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Christchurch Cardioendocrine Research Group, University of Otago Christchurch, Christchurch 8140, New Zealand1 National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan
(Correspondence should be addressed to N Minamino; Email: minamino{at}ri.ncvc.go.jp)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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To date, there have been limited studies examining the bioactivity of CRSP-1 in vivo. Central administration of porcine CRSP-1 to rats suppressed food intake, increased body temperature, and decreased locomotor activity (Sawada et al. 2006). Systemic administration of porcine CRSP-1 in anaesthetized rats induced no significant change in arterial pressure but did induce a significant reduction in plasma Ca (Katafuchi et al. 2003a). There have been no in vivo studies of the biological actions of a CRSP-1 peptide administered to the species of origin. Accordingly, we here report the identification and in vitro characterization of ovine/caprine CRSP-1 and caprine CRSP-2. Furthermore, we have examined the integrative hemodynamic, hormonal, electrolyte, and renal effects of ovine CRSP-1 in normal conscious sheep.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Total RNAs were prepared from ovine thyroid gland and caprine hypothalamus using RNA-Bee reagent, and poly(A+) RNA was purified from the RNAs using an mRNA purification system (Takara, Shiga, Japan). Libraries were constructed from cDNAs synthesized from 3 µg poly(A+) RNAs using TimeSaver cDNA synthesis kit according to the manufacturer's protocol. Approximately, 3x105 independent clones were screened by the standard protocol using 32P-labeled porcine full-length CRSP-1-cDNA. Hybridization and washing were performed at 42 °C in 50% formamide, 6x SSC (pH 7.0), 5x Denhardt's solution and 0.5% SDS, and 65 °C in 0.5x SSC and 0.1% SDS respectively. The isolated positive clones were rescued as pBluescript SK- using Escherichia coli strain SOLR and helper phage. DNA sequences of these clones were determined by the dideoxynucleotide chain termination method using a DNA sequencer (310 Genetic Analyzer; Applied Biosystems, Foster City, CA, USA).
Peptide synthesis
Ovine/caprine CRSP-1 and caprine CRSP-2 were synthesized on an Abacus peptide synthesizer (Sigma Genosys) using Fmoc (N-(9-fluorenyl) methoxycarbonyl) strategy, purified by reversed phase HPLC. Correct synthesis of the peptides was verified by mass spectrometry and amino acid analysis, and their purity (more than 90%) was confirmed on a separate HPLC system.
In vitro studies: measurement of cAMP production
COS-7 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 100 µg/ml penicillin, 100 U/ml streptomycin, and 10% BSA (FBS) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. cDNAs of porcine CT-R, CL-R, and three RAMPs were ligated to a pcDNA3.1 mammalian expression vector as reported previously (Katafuchi et al. 2003a). Each combination of cDNAs of CT-R or CL-R, and one of three RAMPs was mixed at a ratio of 1:3. Transfection of mixed cDNAs was performed using Lipofectamine LTX with Plus reagent according to the manufacturer's protocol (Invitrogen). Following the transfection and further incubation for 24 h, the cells were washed twice with DMEM plus 20 mM HEPES (DMEM/HEPES (pH 7.4)) and incubated with DMEM/HEPES plus 0.5 mM 3-isobutyl-1-methyl xanthine (IBMX). Following a 15-min incubation, the medium was replaced into DMEM/HEPES plus IBMX containing 0.05% BSA and a peptide of interest, and further incubated for 30 min. Then, the incubation medium was collected, and an aliquot of it was succinylated, evaporated, and submitted to RIA for cAMP, as described previously (Katafuchi et al. 2003a).
Bioactivity of ovine CRSP-1 in conscious sheep
The Animal Ethics Committee of the University of Otago, Christchurch approved the study protocol. Six Coopworth ewes weighing 52–66 kg (Lincoln University Farm, Christchurch, New Zealand) were housed in an air-conditioned light-controlled room and received a diet of lucerne chaff and food pellets providing 75 mmol sodium and 150 mmol potassium per day. Under general anesthesia (induced by 17 mg/kg thiopentone sodium and maintained by a mixture of halothane, nitrous oxide, and oxygen), a carotid artery was cannulated (16 G Angiocath; Becton Dickinson, Sandy, UT, USA) for direct measurement of arterial pressure and heart rate. Polyethylene catheters were placed in the jugular veins for blood sampling and measurement of right atrial pressure (RAP), and a Swan-Ganz thermodilution catheter (Edwards Life Sciences, Irvine, CA, USA) was placed in the pulmonary artery via the jugular vein for measurements of cardiac output. A Foley catheter was placed per urethra in the bladder to allow continuous collection of urine. The animals recovered for at least 7 days before experiments.
