Vascular reactivity to calcitonin gene-related peptide is enhanced in subtotalnephrectomy-salt induced hypertension

Scott C. Supowit,1 Khurshed A. Katki,2 Travis W. Hein,3 Prakash Gupta,1 Lih Kuo,4 Ian M. Dickerson,5

and Donald J. DiPette6

Departments of 1Cell Biology and Anatomy and 6Medicine, University of South Carolina School of Medicine, Columbia,South Carolina; Departments of 2Medicine, 3Surgery, and 4Systems Biology and Translational Medicine, Texas A&M HealthScience Center College of Medicine, Temple, Texas; and 5Department of Neurobiology and Anatomy, University of Rochester,Rochester, New York

Submitted 6 July 2009; accepted in final form 19 May 2010

Supowit SC, Katki KA, Hein TW, Gupta P, Kuo L, DickersonIM, DiPette DJ. Vascular reactivity to calcitonin gene-related peptideis enhanced in subtotal nephrectomy-salt induced hypertension. Am JPhysiol Heart Circ Physiol 301: H683–H688, 2011. First publishedJune 10, 2011; doi:10.1152/ajpheart.00598.2009.—In subtotal ne-phrectomy (SN)- and salt-induced hypertension, calcitonin gene-related peptide (CGRP) plays a compensatory role to attenuate theblood pressure increase in the absence of an increase in the neuronalsynthesis and release of this peptide. Therefore, the purpose of thisstudy was to determine whether the mechanism of this antihyperten-sive activity is through enhanced sensitivity of the vasculature to thedilator actions of this neuropeptide. Hypertension was induced inSprague-Dawley rats by SN and 1% saline drinking water. Controlrats were sham-operated and given tap water to drink. After 11 days,rats had intravenous (drug administration) and arterial (continuousmean arterial pressure recording) catheters surgically placed and werestudied in a conscious unrestrained state. Baseline mean arterialpressure was higher in the SN-salt rats (157 � 5 mmHg) comparedwith controls (128 � 3 mmHg). Administration of CGRP (andadrenomedullin) produced a significantly greater dose-dependent de-crease in mean arterial pressure in SN-salt rats compared with controls(�2.0-fold for both the low and high doses). Interestingly, isolatedsuperior mesenteric arterioles from SN-salt rats were significantlymore responsive to the dilator effects of CGRP (but not adenomedul-lin) than the controls (pEC50, SN-salt, 14.0 � 0.1 vs. control, 12.0 �0.1). Analysis of the CGRP receptor proteins showed that only thereceptor component protein was increased significantly in arteriolesfrom SN-salt rats. These data indicate that the compensatory antihy-pertensive effects of CGRP result from an increased sensitivity of thevasculature to dilator activity of this peptide. The mechanism may bevia the upregulation of receptor component protein, thereby providinga more efficient coupling of the receptor to the signal transductionpathways.

experimental hypertension; neuropeptide receptors; sensory nervoussystem

�-CALCITONIN GENE-RELATED peptide (CGRP) is a 37-amino acidneuropeptide derived from the tissue-specific splicing of theprimary transcript of the calcitonin/CGRP gene (1, 19). Calci-tonin is produced mainly in the C cells of the thyroid whileCGRP synthesis occurs almost exclusively in regions of thecentral and peripheral nervous systems (1, 8, 13, 19). CGRPand its receptors are widely distributed in the nervous andcardiovascular systems (1). In the peripheral nervous system,common sites of CGRP synthesis are the dorsal root ganglia.

These structures contain the cell bodies of sensory nerves thatterminate centrally in the spinal cord and peripherally in alltissues (1, 8, 13, 19). Blood vessels in all vascular beds aresurrounded by a dense perivascular network of CGRP-contain-ing nerves. Receptors for CGRP have been identified in themedia, intima, and endothelial layer of resistance vessels (1, 8,13, 19).

