at the dose of 10 nmol. CARTp (0.1–10 nmol) dose-dependently potentiated the sympathoexcitatory effect of glutamate given intrathecally

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Fig. 3. Representative tracings of intrathecal saline and CARTp on cardiovascular responses elicited by intrathetcal glutamate (1 nmol) in urethane-anesthetized rats. (A) Saline injection, (B) 0.1 nmol CARTp, (C) 1 nmol CARTp and (D)10 nmol CARTp on glutamate responses. Glutamate was administered beforehand as a control. After mean arterial pressure and heart rate had recovered to baseline 30 min later, CARTp or saline were injected. A second injection of glutamate was made 5 min after CARTp or saline. Arrows indicate time of injection. Horizontal bar = 5 min.


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was given every 30 min for 120 min. The effect of CARTp was long-lasting, as the glutamate responses remained elevated 90 min after administration of CARTp (Fig. 5). 3.4. CARTp antiserum or normal rabbit serum injection CARTp receptor antagonists are currently not available. For this reason, CARTp antiserum was used to evaluate the specificity of the effects observed after CARTp administration [22]. Following a control response of glutamate (1 nmol) and CARTp (1 nmol) plus glutamate (1 nmol), CARTp antiserum (1:500 dilution) was administrated intrathecally, which did not produce a significant change of blood pressure or heart rate. Thereafter, a second intrathecal injection of CARTp plus glutamate produced a pressor and tachycardiac response, with a magnitude similar to that produced by glutamate alone. The increase in blood pressure and heart rate caused by CARTp plus glutamate,

Fig. 4. Histograms of percent changes of glutamate responses before and after treatment with saline or CARTp (0.1–10 nmol). (A) Changes in mean arterial pressure (MAP). (B) Changes in heart rate (HR). Numbers are reported as percent of the control, which is the response caused by glutamate before treatment with either CARTp or saline. Saline (n=4), 0.1 nmol CARTp (n=4) and 0.5 nmol CARTp (n=4) failed to produce any significant changes in glutamate-induced increases in MAP and HR. CARTp at 1 nmol (n=4), 5 nmol (n=4) and 10 nmol (n=4) dose-dependently augmented the pressor effect. CARTp at 1 nmol (n=4) and 5 nmol (n=4) increased the glutamate-induced tachycardia, whereas 10 nmol CARTp (n=4) inhibited glutamate-induced tachycardia. Vertical bars are meansFS.E.M. Asterisks denote statistical significant difference of Pb0.05.

to the rat spinal cord, whereas saline had no effect (Fig. 4). CARTp at 1, 5 and 10 nmol significantly increased the pressor response to intrathecal glutamate. The maximum increase of tachycardic response was seen from pretreatment with 1 nmol CARTp. At 5 nmol, the peptide significantly augmented the chronotropic effect, but to a lesser extent than 1 nmol CARTp. The tachycardic effect induced by glutamate was significantly lowered by a higher dose of CARTp (10 nmol). 3.3. Long-lasting potentiation by CARTp In this series of experiments, the effect of intrathecal CARTp on pressor responses to glutamate was evaluated over a 2-h period. Control glutamate responses were established prior to CARTp administration. After pretreatment with CARTp (0.5 or 2.5 nmol), intrathecal glutamate

Fig. 5. Duration of activity of intrathecally injected CARTp in urethaneanesthetized rats. (A) Changes in mean arterial pressure (MAP). (B) Changes in heart rate (HR). Glutamate-induced cardiovascular effects before saline or CARTp injection served as the control. After rats were intrathecally injected with CARTp (0.5 and 2.5 nmol) or saline, glutamate was administered intrathecally every 30 min for 120 min to assess persistence of CARTp activity. CARTp at 0.5 nmol (n=4) and saline (n=4) did not significantly augment pressor or positive chronotropic effect of glutamate. However, 2.5 nmol CARTp (n=4) enhanced the effects of glutamate. Vertical bars are meansFS.E.M. Asterisks denote statistical significant difference of Pb0.05.

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and antiserum followed by CARTp plus glutamate was 118F4% and 146F8%, and 96F4% and 103F6%, respectively (n=4). The response induced by glutamate (1 nmol) was taken as 100%.