Each animal was studied on two occasions 2 days apart receiving vehicle control (0.9% saline) and ovine CRSP-1. CRSP-1 was administered in incremental doses as i.v. infusions of 5 pmol/kg per min followed immediately by 50 pmol/kg per min each, for 60-min duration. Control and CRSP-1 were administered in a balanced, random-order design.
Arterial pressure and RAP were recorded using an in-house online data acquisition system, commencing 30 min before infusions and continuing for 60 min beyond end of the highest dose infusion. Heart rate and pressures were digitally integrated in 5-min recording periods and data recorded at preset intervals throughout the study. Cardiac output (thermodilution) was measured in triplicate (three values within 10%) at preset intervals for the duration of infusions. Calculated total peripheral resistance was calculated as mean arterial pressure (MAP) divided by cardiac output.
Venous blood was drawn at preset intervals during the study protocol. Blood was taken into chilled EDTA tubes, centrifuged, and the plasma stored at –80 °C before assay for aldosterone (Lun et al. 1983), catecholamines (Goldstein et al. 1981), cortisol (Lewis et al. 1992), plasma renin activity (PRA) (Dunn & Espiner 1976), amino-terminal pro-C-type natriuretic peptide (NT-CNP) (Prickett et al. 2001), and cAMP by commercially available kit (Biotrak, Amersham). Hematocrit was measured with each blood sample. Additional plasma samples were drawn into heparin tubes for routine biochemical analysis of calcium, sodium, potassium, creatinine, and albumin.
Statistical analysis
Results are expressed as the mean±S.E.M. Two-way ANOVA with time as a repeated measure was used to determine time and treatment differences between CRSP-1 and control arms of the study. Repeated-measures ANOVA is appropriate as it simultaneously incorporates the time and treatment (within animal) effects without direct recourse to numerous pairwise comparisons, and is thus routinely used in this context with group sizes of five to eight using similar experimental design (Charles et al. 1990, 1991, 2001, 2006). The appropriateness of the parametric analysis has been confirmed with attention to graphical displays of the residuals from the models confirming appropriate normality. Statistical significance was assumed at P<0.05. Where significant differences were identified by ANOVA, a priori Fisher's protected least-squares difference (LSD) tests were used to identify time points significantly different from time-matched control. Fisher's LSD in this context is a corrected pairwise comparison involving within-animal effects.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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To determine amino acid sequences of ovine and caprine CRSP precursors, ovine and caprine cDNA libraries were constructed, and were used for screening with 32P-labeled porcine CRSP-1 cDNA as a probe. Out of 3x105 clones of ovine and caprine cDNA libraries respectively, 35 and 14 positive clones were obtained. By nucleotide sequence analysis, all the 35 ovine positive clones were shown to have an identical sequence. Among the 14 caprine positive clones, 9 clones encoded one identical sequence, while the other 5 clones encoded a second sequence. Based on the similarity in amino acid sequences deduced from the nucleotide sequences along with biological activities, as described below, the ovine clone and one of the caprine clones were categorized as CRSP-1, while the other caprine clone was categorized as CRSP-2. The nucleotide sequences and their deduced amino acid sequences are shown in Fig. 1. All CRSPs identified in this study have typical features of peptides in the CGRP family with a signal sequence and N-terminal flanking peptide, but all sequences lack a donor glycine for the C-terminal amidation which exists in the members of CGRP family, in between mature peptide and C-terminal flanking peptide.
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Using synthetic peptides of ovine/caprine CRSP-1 and caprine CRSP-2, we first evaluated effects of CRSPs on porcine CT-R, which has strong affinity to porcine CRSP-1 and mediates its signal into cells by activating adenylyl cyclase. Porcine CT-R was transiently expressed in COS-7 cells, and then those cells were stimulated with ovine/caprine CRSP-1 or caprine CRSP-2 and compared with stimulation with porcine CRSP-1 and CT. As shown in Fig. 3A, all CRSP-1 peptides and porcine CT stimulated cAMP production in a dose-dependent manner. By contrast, caprine CRSP-2 did not alter cAMP production at all, results similar to those seen with CRSP-2 peptides from other species such as pig and dog. The potency of ovine/caprine CRSP-1 in the porcine CT-R activation was almost as strong as that of porcine CRSP-1, and much stronger than that of porcine CT. We then evaluated the effect of CRSPs on CT-R-RAMP or CL-R-RAMP co-expression systems to observe the effect of those peptides on CT, CGRP, or AM receptors. As described by McLatchie et al. (1998), CL-R forms CGRP receptor when it is co-expressed with RAMP1, and forms AM receptor when it is co-expressed with RAMP2 or RAMP3. Figure 3B and C shows the effect of the CRSPs, CT, CGRP, and AM, on cAMP production in cells that were transiently expressing CT-R or CL-R either alone or with one of three RAMPs. These peptides did not alter cAMP level in COS-7 cells with pcDNA3.1 empty vector (data not shown). Ovine/caprine CRSP-1 stimulated cAMP production from cells co-expressing CT-R and the RAMPs, no matter which RAMP was co-expressed. However, none of the CRSPs stimulated cells expressing CL-R with or without any of RAMPs. These results indicate that ovine/caprine CRSP-1 stimulates CT-R with potency similar to that of porcine CRSP-1 but does not stimulate CGRP or AM receptor, and that CRSP-2 does not stimulate either CT-R or CL-R at all, even in the presence of any RAMPs.
