| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, Missouri
Submitted 3 November 2004 ; accepted in final form 25 November 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
calcitonin gene-related peptide; heart rate; brain
IMD/AM2 is a novel member of the calcitonin peptide family, which includes calcitonin, calcitonin gene-related peptide (CGRP), AM, and amylin. IMD can be processed into two biologically active forms, a 47-amino acid COOH-terminally amidated peptide, IMD-long (IMDL), and a 40-amino acid peptide resulting from proteolytic cleavage of the seven NH2-terminal residues, IMD-short (IMDS; Ref. 20). Whereas IMDS was more potent at the cellular level, IMDL was demonstrated to be more potent than IMDS in vivo (20). In addition to being produced in the intermediate lobe of the pituitary, the original reports showed that IMD expression was localized by RT-PCR to the kidneys, lungs, gastrointestinal system, thymus, and brain (26). Peptide levels in various tissues and plasma, however, were not reported.
Roh and colleagues (20) not only identified the peptide but also demonstrated that IMD binds to and activates both the CGRP and AM receptors. The calcitonin receptor-like receptor (CRLR) in association with one of three known receptor activity-modifying proteins (RAMPs) comprises the receptors for both AM and CGRP. CRLR in association with RAMP1 is a CGRP-specific receptor, whereas CRLR in association with RAMP2 and -3 are AM-specific receptors. Although IMDS was less potent at eliciting cAMP accumulation in cells than AM and CGRP (20), it could still be hypothesized that IMD would illicit biological responses similar to both AM and CGRP. Indeed, like AM (7) and CGRP (5), IMD had potent hypotensive effects when administered intraperitoneally (ip) (20) or intravenously (iv) (26), which could be at least partially blocked by AM and CGRP receptor antagonists (20).
We demonstrate here that immunoreactive IMD protein is present in plasma as well as in heart, lung, kidney, stomach, pituitary, and brain. Because IMD is present in brain and both AM and CGRP have potent central nervous system (CNS) effects, we examined the ability of IMD to act within brain to regulate blood pressure and ingestive behaviors. Both AM and CGRP elevate blood pressure when given intracerebroventricularly (icv) through activation of the sympathetic nervous system (8, 22). In addition, both peptides have inhibitory effects on ingestive behaviors, CGRP on food (14) and AM on both food and water (16, 27). Here, we present studies verifying the action of IMDL administered iv to lower blood pressure and show that the effects of the peptide are independent of NO formation. We then demonstrate that IMDL has potent effects within brain to elevate blood pressure and heart rate. These effects are similar to those of CGRP and are partially blocked by CGRP(8–37). In addition, we demonstrate that IMDL potently inhibits both food and water intake in sated and in food- and water-restricted rats, suggesting additional actions in brain similar to those of AM and CGRP.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
IMD measurements. Animals were killed by rapid decapitation. Trunk blood was collected into heparinized tubes and placed on ice. Whole heart, kidney, adrenal, stomach, lung, and anterior pituitary were collected and rapidly frozen on dry ice. Neural tissue was dissected on the basis of the following coordinates and frozen on dry ice: hypothalamus: rostral—optic chiasm, caudal—mammillary bodies, lateral—optic tracts, and dorsal—roof of third cerebroventricle; brain stem: rostral—dorsal raphe, caudal—area postrema and nucleus tractus solitarii, and dorsal—floor of fourth cerebroventricle; cerebellum: lateral hemisphere to include simple lobule, crus I ansiform lobule, paraflocculus, flocculus, and lateral aspects of lobules 1–5. Posterior and intermediate lobes of the pituitary were collected together. Blood samples were spun (600 g, 10 min, 4°C) to obtain plasma, and the plasma was stored in a –20°C freezer. Before peptide measurement, 1 ml of plasma was acidified with an equal volume of 0.1 N HCl and centrifuged to pellet large plasma proteins. Remaining proteins were extracted on C-18 extraction columns (PrepSep SPE tubes; Fisher Scientific), eluted in an acetonitrile-acetic acid solution, and dried down in a vacuum centrifuge. Tissues were homogenized and extracted in 1.0 N HCl and then centrifuged to pellet nonsoluble matter. The supernatant from the tissues was also dried in a vacuum centrifuge. Samples were resuspended in RIA buffer, and IMD levels were determined with a RIA kit from Phoenix Pharmaceuticals (Belmont, CA). The limits of sensitivity for the IMD RIA were 20–1,280 pg, with an IC50 of 474 pg. There was 100% cross-reactivity with both IMDL and IMDS but no cross-reactivity with AM or CGRP. The intra-assay variability was 3.9%. Tissue IMD levels determined by RIA were normalized for total protein in each sample (total protein determined with Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA).