CGRP dilates multiple vascular beds, with the coronaryvasculature being a particularly sensitive target (2, 3, 13).Adrenomedullin (AM), a CGRP-related protein, is also a po-tent vasodilator that has antihypertensive activity (1, 13, 19).Systemic administration of CGRP (and AM) decreases bloodpressure in normotensive and hypertensive animals and hu-mans (1–3, 13). The primary mechanism responsible for thisreduction in blood pressure is peripheral arterial dilation (1–3,13, 19). The receptor for CGRP and AM is a complex ofproteins with a common ligand-binding component namedcalcitonin-like receptor (CLR). Ligand specificity is deter-mined by coexpression of either of three chaperone proteinsRAMP1 (CGRP) or RAMP2/3 (AM; see Ref. 5). Three func-tions for RAMPs have been defined: they aid CLR traffickingto the cell surface, define the pharmacological specificity ofCLR, and determine the glycosylation state of CLR. A thirdaccessory protein, named CGRP-receptor component protein(RCP), is also required (4). RCP is an intracellular peripheral-membrane protein that appears to couple the CLR/RAMPheterodimer to the cellular signal transduction pathways (4, 5).The CGRP and AM receptors are coupled to G proteins and ina number of tissues, including vascular smooth muscle, in-crease intracellular cAMP (1, 13, 19).

We have reported that CGRP acts as a depressor mechanismto partially attenuate the blood pressure increase in threemodels of experimental hypertension, deoxycorticosterone-salt(DOC-salt; see Ref. 16), subtotal nephrectomy-salt (SN-salt;see Ref. 17), and NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension in pregnancy (6). In the DOC-salt model,the antihypertensive activity of CGRP was mediated primarilyby an upregulation of neuronal CGRP synthesis and release. Incontrast, in SN-salt hypertension, the compensatory depressoractivity occurred in the absence of any changes in CGRPexpression and release, suggesting that the vasculature mightbe hyperresponsive to the vasodilator activity of CGRP andperhaps AM. Therefore, the purpose of this current study wasto determine if there is a significant in vivo increase in vascularreactivity to CGRP or AM in SN-salt hypertension and todetermine if this hyperresponsiveness could be recapitulated inisolated arterial preparations. Expression of CGRP receptor

Address for reprint requests and other correspondence: S. C. Supowit, 6439Garners Ferry Road, Bldg. 1, Columbia, SC 29208 (e-mail: Scott.Supowit@uscmed.sc.edu).

Am J Physiol Heart Circ Physiol 301: H683–H688, 2011.First published June 10, 2011; doi:10.1152/ajpheart.00598.2009.

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proteins was monitored in parallel with the physiologicalstudies to determine if there was a relationship between recep-tor protein expression and CGRP efficacy.

METHODS

Animals. Animal protocols were approved by the InstitutionalAnimal Care and Use Committee. Male Sprague-Dawley rats (HarlanSprague-Dawley, Indianapolis, IN), initially weighing 150 g, wereused for this study. For the surgical procedures, the rats were anes-thetized with ketamine and xylazine (80 and 4 mg/kg ip). SN-salthypertension was induced by the removal of the right kidney and�25% of the left kidney (17). Animals received 1% saline to drink.Sham-operated animals that received tap water were used as controls.The tail-cuff method (Narco Bio Systems, Austin, TX) was used torecord blood pressures before the surgeries and every 3 days thereaf-ter. The animals were studied 11 days after the surgical procedure.

Mean blood pressure determinations. The rats that received exog-enous CGRP or AM were anesthetized as described above. The leftcarotid artery was cannulated for continuous measurement of meanarterial pressure (MAP) and heart rate with a pressure transducerlinked to a recorder (Gould Instruments, Valley View, OH; see Ref.16). The right jugular vein was also cannulated for infusion of eithervehicle (saline), CGRP, or AM (American Peptide, Sunnyvale, CA).Hemodynamic studies were performed �3 h after surgery, with therats fully awake and unrestrained. Values for the observed bloodpressure changes following CGRP or AM administration were deter-mined from the integrated areas under the curve. These values werethen normalized to the baseline MAP of either the SN-salt or controlgroups.