4. Discussion In vivo experiments were conducted to examine the effects of CARTp on spinal sympathetic outflow to the vasculature and heart alone and in combination with glutamate. Our results indicate that CARTp in lower concentrations (b1 nmol), which administered alone elicits little or no change of MAP and HR, significantly potentiates the pressor response of glutamate injected intrathecally. The peptide at higher concentrations (N10 nmol) by intrathecal injection produces a modest increase of blood pressure and heart rate. In this and a previous study [14], intrathecal glutamate (10 nmol–1 Amol) raised arterial pressure and heart rate in urethane-anesthetized Sprague–Dawley rats. It is worth noting that the percent increase of heart rate was higher than that of blood pressure following intrathecal glutamate. This is probably due to the fact that the intrathecal catheter was placed at the T1–T2, where the majority of sympathetic preganglionic neurons project to the stellate ganglion and middle cervical ganglion whose neurons innervate the heart, as determined by anatomical tracing and functional studies [2,21,24,26]. Pretreatment with CARTp (1–10 nmol) enhanced the pressor response of a submaximal dose of glutamate (1 nmol). For example, CARTp (10 nmol) increased the MAP by 50F24 mm Hg, which is 241F40% of the response induced by glutamate alone. For heart rate, a maximal potentiation was seen with 1 nmol CARTp. While a statistically significant increase of glutamate-induced tachycardia is evident with CARTp (5 nmol), the increase is smaller as compared to that induced by a lower dose of CARTp. At the dose of 10 nmol, the peptide attenuated the glutamate-induced tachycardia as compared to that caused by glutamate alone. It is possible that large pressor responses caused subsequent activation of the baroreflex, which consequently reduced the increase in HR. CARTp receptor antagonists are currently not available. We therefore evaluated the specificity of the response with the use of CARTp antiserum [22]. CARTp antiserum was a rabbit polyclonal, which exhibits 100% cross-reactivity with CARTp used in this study [10,11]. CART antiserum administrated immediately before CARTp plus glutamate produced a pressor response that is comparable to that caused by glutamate alone, indicating that the antiserum neutralizes the effect of CARTp. On the other hand, normal rabbit serum caused little or no change of the pressor response of CARTp plus glutamate. CARTp by intrathecal administration exerted a long lasting effect, as it continued to enhance glutamate-induced

responses for over 90 min after administration. Another interesting feature is that CARTp at doses of 0.1–5 nmol by itself did not elicit any significant cardiovascular responses, whereas at the dose of 10 nmol it significantly increased the HR as compared to saline injection. The potentiation of glutamate-induced pressor response by CART is in many respects similar to the enhancement of spinothalamic neuron responses to chemical and mechanical stimuli following combined micro-iontophoretic application of N-methyl-daspartic acid (NMDA) and substance P [8]. In both cases, putative transmitters act cooperatively to enhance the physiological response. The mechanism whereby one transmitter enhances the response of another transmitter is largely not known. It may interact at the receptor level or at the signaling pathway. For example, pituitary adenylate cyclase activating polypeptide enhances NMDA currents probably by elevating cAMP levels in rat sympathetic preganglionic neurons [28]. CARTp is present in a wide range of locations such as the hypothalamus, nucleus accumbens, amygdala, intermediolateral cell column, vagal afferent fibers, adrenal medulla and cardiac ganglia [3,4,10,11,16,17]. The peptide has been implicated in a variety of functions within the central nervous system. CARTp increases anxiety-like behavior [5], decreases feeding [1,23,27] and increases sympathetic activity upon intracerebroventricular injection [20]. Previously, we showed that CARTp acts at the level of the nucleus tractus solitarius to attenuate baroreflex in rats [22]. The present results indicate that CARTp augments sympathetic outflow by acting on sympathetic preganglionic neurons, supporting the contention that CARTp may mediate pathway specific aspects of cardiovascular function. In summary, the sympathoexcitatory actions of CARTp were only slightly self-evident at doses of 5 and 10 nmol. On the other hand, CARTp (0.1–10 nmol) dose-dependently augmented glutamate-induced increases in mean arterial pressure and significantly increased chronotropic effects of glutamate at moderate doses. At a higher dose (10 nmol), glutamate-induced tachycardia was inhibited. The mechanisms involved are still unclear, as the putative CARTp receptor has not been characterized.

Acknowledgement This study was supported by NIH Grant NS18710 from the Department of Health and Human Services.

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