Surgery. Under ketamine (Ketaset; Fort Dodge Animal Health, Fort Dodge, IA)-xylazine (TranquiVed; Vedco, St. Joseph, MO) anesthesia (60 mg-8 mg mixture/ml, 0.1 ml/100 g body wt ip), rats were placed in a stereotaxic device and a 23-gauge stainless steel cannula (17 mm) was implanted into the right lateral cerebroventricle as previously described (22). Patency of the icv cannula was verified by a dipsogenic response to 50 pmol of angiotensin II the day after experimentation. After a return to preimplantation body weight, animals to be used for blood pressure monitoring were implanted with an indwelling left carotid cannula (PE-50) under ketamine-xylazine anesthesia. The cannula was exteriorized at the back of the neck and sealed with heparinized saline (200 U/ml 0.9% NaCl). In another group of animals, a jugular cannula was implanted as previously described (10) at the same time as a carotid cannula. Before experimentation, the exteriorized carotid cannula was flushed with heparinized saline and attached to a blood pressure transducer (Digi-Med blood pressure analyzer; Micro-Med, Louisville, KY). Jugular cannulas were also flushed with heparinized saline and attached to an extension tubing to permit iv injections without disturbing the animal. The rats were left undisturbed for at least 1 h before experimentation began. Mean arterial pressure and heart rate were digitally averaged and recorded every 60 s during the test period.
Intravenous blood pressure studies. Rats were administered a 100-µl bolus of 0.9% NaCl vehicle (iv), followed by continuous recording of mean arterial pressure and heart rate for 1 h. Animals then received a series of iv injections minimally 1 h apart containing increasing doses of rat IMDL (0.1, 1.0, and 3.0 nmol/kg in 100 µl saline; Phoenix Pharmaceuticals) while blood pressure and heart rate were being recorded. To compare the blood pressure and heart rate effects of IMDL, AM, and CGRP (all rat; Phoenix Pharmaceuticals), each peptide was administered iv in random order at a dose of 1 nmol/kg in 100 µl of saline. Another group of animals were administered 20 mg/kg NG-monomethyl-L-arginine (L-NMMA; EMD Biosciences, La Jolla, CA) iv 10 min before receiving an iv injection of saline vehicle or vehicle containing 1.0 nmol/kg IMDL. Baseline blood pressure and heart rate were calculated for each animal as the average readings over the 10 min before injection. Mean arterial pressure and heart rate in each rat were then averaged every 5 min after injection.
Intracerebroventricular blood pressure studies. After the stabilization period, rats received an icv injection of 2 µl of saline vehicle followed by continuous blood pressure and heart rate monitoring. Minimally 1 h later animals received a series of icv injections at least 1 h apart (or until the animal's blood pressure returned to baseline values) containing increasing doses of IMDL (65, 195, 585 pmol in 2 µl saline, chosen based on the previously reported effective doses of AM; Ref. 22), and blood pressure and heart rate were recorded. A second group of animals received a series of three icv injections in random order containing 195 pmol of AM, 195 pmol of CGRP, or 195 pmol of IMDL in 2 µl of saline vehicle. Injections were made minimally 1.5 h apart, and blood pressure and heart rate were continuously monitored throughout the entire experiment. A third group of rats, after the stabilization period, were given icv 2 µl of saline vehicle or 2.93 nmol of CGRP(8–37) (Phoenix Pharmaceuticals) in 2 µl of saline. Ten minutes later the rats were given a second icv injection containing saline, 195 pmol of CGRP, or 195 pmol of IMDL as before. Another group of rats was administered phentolamine (10 µg/kg in 100 µl saline; Sigma, St. Louis, MO) or saline via the carotid cannula. Ten minutes later the rats were administered 2 µl of saline vehicle alone or saline containing 195 pmol of IMDL via the icv cannula. Baseline and experimental blood pressures and heart rates were calculated as before for each animal.