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Experiments were completed without mishap and data collection was complete. There was no significant difference in baseline levels of most variables between study days apart from plasma corrected Ca that was lower at baseline (–30 and 0 min) on the day CRSP-1 was infused compared with control day (2.52±0.054 vs 2.59±0.028, P<0.01). Therefore, plasma corrected Ca data are presented as percentage change from baseline.
Infusion of CRSP-1 induced significant decreases in plasma total Ca (P=0.02) and corrected Ca levels (P=0.017) (Fig. 4). Reduction in Ca was apparent at both doses of CRSP-1 and the levels remained lower than time-matched control levels for at least 60 min following the cessation of CRSP-1 infusions. Plasma ionized Ca tended to be lower throughout the infusion of CRSP-1 but these changes did not achieve statistical significance (Fig. 4). CRSP-1 infusions induced dose-dependent increases in plasma cAMP (P=0.002) with levels being elevated twofold over time-matched control by the end of the higher dose of CRSP-1 and levels returning toward control by 60 min post-infusion (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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The cDNA and deduced amino acid sequences of ovine and caprine CRSP-1 are shown in Fig. 1A and B. In evolutionarily closely related animals, amino acid sequences of CRSP-1 peptides are similar to each other. Deduced mature amino acid sequences of ovine and caprine CRSP-1 are identical to each other, almost identical to that of bovine CRSP-1 peptide, and show 83% identity with that of porcine CRSP-1. Like canine and bovine CRSP-1, ovine/caprine CRSP-1 does not have a C-terminal amide that exists in other members of the CGRP family. Despite having the non-amidated C-terminus, ovine/caprine CRSP-1 potently stimulated CT-R in a manner similar to porcine CRSP-1. Of note, the C-terminal glycine extended form of porcine CRSP-1 has been shown to be just as potent in stimulating CT-R as CRSP-1 (Katafuchi et al. 2005), thus supporting the idea that the C-terminal sequence and/or amidation are not essential for the interaction between CRSP-1 and its receptor, in contrast to other members of the CGRP family. Neither CT nor CRSP-1 stimulated CGRP receptor and AM receptor composed of CL-R and RAMPs, both of which require the C-terminal amidation of ligand peptides. The expression of any RAMP did not alter the specific stimulation of CT-R by CRSP-1, regardless of their differences in the sequence and C-terminal amidation. Taken together, ovine and caprine CRSP-1 are confirmed to share the structural and biological features of CRSP-1 so far identified. By contrast, caprine CRSP-2 did not stimulate either CT-R or CL-R, even in the presence of any of RAMPs. Similarly, neither porcine nor canine CRSP-2 stimulated any combination of these receptors and RAMPs. Unlike precursors for porcine and canine CRSP-2 peptides that have no typical prohormone convertase processing site on either end of the deduced mature peptides, the precursor of caprine CRSP-2 has a typical prohormone convertase processing site between the N-terminal flanking peptide and the deduced mature CRSP-2. Nevertheless, the synthetic CRSP-2 peptides used for this and previous studies might not be endogenous forms of CRSP-2 peptides (Katafuchi et al. 2003b). Purification and structural determination of native CRSP-2 peptides is a prerequisite for evaluating the function and biological activity of CRSP-2 peptides.