Functional assessment of isolated mesenteric arterioles. For thesestudies, additional groups of SN-salt (n � 10) and control (n � 11)rats were used. The tail-cuff method was used to determine bloodpressures before initiation of the SN-salt protocol and every 3 daysthereafter as described above. After 11 days, the SN-salt hypertensiveand control rats were anesthetized, and the small intestine wasremoved. Third-order arterioles (�1 mm in length; 40–60 �m inter-nal diameter) were isolated from the mesentery. The arteriole was thentransferred to a chamber containing physiological salt solution (PSS)with 1% albumin and equilibrated at ambient temperature. Both endsof the arteriole were then cannulated using glass micropipettes filledwith PSS-albumin solution (10). The vessel was transferred to thestage of an inverted microscope coupled to a video camera and videomicrometer for continuous measurement of the internal diameter. Themicropipettes were connected to pressure reservoirs, and the vesselwas pressurized to 60 cm H2O intraluminal pressure without flow.Arterioles were bathed in PSS-albumin at 36–37°C to allow devel-opment of basal tone (�60 min). The concentration-dependent re-sponses to CGRP (0.1 aM to 10 nM; American Peptide), AM (0.1 pMto 1 �M; American Peptide), or sodium nitroprusside (SNP, 1 nM to0.1 mM; Sigma-Aldrich, St. Louis, MO) were examined. In somevessels, dilation in response to CGRP and SNP was evaluated in theabsence and presence (30-min pretreatment) of the CGRP receptorantagonist CGRP8–37 (1 �M; American Peptide). Arterioles wereexposed to each dose of the vasodilator agents for 1–2 min until astable diameter was established. At the end of each experiment, thevessel was relaxed with 0.1 mM SNP to obtain its maximal diameterat 60 cm H2O intraluminal pressure. Diameter changes in response toagonists were normalized to this maximal dilation and expressed as apercentage of maximal dilation (10). Data are reported as means �SE. The EC50 values are presented as the negative logarithm (pEC50)and calculated using GraphPad Prism software.

Western blot analysis. The RAMP1, RCP, and CLR antibodieswere provided by one of the authors (Dickerson; see Refs. 4 and 14).RAMP1 is a rabbit polyclonal antibody against the synthetic peptideMVTACRDPDYGTL of mouse RAMP1. RCP is a chicken poly-clonal antibody against mouse RCP sequence EEQIEALLHTVT

conjugated to keyhole limpet hemocyanin. CLR is a rabbit polyclonalantibody against the mouse CLR sequence GYSHDCPTEHLNGK.The RAMP2 and RAMP3 antibodies were purchased from Santa CruzBiotechnology (Santa Cruz, CA), and the anti-actin antibody wasobtained from Sigma Aldrich. For this series of experiments, separategroups of SN-salt (n � 12) and control (n � 12) were used. Again,systolic blood pressures were determined by the tail-cuff method asdescribed previously. Superior mesenteric arteries from SN-salt andcontrol rats were isolated and frozen. Protein content of the membranepreparations was measured by the Bradford method (Bio-Rad Labo-ratories, Hercules, CA), and equal amounts of cell lysates weresubjected to 12.5% SDS-PAGE and transferred to membranes. Eachsample was analyzed a minimum of three times. Membranes wereincubated for 3 h with the indicated antiserum, washed, and thenincubated again with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Arlington Heights, IL). Immuno-complexes were then visualized using chemiluminescence detection.For all of the blots, the linearity of the chemoluminescence signal withinput protein was verified.

Statistical analysis. Statistical comparisons of data were performedby Student’s t-test or by two-way ANOVA followed by the Bonfer-roni multiple-range test, as appropriate. A value of P � 0.05 wasconsidered significant.

