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1Research Equipment Center and 3Department of Pharmacology, School of Medicine, Kagawa University, Kagawa; and 2Department of Applied Pharmacology and Therapeutics, Osaka City University Medical School, Osaka, Japan
Submitted 6 May 2005 ; accepted in final form 12 October 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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hemodynamics; sympathetic outflow; vasodilation
Intrarenal infusion of human AM2 decreased BP in a dose-dependent manner, and a subdepressor dose of the peptide increased renal blood flow (RBF) and caused diuretic and natriuretic actions without altering the glomerular filtration rate in anesthetized rats (5). Administration of IMD into the lateral cerebroventricle caused an elevation of BP that was inhibited by phentolamine and also inhibited water intake in rats (27). Thus AM2/IMD could play an important role in controlling fluid homeostasis and cardiovascular and sympathetic nervous system function. However, the biological activity of AM2 has not been fully explored.
It has been reported that AM also lowers BP and causes a reflex increase in heart rate (HR) and sympathetic outflow (6, 24), as well as an increase in peripheral blood flow (8, 11). In addition to AM, proadrenomedullin contains a unique 20-amino acid peptide, the proadrenomedullin NH2-terminal 20 peptide (PAMP), which is also biologically active. Therefore, to verify the biological properties of AM2, the present study compared the effects of synthesized rat AM2 (rAM2) on BP, HR, renal sympathetic nerve activity (RSNA), and RBF with the effects of synthesized rat AM (rAM) and rat PAMP (rPAMP). The study was done in conscious freely moving rats, because anesthesia and surgical stress strongly modify, and occasionally reverse, cardiovascular and sympathetic nervous system responses (13, 29).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Male 9- to 11-wk-old Sprague-Dawley rats (290–350 g body wt; CLEA JAPAN, Tokyo, Japan) were housed in separate cages in a temperature-controlled room under a 12:12-h light-dark cycle. They were fed a standard laboratory diet and water ad libitum. All surgical and experimental procedures were approved by the Animal Care and Use Committee of Kagawa University and conformed to the Guidelines for Animal Experimentation of Kagawa University.
Under pentobarbital sodium anesthesia, a polyethylene catheter (PE-60) was inserted into the abdominal aorta via the right femoral artery for BP measurements. Another catheter (PE-50) was inserted into the inferior vena cava via the right femoral vein for administration of saline solution and drugs. HR was triggered by the BP pulse waveform. All catheters were filled with heparinized saline (100 U/ml). The left kidney was exposed through a retroperitoneal flank incision. The renal artery was carefully isolated from the tissue connecting the renal hilum cephalic. A Doppler flow probe (model HDP 10.20R, Crystal Biotech) was placed around the renal artery for RBF measurements. The electrodes for RSNA recording were implanted as described below (see Renal nerve recording and signal processing). After completion of the surgery, the catheters and the lead wires from a flow probe and an electrode were tunneled under the skin to be exteriorized at the nape of the neck. Each rat was then placed in a cage and allowed to recover for 2 days.
Renal nerve recording and signal processing. RSNA was recorded from the left renal nerve branch as previously described (4). Briefly, the renal nerve was isolated near the aortic-renal arterial junction and placed on a Teflon-coated stainless steel bipolar electrode with the aid of an operation microscope. The renal nerve and electrode were then covered with silicone rubber (Semicosil 932 A and B, Wacker-chemie, Munich, Germany). The renal nerve discharge was amplified using a differential amplifier (model AVB-10, Nihonkohden, Tokyo, Japan) with a band-pass filter (low: 50 Hz; high: 1 kHz). The amplified and filtered signal was visualized on a dual-beam oscilloscope (model VC-10, Nihonkohden) and monitored by an audio speaker. The output from the amplifier was integrated by an integrator (model 1322, Nihondenki-Sanei) with 1-s resetting. The baseline noise, determined when nerve activity was eliminated by elevation of BP with phenylephrine, was subtracted from the integrated RSNA. For quantification of RSNA, the height of the integrated nerve discharge was measured for 30 s in each experiment. The changes in nerve activity were expressed as percentages of the control resting spontaneous nerve activity.
Experimental Protocols
All experiments were performed on conscious and freely moving rats. During the experiments, the arterial catheter was connected to a pressure transducer (model P-23ID, Statham). All chemical agents were dissolved in saline solution. The three peptides (rAM2, rAM, and rPAMP) were purchased from the Peptide Institute (Osaka, Japan).