Ingestion studies. Rats implanted with icv cannulas only were placed into metabolic cages and allowed free access to food and water. Daily food and water consumption and animal weights were monitored for minimally 3 days before experimentation. On the day of experimentation, rats were injected icv with 2 µl of saline vehicle or vehicle containing 195 pmol of IMDL. Ten minutes later rats were allowed free access to food and water. Cumulative intakes were recorded every 30 min for 5 h and again at 24 h. A second set of animals was acclimated to metabolic cages as before. The evening before experimentation, rats were denied access to food and water. On the day of experimentation, IMDL (195 pmol) or saline vehicle was administered as above. Ten minutes later food and water were returned to the animals, and cumulative intakes were measured as above. A third group of animals was denied access to food and water for 18 h before experimentation as above. Ten minutes after a central injection of IMDL (195 pmol) or saline vehicle icv, rats were given water bottles, but no food, and water intake was measured for every 30 min for 5 h, after which food was returned to the animals. Cumulative water intake was again measured at 24 h after injection. To determine the dose relationship, another group of rats was food and water restricted as before and animals received icv injections containing saline vehicle or 65, 195, or 585 pmol of IMDL. Cumulative water intake was measured as before.
Statistics. Significance in the blood pressure studies was examined by ANOVA with Bonferroni post hoc testing within groups across time. In addition, ANOVAs or independent t-tests were run between test groups at each time. Significance in the ingestion studies was examined by independent t-tests or ANOVAs with Bonferroni post hoc testing at each time point. A probability <5% was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
|
|
|
|
-adrenergic receptor blocker phentolamine completely blocked the blood pressure rise induced by central administration of 195 pmol of IMDL (Table 2).
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
200 pg/ml, which is higher than the reported levels of circulating AM (10–100 pg/ml; Ref. 22) and CGRP (2–25 pg/ml; Ref. 9), but may be a reflection of the properties of the RIA used. IMD in plasma may result from spillover of locally produced IMD into the circulation, or the peptide may be made by endothelial and vascular smooth muscle cells like AM (24) or released by neurons into the circulation like CGRP (31). The highest IMD levels within tissues were found in kidney and stomach, a finding in agreement with RT-PCR expression studies reported in the original description of the peptide (26). IMD immunoreactivity was found in extracts of both the anterior and posterior/intermediate lobes of the pituitary, confirming the initial immunohistochemical staining (20). IMD was also detected in heart and lung, but no peptide was detected in adrenal tissue, again in agreement with expression studies (26). IMD immunoreactivity was detected throughout the CNS, with highest peptide levels in the hypothalamus. It remains to be determined whether the high levels of IMD detected in hypothalamic extracts reflect uptake of circulating IMD of peripheral origin or mirror peptide synthesized locally within the hypothalamus. Nevertheless, the presence of immunoreactive IMD in brain suggests that the peptide may have a physiological role in the regulation of CNS function. Overall, these findings suggest that IMD could act as a local factor in the tissue in which it is produced or that IMD may act as a circulating factor. It will be important in future studies to examine whether IMD levels in plasma and tissue fluctuate in concert with various physiological and pathophysiological stimuli. The ability of IMD to activate both AM and CGRP receptors suggested that IMD may have biological effects similar to those of CGRP and AM. Initial studies described the potent effect of peripheral (iv and ip) administration of IMDL/AM2 to lower blood pressure and elevate heart rate in mice (26) and rats (20). This finding is not surprising because both AM, through CRLR and RAMP2/3, and CGRP, through CRLR and RAMP1, lower blood pressure (5, 7). In concurrence with the original findings, we demonstrate the ability of IMDL administered iv to dose-dependently lower mean arterial pressure and elevate heart rate at lower doses than originally described (Fig. 1). In addition, we demonstrate that the effects of IMDL on blood pressure are similar to those of CGRP and significantly less potent than those of AM given iv (Fig. 2A). The blood pressure effect of IMDL is slightly greater than that of CGRP, however, and may represent a combined effect of IMDL at both the AM and CGRP receptors. There was no difference in the effects of three peptides on heart rate at the dose we used (Fig. 2B); however, the effects of IMDL given ip on heart rate were greater than those of AM (20), suggesting that like AM (18) and CGRP (3) IMDL may exert direct inotropic and chronotropic effects in vivo. Roh et al. (20) demonstrated that the blood pressure-lowering effects of IMDL could be blocked with the CGRP receptor antagonist CGRP(8–37) and partially blocked with AM(22–52). All of AM's (7) and some of CGRP's (through spillover onto CRLR/RAMP3; Refs. 1, 12, 13) effects to lower blood pressure are thought to be mediated via the production of NO. We therefore pretreated rats with a NO synthase inhibitor, L-NMMA, to determine whether the effects of IMDL on blood pressure were mediated through NO formation. Blockade of NO production did not alter the effects of IMDL on blood pressure (Fig. 3). This finding, in addition to the ability of CGRP(8–37) to completely inhibit IMDL's hypotensive effects (20), would indicate that the actions of iv administered IMDL on blood pressure are likely mediated by the CGRP receptor CRLR/RAMP1. Alternatively, it is possible that IMDL interacts with an as yet unidentified receptor.