RESULTS

Effects of CGRP and AM on blood pressure. Systolic bloodpressures were determined by the tail-cuff method beforesurgery and every three days thereafter. The average systolicblood pressures were 210 � 11 and 143 � 4 mmHg for theSN-salt and control animals, respectively. For the in vivodose-response studies, separate groups of SN-salt (n � 6) andcontrol (n � 6) rats were used for each dose of agonist. On day11 after initiation of the SN-salt protocol, rats had arterial (forcontinuous MAP recording) and intravenous (for drug admin-istration) catheters surgically implanted and were studied in afully awake and unrestrained state. The changes in MAP weredetermined by calculating the integrated areas under the curve.Because the basal MAP levels were different between theSN-salt (157 � 5 mmHg) and control (128 � 3 mmHg)animals, it was necessary to normalize the values obtained forthe CGRP- and AM-induced MAP decreases to the basalMAPs. Therefore, the data in Figs. 1 and 2 are expressed as apercentage decrease in baseline MAP. Administration of saline(0.1 ml iv) did not significantly change MAP in either group(data not shown). In contrast, administration of a bolus dose ofCGRP (0.128 nM/kg) produced a 9.2 � 2.6% decrease in MAP(11 mmHg maximum reduction) in the control rats and an18.3 � 1.7% MAP decrease (27 mmHg maximum reduction)in the hypertensive animals (Fig. 1). Likewise, the higher 0.64nM/kg dose of CGRP produced a 17.5 � 1.0% MAP decrease(20 mmHg maximum reduction) in the control rats and a 25 �2.5% MAP decrease (40 mmHg maximum reduction) in thehypertensive animals. The depressor effects of CGRP (andAM) occurred rapidly (10–15 s) and lasted for �15–20 and30–35 min for the low and high doses, respectively. Similarresults were obtained following administration of AM (Fig. 2).The lower dose of AM (0.5 nM/kg) resulted in a 5.1 � 0.4%decrease in MAP (8 mmHg maximum reduction) in the controlrats and a 12.2 � 1.0% MAP decrease (21 mmHg maximumreduction) in the hypertensive animals. The 1 nM/kg doseproduced a 9.3 � 0.4% decrease in MAP (15 mmHg maximumreduction) in the control rats and a 21.2 � 1.7% baseline

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decrease in MAP (32 mmHg maximum reduction) in theSN-salt rats.

Effect of CGRP and AM on mesenteric arterioles. Mesen-teric arterioles from SN-salt (n � 10) and control (n � 11) ratswere isolated and pressurized without flow. There were nosignificant differences in resting diameter (control, 58 � 3 vs.SN-salt, 58 � 4 �m), maximal diameter (control, 92 � 6 vs.SN-salt, 98 � 5 �m), and basal tone (control, 64 � 3%vs. SN-salt, 58 � 2%) in the vessels from the two groups.Treatment of the arterioles from control and SN-salt rats withCGRP produced concentration-dependent dilations (Fig. 3A);however, the arterioles from the SN-salt rats displayed amarked leftward shift of the curve, indicating a significantincrease in vascular reactivity to CGRP (pEC50, SN-salt,14.0 � 0.1 vs. control, 12.0 � 0.1). To ensure that the effectsof CGRP were mediated through the CGRP receptor, a con-centration-response curve was generated using arterioles fromboth groups in the presence of the CGRP receptor antagonist CGRP8–37. The antagonist inhibited the dilator activity of

vessels from the SN-salt (Fig. 3B) and control (data not shown)rats in response to CGRP without significantly altering theresponse to the highest concentration (10 nM) of CGRP. Incontrast, the mesenteric arterioles from both groups weremarkedly less responsive to AM than to CGRP, and theconcentration-dependent dilation curves were virtually identi-cal between the two groups (Fig. 4).

To determine whether the arteriole preparations would re-spond differently to a receptor-independent vasodilator, bothwere treated with increasing concentrations of the nitric oxidedonor SNP. As shown in Fig. 5A, the concentration-responsecurves were similar (pEC50, SN-salt, 7.0 � 0.2 vs. control,7.1 � 0.2), and treatment of SN-salt vessels with CGRP8–37

did not alter vasodilation to SNP, indicating that the CGRPantagonist did not exert a nonspecific inhibitory effect onvascular reactivity (Fig. 5B). Similar results were obtainedwhen the arterioles from the control rats were treated with theCGRP receptor antagonist (data not shown).

Western blot analysis of the CGRP/AM receptor. The finalseries of experiments used Western blot analysis of mesenteric

Fig. 1. The mean arterial pressure (MAP) of subtotal nephrectomy (SN)-salthypertensive rats is more sensitive to exogenous calcitonin gene-relatedpeptide (CGRP). Groups (n � 6) of SN-salt and control rats were instrumentedfor MAP recording and CGRP administration as described in the text. With therats fully awake and unrestrained, bolus doses of the indicated amounts ofCGRP were given intravenously (0.1 ml in saline). Percent baseline MAPvalues are reported as means � SE. **P � 0.01, SN-salt vs. control rats at thelower CGRP dose; SN-salt vs. control rats at the higher CGRP dose. *P �0.05, SN-salt rats at the higher CGRP dose vs. SN-salt rats at the lower CGRPdose.