In the dose-response study, rats were infused with saline solution at a rate of 50 µl/min via the femoral vein, and BP, HR, and RSNA were continuously recorded for 60 min (n = 5). Intravenous infusion of increasing doses (1, 2, 5, and 10 nmol/kg) of rAM2 was then started at 30-min intervals, and each parameter was continuously recorded.
The systemic BP, HR, RBF, and RSNA were recorded continuously. Saline solution was infused into the femoral vein at a rate of 50 µl/min. Each parameter was stabilized, and its control value was measured. Subsequently, rAM2 (5 nmol/kg), instead of saline solution, was infused for 10 min. After cessation of the peptide infusion, saline solution was infused again, and the measurements were continued for an additional 60 min (n = 9). In another series of experiments, rAM and rPAMP were infused at 5 and 200 nmol/kg, respectively, for 10 min, and the responses were compared with those of rAM2 (n = 7). All other procedures were the same as those used for the rAM2 infusion experiments.
To evaluate the influence of rAM2 on the baroreflex, sodium nitroprusside (SNP) was intravenously infused at a rate of 20 µg/kg for 10 min, and the BP, HR, and RSNA responses to SNP were compared with the responses to rAM2 and rAM infusion (n = 7). Furthermore, the reflex increases in HR and RSNA to rAM2 were examined in intact (n = 7) and baroreceptor-denervated rats (n = 7), respectively. The sinoaortic baroreceptors were denervated by bilateral removal of the superior cervical ganglia, sectioning of the superior laryngeal and aortic depressor nerves, and stripping of all fibers from the carotid bifurcations, which were painted with 10% phenol (in 95% ethanol). Animals were used 10 days after denervation. The highest value obtained for each parameter during the infusion period was used for comparisons.
Statistical Analysis
Values are means ± SE. Data were analyzed by paired or unpaired Student's t-test or two-way analysis of variance for repeated measurements with a posteriori (Bonferroni's) comparison. P < 0.05 was regarded as significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Table 1 shows the dose responses of rAM2 on mean BP (MBP), HR, and RSNA. Intravenous infusion of rAM2 at 1–10 nmol/kg decreased BP and increased HR and RSNA in a dose-dependent fashion. The HR and RSNA responses to rAM2 (5 and 10 nmol/kg) were similar. On the basis of these results, 5 nmol/kg was selected as the experimental dose of rAM2. Figure 1 shows typical BP, HR, RSNA, and RBF responses to intravenous infusion of rAM2. Infusion of rAM2 decreased BP and increased HR, RSNA, and RBF. Time courses of MBP, HR, RSNA, and RBF responses to rAM2 are shown in Fig. 2. Intravenous infusion of rAM2 resulted in a significant decrease in MBP (from 108 ± 3 to 100 ± 2 mmHg) accompanied by a simultaneous significant increase in HR (from 356 ± 8 to 411 ± 11 beats/min), RSNA (from 100 to 164 ± 11%), and RBF (from 7.5 ± 0.7 to 10.4 ± 0.9 ml/min). Immediately after cessation of the infusion, BP returned to basal level; HR and RSNA decreased within 30 min after cessation. However, a significant increase in RBF was still evident even 60 min after cessation of the peptide infusion.
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Figure 3 shows typical BP, HR, RSNA, and RBF responses to rAM. As shown in Fig. 4, infusion of rAM resulted in a significant decrease in MBP (from 109 ± 3 to 99 ± 4 mmHg) accompanied by simultaneous increases in HR (from 349 ± 6 to 448 ± 6 beats/min), RSNA (from 100 to 167 ± 19%), and RBF (from 7.7 ± 0.4 to 10.6 ± 0.5 ml/min). Moreover, the changes in BP, HR, RSNA, and RBF during infusion of rAM2 or rAM were similar, although the BP, HR, and RSNA responses to rAM after cessation of the infusion continued for a longer period of time than the responses to rAM2. Hypotension and tachycardia induced by rAM were still evident even 60 min after cessation of the infusion. The increase in RSNA induced by rAM2 was diminished 20 min after cessation of the infusion, whereas the increase induced by rAM was still significant. On the other hand, the change in RBF was similar for rAM2 and rAM, and the increase was maintained even 60 min after cessation of the corresponding peptide infusion. Figure 5 shows typical BP, HR, RSNA, and RSNA responses to rPAMP. Intravenous infusion of rPAMP caused a transient and prompt biphasic response in BP; i.e., BP decreased immediately after the start of the infusion and then returned to basal level within 1 min. The HR and RSNA responses to rPAMP were significant (from 350 ± 7 to 469 ± 10 beats/min and from 100 to 179 ± 15% respectively), even though the reduction in BP was minor. These changes disappeared within 30 min after cessation of the peptide infusion, which was similar to the response to rAM2. The RBF response was not detectable.