The CRLR and RAMPs are expressed throughout the CNS (17), and both AM and CGRP exert effects within the brain (21, 28, 29). Furthermore, we have demonstrated the presence of immunoreactive IMD in the CNS (Table 1), suggesting that IMDL may have actions within brain. When given icv, AM (22) and CGRP (8) elevate mean arterial pressure and heart rate through activation of the sympathetic nervous system. We therefore hypothesized that central administration of IMDL would also activate the sympathetic nervous system, leading to elevated blood pressure and heart rate. Indeed, icv administration of IMDL resulted in dose-related elevations of mean arterial pressure and heart rate (Fig. 4). Although all three doses of IMDL resulted in elevated heart rate when given centrally, the delayed heart rate response to the two higher doses of IMDL may have reflected an initial baroreflex-mediated dampening of the heart rate response. The effect of IMDL on mean arterial pressure was abrogated by the -adrenergic receptor antagonist phentolamine (Table 2), suggesting that the actions of IMDL centrally on blood pressure are through the sympathetic nervous system. The effects of icv administration of IMDL are most like those of CGRP (i.e., potent, long-lasting elevations in both mean arterial pressure and heart rate; Fig. 5); therefore, it is not surprising that CGRP(8–37), at a dose that can block the effects of CGRP (data not shown), was able to partially attenuate the effects of central administration of IMDL on blood pressure and heart rate (Fig. 6). The reversal was not complete, however, and it may be that some of the effects of IMDL administered icv on blood pressure and heart rate were through the AM receptors or an unidentified receptor. We were unable to test whether IMDL was acting through the AM receptors centrally because the only available commercial antagonist for the receptors, AM(22–52), must be given in extremely high amounts. The antagonist is not sufficiently soluble, in our hands, at those concentrations to permit use of a reasonable volume for icv injections.
CGRP, when given peripherally or centrally, is a potent inhibitor of feeding (14, 15, 19). Central or peripheral administration of high doses of AM can also cause a small inhibition of food intake (19, 27) likely by spillover onto CGRP receptors. Roh et al. (20) reported that ip administration of IMDL inhibited food intake. Like CGRP and AM, the IMDL-induced inhibition of feeding may be due in part to suppression of gastric emptying (19, 20). Here, we examined the effects of icv administration of IMDL on food intake. As predicted based on the actions of CGRP and AM on food intake, central administration of IMDL inhibited food intake in sated rats (Fig. 7A) as well as in rats restricted from food and water overnight (Fig. 8A). Central IMDL-treated rats also consumed less water than saline-injected controls in both the sated (Fig. 7B) and restricted (Fig. 8B) states. It is interesting that the major inhibitory effect of IMDL on food and water intakes in sated rats appeared to occur within the first hour of experimentation, after which the two groups' consumption patterns were parallel. On the other hand, in the restricted state, where animals were driven to eat and drink, both groups consumed similar amounts of food and water initially and the inhibitory effect of IMDL did not appear until later in the experimental protocol. Other than food and water intake, IMDL-treated rats did not have any readily apparent changes in behavior (i.e., grooming, locomotion) compared with controls. It was somewhat surprising that IMDL inhibited both food and water intake because, at least in terms of blood pressure, IMDL appears to have actions similar to those of CGRP and it has been reported that although CGRP inhibits food intake it has no effect on water intake (15). However, central administration of AM inhibits water intake in overnight water-restricted, but not sated, rats (22), and thus the actions of IMDL centrally on water intake could be due to an action on the CRLR and RAMP2/3 (AM receptors). Again, we were not able to test this because of the lack of an appropriate, commercially available AM antagonist for CNS studies. The actions of IMDL given icv on water intake are dose dependent and are extremely potent, with inhibition observed even after administration of only 65 pmol of IMDL (Fig. 9B). To dissect whether the decrease in water intake was due to the inhibition of food intake, we gave IMDL or vehicle to animals denied food and water overnight. After the icv injection, rats were allowed access to water but not to food. In this experiment, IMDL-treated rats continued to ingest less water than controls (Fig. 9A), indicating that the effects of IMDL centrally to inhibit water intake were, in all likelihood, independent from the peptide's effects on food intake. Interestingly, like AM (6, 30) IMDL has been reported to have further actions on fluid and electrolyte homeostasis by acting within kidney to increase renal blood flow, diuresis, and natriuresis (9). These findings further support the hypothesis that IMDL can elicit combined actions at both the AM and CGRP receptors.