Fig. 2. The MAP of SN-salt hypertensive rats is more sensitive to exogenousadrenomedullin (AM). Groups (n � 6) of SN-salt and control rats wereinstrumented for MAP recording and AM administration as described in thetext. With the rats fully awake and unrestrained, bolus doses of the indicatedamounts of AM were given intravenously (0.1 ml in saline). Percent baselineMAP values are reported as means � SE. **P � 0.01, SN-salt vs. control ratsat the lower AM dose; SN-salt vs. control rats at the higher AM dose; SN-saltrats at the higher AM dose vs. SN-salt rats at the lower AM dose.

Fig. 3. Isolated mesenteric arterioles from SN-salt hypertensive rats arehyperresponsive to CGRP. A: mesenteric arterioles isolated and pressurizedwithout flows from SN-salt (Œ, n � 10) and control (�, n � 11) rats weretreated with increasing concentrations of CGRP. B: arterial preparations fromSN-salt rats (n � 6) were treated with increasing concentrations of CGRP inthe absence (�) and presence (Œ) of CGRP8–37. Values shown are means � SE.*P � 0.05, SN-salt vs. control (A) and control in the presence of CGRP8–37 vs.control in the absence of the antagonist (B).

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artery membrane preparations from control (n � 12) andSN-salt (n � 12) animals to determine whether the increase invascular reactivity to CGRP (and AM) in SN-salt hypertensionwas associated with alterations in the levels of one or more ofthe components of the CGRP/AM receptor. Figure 6 is arepresentative composite blot that indicated that there were nosignificant alterations in the levels of RAMPs 1–3 between theSN-salt and control groups. Figure 7 is a representative com-posite blot comparing the levels of CLR and RCP between thetwo groups. CLR content was unchanged in the SN-salt arterialpreparations compared with controls. In contrast, the expres-sion of RCP was increased 1.4-fold in the hypertensive rats.

DISCUSSION

This paper reports the following three new observations:1) the in vivo hypotensive effects of exogenous CGRP and AMwere significantly greater in SN-salt hypertensive rats than incontrols; 2) the in vitro vascular reactivity of mesentericarterioles to CGRP, but not AM, was enhanced significantly inSN-salt hypertension; and 3) RCP content was increased sig-nificantly in the SN-salt arterioles in the absence of anychanges in CLR or RAMPs 1–3. These findings are supportedby other studies showing that CGRP (and AM) is a potentregulator of smooth muscle (vascular, uterine, gastrointestinal

tract) function (4, 7, 12, 14, 18, 20). These activities areregulated at the level of the vasculature via mechanisms thatare, in most cases, correlated with alterations in the CGRPreceptor (7, 9, 11, 12, 15, 18, 20). For example, alterations inCGRP potency in the absence of any changes in CGRPreceptor number have been reported. Using isolated hypoxicporcine intramyocardial arteries, Hasbak et al. (7) demon-strated increased affinity of the receptor for CGRP, increasedvascular cAMP, and enhanced vasorelaxant effects of CGRP,but not AM, without any changes in CLR or RAMP1 and

Fig. 6. RAMP1–3 levels do not change in the vasculature of SN-salt hyper-tensive and control rats. Protein extracts isolated from SN-salt (n � 6) andcontrol (n � 6) superior mesenteric arteries were fractionated by PAGE andtransferred to a nylon membrane. Separate blots were performed for incubationwith RAMPs 1–3. Each blot was stripped followed by incubation with the actinantibody. The blot shown is a representative composite blot. RAMP-to-actinratios were determined by scanning laser densitometry. Values are expressedas means � SE.

Fig. 4. Isolated mesenteric arterioles from SN-salt hypertesnsive rats are nothyperresponsive to AM. Mesenteric arterioles isolated and pressurized withoutflow from SN-salt (Œ, n � 7) and control (�, n � 8) rats were treated withincreasing concentrations of AM. Values shown are means � SE.