The existence of CT, a peptide that lowers plasma Ca and potently inhibits bone resorption, has been known for some 40 years (Zaida et al. 2002). We have recently identified a series of CRSP-1 peptides in mammalian species, which more potently activate the CT-R in vitro (Katafuchi et al. 2003a,b, 2004). Early in vivo studies showed that porcine CRSP-1 lowers plasma Ca when administered to anaesthetized rats (Katafuchi et al. 2003a). The present study extends this finding by demonstrating that ovine CRSP-1 administered to its species of origin exhibits potent, long-lasting actions to lower plasma Ca levels, presumably by activation of the CT-R. It is likely that CRSP-1 acts via stimulation of cAMP as circulating levels of cAMP showed a dose-dependent rise to approximately twofold time-matched control levels during the high-dose infusion. This is consistent with potent stimulation of cAMP production observed by the CRSP-1 peptides in vitro both in the present study and in the previous reports (Katafuchi et al. 2003a,b, 2004). CRSP-1 has been previously shown to dose dependently inhibit the formation and activity of osteoclast-like cells in vitro (Notoya et al. 2007). Of interest in the present study, plasma levels of NT-CNP were subtly but significantly suppressed during CRSP-1 infusion with rebound activation above time-matched control post-infusion. NT-CNP is strongly correlated with skeletal growth and markers of bone formation and as such is an emerging marker of growth plate activity in humans and other mammals (Prickett et al. 2005). Taken together, these results suggest that CRSP-1 is likely to be involved in bone turnover by inhibition of osteoclast formation and activity.
As stated earlier, CRSP-1 is a member of the CGRP family that includes CT, CGRP, and AM. Given the sequence homology between peptides in this family, it is of interest to speculate whether CRSP-1 may activate not only the CT-R but also the CL-R-RAMP complexes. CGRP activates CL-R-RAMP1 while AM and AM-2 activate the CL-R-RAMP2 and CL-R-RAMP3 receptor complexes. Activation of these receptor complexes by CGRP (Gnaedinger et al. 1989), AM (Charles et al. 2001), and AM-2 (Charles et al. 2006) results in a number of hemodynamic actions including lowering blood pressure and peripheral resistance while increasing heart rate and cardiac output. These changes are also associated with significant activation of PRA. Of note in the present study, CRSP-1 had no effect on any of these hemodynamic indices and actually lowered PRA. Thus, results of this in vivo study suggest that CRSP-1, at least at these doses in sheep, does not bind to and/or signal via CL-R-RAMP receptor complexes. This is in agreement with the in vitro studies of this and previous studies which show that the CRSPs appear not to be ligands for any of the combinations of the CL-R-RAMP complexes (Katafuchi et al. 2003a,b, 2004). This is the first report of an effect of CRSP-1 on PRA, but the significance of this finding requires further studies.
While reduction of plasma Ca is the major biological effect attributable to CT, administration of CT to men has also been shown to induce a number of renal effects including increased urinary volume, cAMP, electrolyte excretion, and glomerular filtration (Gnaedinger et al. 1989). This study showed no evidence for such renal effects of CRSP-1, at least at these doses in normal conscious sheep. In the rat, intracerebroventricular injection of porcine CRSP-1 is more potent than porcine CT in its effects on feeding and body temperature, whereas systemic administration of porcine CRSP-1 is less potent than porcine CT in reducing plasma Ca (Katafuchi et al. 2003a, Sawada et al. 2006). These results indicate that there might be two different states of CT-R: one that transduces a CRSP-1 signal more potently than CT, while the other transduces a CRSP-1 signal more weakly than CT. On the other hand, there are marked differences in the tissue expression level of CRSP-1 and CT in the central nervous system and thyroid gland. Therefore, it is necessary to take all these points into account when postulating a physiological role for CRSP-1.
In conclusion, we have identified ovine/caprine CRSP-1 and caprine CRSP-2. This newly identified CRSP-1 has been shown to share the structural and biological features of CRSP-1 peptides identified to date. In vivo studies confirm that CRSP-1 reduces plasma Ca levels, presumably via a cAMP-mediated mechanism, along with several new effects on the PRA and NT-CNP. By contrast, caprine CRSP-2 did not stimulate any combination of CT-R, CL-R, and RAMPs. Purification and structural determination of native CRSP-2 peptides is essential for searching and evaluating the function and biological activity of CRSP-2.
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Declaration of Interest |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Funding |
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Charles CJ, Espiner EA, Yandle TG, Cameron VA & Richards AM 1991 Biological actions of cleaved atrial natriuretic factor (ANF101-105/106-126) in conscious sheep. Journal of Cardiovascular Pharmacology 17 403–410.[Web of Science][Medline]
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Received in final form 11 May 2008
Accepted 15 May 2008
Made available online as an Accepted Preprint 15 May 2008
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), caprine CRSP-2 (
), porcine CRSP-1 (
), or porcine CT (
). Effects of CRSPs on cAMP production via porcine (B) CT-R or (C) CL-R in the presence or absence of RAMPs. COS-7 cells were co-transfected with porcine CT-R or CL-R cDNA and one of porcine RAMP1, 2, 3 cDNA, or pcDNA using Lipofectamine LTX with Plus reagent, cultured for 24 h, and then used for the experiments. The cells were stimulated with 100 nM of each peptide. o, ovine; c, caprine; p, porcine; h, human. Each point represents the mean±S.D. of (A) three and (B and C) four separate determinations.
P<0.01.