Clearly, IMD has the potential to be an important physiological regulator of blood pressure, appetite, and fluid balance. It will be important to examine whether IMD levels in plasma and tissues change under physiological conditions such as hypertension, hunger/satiety, and volume overload. Another priority will be to determine whether IMD binds to the AM/CGRP receptors in vivo as the work of Roh et al. (20) suggests or whether there are as yet unidentified IMD receptors. If AM, CGRP, and IMD all activate the same receptor systems, examination of the interactions of the three peptide systems is necessary. For example, do all three peptide levels change in concert with one another and can one peptide compensate for the loss of another? To truly understand the physiological role of IMD it will be important to create animal models in which the peptide's production or action (i.e., embryonic knockout, antisense, small interfering RNA, etc.) has been compromised.
In summary, we have shown that IMDL given iv lowers blood pressure through an NO-independent mechanism. Central administration of IMDL elevated blood pressure and heart rate by activating the sympathetic nervous system. Central administration of IMDL also inhibited food and water intake. The actions of IMDL appear to be related to the combined effects of CGRP and AM. It will be important to examine what cells in brain make IMDL and, more importantly, what controls IMDL release. We also need to determine what other actions the peptide may express in brain. In addition, the actions of IMDL in the periphery should be examined more closely because multiple pharmacological and, in some cases physiological, actions of AM and CGRP have been described (reviewed in Ref. 4).
Perspectives
AM has been recommended for use clinically in congestive heart failure and septic shock and is being investigated as a potential therapeutic for myocardial infarction and other cardiovascular disorders (4). Clinical applications of CGRP have also been examined (4, 29). CGRP, like AM, has been shown to be beneficial in treating congestive heart failure in humans and has been demonstrated in rats to provide tissue protection after ischemic injury. In addition, the roles of CGRP in appetite regulation and migraines are being examined for potential clinical applications. Because of IMDL's ability to activate both the AM and CGRP receptors and the apparent effects of the peptide through both sets of receptors, this peptide may also represent a promising therapeutic agent. It will be important to closely examine the potential regulatory functions IMD may play in normal physiology and in the pathophysiology of all these disease states.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
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 |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. K. Samson, M. M. White, C. Price, and A. V. Ferguson Obestatin acts in brain to inhibit thirst Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R637 - R643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Chauhan, U. Yallampalli, L. Reed, and C. Yallampalli Adrenomedullin 2 Antagonist Infusion to Rats During Midgestation Causes Fetoplacental Growth Restriction Through Apoptosis Biol Reprod, December 1, 2006; 75(6): 940 - 947. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Dong, M. M. Taylor, W. K. Samson, and J. Ren Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes J Appl Physiol, September 1, 2006; 101(3): 778 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fujisawa, Y. Nagai, A. Miyatake, K. Miura, T. Shokoji, A. Nishiyama, S. Kimura, and Y. Abe Roles of adrenomedullin 2 in regulating the cardiovascular and sympathetic nervous systems in conscious rats Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1120 - H1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hashimoto, S. Hyodo, M. Kawasaki, T. Mera, L. Chen, A. Soya, T. Saito, H. Fujihara, T. Higuchi, Y. Takei, et al. Centrally administered adrenomedullin 2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats Am J Physiol Endocrinol Metab, November 1, 2005; 289(5): E753 - E761. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