Fig. 5. SN-salt hypertension does not globally in-crease vascular responsiveness. A: mesenteric arteri-oles isolated and pressurized without flow from SN-salt (Œ, n � 7) and control (�, n � 8) rats were treatedwith increasing concentrations of sodium nitroprus-side (SNP). B: arterioles from SN-salt rats (n � 8)were treated with increasing concentrations of SNP inthe absence (�) and presence (Œ) of CGRP8–37. Val-ues are expressed as means � SE.

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RAMP2 levels. Having performed experiments to rule out theparticipation of other vasodilator agents or peptidases, theauthors concluded that the increased affinity of the receptor forCGRP was likely due to a conformational change at the CGRPreceptor site. RCP was not assessed in these studies, thus it ispossible to postulate a stimulatory role for RCP in this setting.RCP may act as an allosteric modifier to influence CLRconformation by increasing the affinity of CLR for its ligandand/or increasing the signaling capacity of the receptor.

It is interesting that our isolated vessel results agree with thefindings described above; in both cases, the vessels becamehyperresponsive to CGRP but not AM. Based on our in vivostudies and observations that CLR and RAMP1–3 did notchange appreciably, the lack of a similar increase in AMresponsiveness is puzzling. However, these experiments onlylooked at expression of receptor proteins and not their inter-actions. It is possible that RAMP2 and -3 may be segregatedfrom CLR under conditions of hypoxia or hypertension, lead-ing to a preferential coupling of CLR with RAMP1. In thisscenario, an increase in RCP would enhance CLR activity, and,if CLR was primarily coupled with RAMP1, then an increasedresponsiveness to CGRP, but not AM, would be observed.Alternatively, differences in the responsiveness of differentvascular beds to CGRP and AM under normal physiologicalconditions have been demonstrated (9). In this report, CGRP orAM was found to cause changes in systemic and regionalhemodynamics in rats assessed by fluorescent microspheres.Although CGRP and AM both produced a similar globalhypotensive response, there were significant differences in

regional vasodilator effects of the two peptides. Indeed, bloodflow to the gut was increased by CGRP to a much greaterdegree than by AM. Our results are consistent with these data,but the mechanism underlying differences in regional organblood flows evoked by CGRP and AM are not clear.

Our finding that the enhanced vascular responsiveness toCGRP (and AM) was associated with increased levels of RCPis also consistent with other reports demonstrating that RCP isa potential regulator of CLR/RAMP function in vivo. It waspreviously reported that RCP in dorsal root ganglia and dorsalhorn neurons of the rat can be regulated by CGRP receptorblockade and pain-related stimuli (11). From these studies, itwas concluded that modulation of RCP expression was directlyrelated to altered signaling capacity at the CGRP receptor. Inaddition, CGRP also inhibits the contraction of the myome-trium during pregnancy and the estrous cycles (12, 18, 20). Inthe pregnant mouse, there was an increase in myometrialreactivity to CGRP that correlated with an increase in uterineRCP protein. At parturition, there was a rapid decline in RCPcontent that coincided with the loss of CGRP inhibition ofacetylcholine-induced uterine contractions. Similarly, sensitiv-ity of the myometrium to CGRP was found to vary during themouse estrous cycle in a progesterone-dependent manner, andsensitivity to CGRP correlated with RCP expression. In thesestudies, CLR and RAMP1 expression was measured but didnot correlate with CGRP responsiveness. In this current study,we have demonstrated that the vascular responsiveness toCGRP increased with the increased expression of RCP, similarto that observed previously in the uterus. Interestingly, thisenhanced vascular response to CGRP was not accompanied byan increase in either CLR or RAMP1. Thus, it appears that theincreased vascular response to CGRP by an upregulation ofRCP expression is a novel method of counterregulation forincreased blood pressure that results in increased signalingfrom a constant population of CGRP receptors.

If RCP does play a role in mediating the enhanced vascularreactivity to CGRP (and AM) in SN-salt hypertension, then thequestion arises as to its function. RCP is a 148-amino acidintracellular protein (4, 14). RCP has no homology to CLR, theRAMPs, or other sequences in GenBank and contains nodefined protein motifs that can be used to predict its function(4, 14). Structural analysis of RCP shows that it lacks hydro-phobic regions that could serve as transmembrane domains andcontains no consensus sequences for lipid attachment, indicat-ing that RCP associates with the membrane through ionicinteractions (4, 14). In addition, subcellular localization studiessuggest that RCP is a peripheral rather than an integral mem-brane protein, and it coimmunoprecipitates in a protein com-plex with CLR and a RAMP protein (4, 14). In vitro analysisof cultured cells that express functional CGRP and AM recep-tors using an antisense RCP construct for transfection demon-strated that inhibition of RCP expression sharply reducedCGRP- and AM-mediated cAMP production. Loss of RCP didnot affect ligand binding to the receptor or receptor density,indicating that RCP did not function as a chaperone, like theRAMP proteins, to route CLR to the cell surface but rather tocouple the receptor complex to the cellular signaling pathways.Furthermore, inhibition of RCP expression did not alter signaltransduction of other G protein-coupled receptors such as the�2-adrenergic and A2b adenosine receptors (4, 14).

Fig. 7. RCP but not CLR content is increased in the vasculature of SN-salthypertensive compared with control animals. Protein extracts isolated fromSN-salt (n � 12) and control (n � 12) superior mesenteric arteries werefractionated by PAGE and transferred to a nylon membrane. The membraneswere treated sequentially with the CLR, RCP, and actin antibodies. The blotshown is a representative composite blot. CLR-to-actin and RCP-to-actin ratioswere determined by scanning laser densitometry. Values are expressed asmeans � SE. *P � 0.05.

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To date, however, very little is known about the regulationof RCP levels in studies where changes in RCP content andCGRP receptor function were examined (11, 12, 18, 20).Likewise, the regulation of CLR and/or RAMP expression isunclear. RAMP1 appears to be induced by dexamethasonetreatment of cultured human coronary artery muscle cells, andthe cytokine tumor necrosis factor-� decreases CLR andRAMPs 1 and 2 in cultured coronary artery smooth muscle(2a). In addition, there is a complex modulation of CLR andRAMP levels following aortic banding and heart failure (2a).Sex steroids appear to increase CLR and RAMP levels in therat during pregnancy, and decreased CLR and RAMP1 proteinswere observed in vascular tissues in preeclamptic placentasalong with decreased CGRP-binding sites (20, 2a).

In summary, these data indicate that the in vivo vascularresponsiveness to the dilator activity of CGRP and AM isenhanced in SN-salt hypertension and provide a mechanism toexplain how CGRP plays a compensatory depressor role topartially attenuate the blood pressure elevation in the absenceof increased neuronal CGRP synthesis and release. Further-more, this increase in vascular sensitivity may be mediated byan increase in RCP, a protein that represents a novel mecha-nism for regulating signal transduction at G protein-coupledreceptors via enhanced coupling between the ligand-bindingcomponent of the receptor and the signaling mechanism(s).

ACKNOWLEDGMENTS

We thank Dr. Xin Xu and Natalie Xu for technical assistance.

GRANTS

This work was supported by National Institutes of Health Grants HL-067472 (D. J. DiPette and S. C. Supowit) and DK-52328 (I. M. Dickerson).

DISCLOSURES

No conflicts of interest are declared by the authors.

REFERENCES

1. Brain SD, Grant AD. Vascular actions of calcitonin gene-related peptideand adrenomedullin. Physiol Rev 84: 903–934, 2004.

2. Brain SD, William TJ, Tippins JR, Morris HR, MacIntyre I. Calci-tonin gene-related peptide is a potent vasodilator. Nature 313: 54–56,1985.

2a.Dickerson I. The CGRP-receptor component protein: a regulator for CLRsignaling. In: The Calcitonin Gene-Related Peptide Family: Form, Func-tion, and Future Perspectives, edited by Hay DL and Dickerson IM. NewYork, NY: Springer Business Media, 2010, p. 59–73.

3. DiPette DJ, Schwarzenberger K, Kerr N, Holland OB. Dose dependentsystemic and regional hemodynamic effects of calcitonin gene-relatedpeptide. Am J Med Sci 297: 65–70, 1989.

4. Evans BN, Rosenblatt MI, Mnayer LO, Oliver KR, Dickerson IM.CGRP-RCP, a novel protein required at CGRP and adrenomedullinreceptors. J Biol Chem 275: 31438–31443, 2000.

5. Foord SM, Marshall FH. RAMPS: accessory proteins for seven trans-membrane domain receptors. TIPS 20: 184–187, 1999.

6. Gangula PR, Supowit SC, Wimalawansa SJ, DiPette DJ, YallampalliC. Calcitonin gene-related peptide is a depressor in NG-nitro-L-argininemethyl ester (L-NAME)-induced preeclampsia. Hypertension 29: 248–253, 1997.

7. Hasbak P, Eskesen K, Schifter S, Edvinsson L. Increased �-CGRPpotency and CGRP-receptor antagonist affinity in isolated hypoxic porcineintramyocardial arteries. Br J Pharmacol 145: 646–655, 2005.

8. Holzer P. Local effector functions of capsaicin-sensitive sensory nerveendings: involvement of tackykinins, calcitonin gene-related peptide andother neuropeptides. Neuroscience 45: 739–768, 1988.

9. Kawai J, Ando K, Shimosawa T, Harii K, Fujita T. Regional hemo-dynamic effects of adrenomedullin in Wistar rats: a comparison withcalcitonin gene-related peptide. Hypertension Res 25: 441–446, 2002.

10. Kuo L, Chilian WM, Davis MJ. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am J PhysiolHeart Circ Physiol 261: H1706–H1715, 1991.

11. Ma W, Chabot JG, Powell K, Jhamandas K, Dickerson IM, QuirionR. Localization and modulation of calcitonin gene-related peptide-recep-tor component protein-immunoreactive cells in the rat central and periph-eral nervous systems. Neuroscience 120: 677–694, 2003.

12. Naghashpour M, Rosenblatt MI, Dickerson IM, Dahl GP. Inhibitoryeffect of calcitonin gene-related peptide on myometrial contractility isdiminished at parturition. Endocrinology 138: 4207–4214, 1997.

13. Poyner DR, Sexton PM, Marshall I, Smith DM, Quirion R, Born W,Muff F, Fischer JA, Foord SM. The mammalian calcitonin gene-relatedpeptides, adrenomedullin, amylin, and calcitonin receptors. Pharmol Rev54: 233–246, 2002.

14. Prado MA, Evans-Bain B, Dickerson IM. Receptor component protein(RCP): a member of a multi-protein complex required for G-protein-coupled signal transduction. 30: 460–464, 2002.

15. Supowit SC, Watts SW, Zhao H, Wang D, DiPette DJ. Vascularreactivity to calcitonin gene-related peptide is enhanced in subtotal ne-phrectomy-salt hypertension (Abstract). Hypertension 36: 701, 2002.

16. Supowit SC, Zhao H, Hallman DM, DiPette DJ. Calcitonin gene-relatedpeptide is a depressor of deoxycorticosterone-salt hypertension in the rat.Hypertension 29: 945–950, 1997.

17. Supowit SC, Zhao H, Hallman DM, DiPette DJ. Calcitonin gene-relatedpeptide is a depressor in subtotal nephrectomy hypertension. Hypertension31: 391–396, 1998.

18. Thota C, Gangula PRR, Dong YL, Yallampalli C. Changes in theexpression of calcitonin receptor-like receptor, receptor activity-modify-ing protein (RAMP) 1, RAMP 2, and RAMP 3 in rat uterus duringpregnancy, labor, and by steroid hormone treatments. Biol Reproduc 69:1432–1437, 2003.

19. Wimalawansa SJ. Calcitonin gene-related peptide and its receptors:Molecular Genetics, physiology, pathophysiology, and therapeutic poten-tials. Endocrine Rev 17: 533–585, 1996.

20. Yallampalli C, Kondapaka SB, Lanlua p Wimalawansa SJ, GangulaPRR. Female sex steroid hormones and pregnancy regulate receptors forcalcitonin gene-related peptide in rat mesenteric arteries but not aorta. Biolof Reproduc 70: 1055–1062, 2003.

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