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Figure 6 shows the reflex increases in HR and RSNA for rAM2, rAM, and SNP. Infusion of SNP resulted in a decrease in BP (from 111 ± 3 to 103 ± 3 mmHg) and an increase in HR and RSNA (from 352 ± 7 to 379 ± 8 beats/min and from 100 to 139 ± 6%, respectively). Although the BP response was similar for rAM2, rAM, and SNP, the reflex increases in HR and RSNA induced by rAM2 or rAM were greater than those induced by SNP. The reflex increases in HR and RSNA were analyzed using the absolute values for the ratio of peak changes in HR and MBP or RSNA and MBP. As shown in Fig. 7, 
HR/MBP and RSNA/MBP were significantly larger for rAM2 (8.3 ± 2.1 and 9.4 ± 1.6, respectively) than for SNP (3.6 ± 1.0 and 5.1 ± 0.6, respectively) in intact rats. Sinoaortic denervation markedly reduced the reflex increase in HR and RSNA to the SNP-induced decrease in BP: HR/MBP and RSNA/MBP for SNP were 0.8 ± 0.3 and 0.8 ± 0.3, respectively. Although the rAM2-induced increases in HR and RSNA were also suppressed by sinoaortic denervation, those responses remained: HR/MBP and RSNA/MBP for rAM2 were 5.9 ± 1.1 and 4.7 ± 1.7, respectively.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Recently, AM-like peptides, AM1–AM5, were identified in the pufferfish (19), and then AM2 was identified in mice, rats, and humans (26). At the same time, a novel calcitonin/CGRP family member, IMD, was identified (23). The precursor and mature sequence of AM2 and IMD were identical. Takei et al. (26) reported expression of AM2 mRNA in various tissues in mice, especially in kidneys, but not in adrenal glands, where AM mRNA is abundantly expressed. Taylor et al. (27) further demonstrated that immunoreactive AM2/IMD was strongly detected in kidneys and stomach. In addition, they reported that the circulating level of AM2/IMD in rats was 
200 pg/ml, which is higher than the level of AM and CGRP. Taken together, it was hypothesized that AM2 may have a biological activity that is different from that of AM or CGRP and may have an important role in the regulation of organ function. We previously reported that intrarenal infusion of human AM2 at a subdepressor dose increased RBF and caused diuresis and natriuresis without altering the glomerular filtration rate in anesthetized rats (5). Therefore, the present study compared the physiological activity of AM2, AM, and PAMP to enhance our understanding of the physiological role of AM2.
Some previous reports suggested that AM has a role in regulating cardiovascular and sympathetic nervous system function. Intravenous administration of human AM decreased BP and increased HR and RSNA in conscious rabbits, and the duration of these responses was relatively long (6). In conscious rats, intravenous injection of rAM also decreased BP and increased HR and RSNA, and the tachycardiac and sympathoexcitatory responses were still evident 30 min after the peptide injection (24). Thus, as a circulating hormone, AM plays a important role in cardiovascular regulation, including the autonomic nervous system, and its action is relatively long lasting. In the present study, we observed the same responses to rAM as mentioned above, but the duration of the responses to rAM2 and rAM was different. In other words, the maximum responses in BP, HR, and RSNA induced by rAM2 were similar to those induced by rAM, but the duration of the responses to rAM2 after cessation of the infusion was shorter than the duration of the responses to rAM, except for the RBF response, which was longer. Thus the cardiovascular and sympathetic actions of rAM2 and rAM were similar, but the duration of the rAM2 effects appears to be shorter than the duration of the rAM effects.
Intravenous infusion of rPAMP at 200 nmol/kg slightly reduced BP but significantly increased HR and RSNA. This RSNA response to PAMP could not be explained by the baroreflex alone, suggesting an additional activity of rPAMP, such as modification of the baroreflex or a direct effect on the central nervous system. We hypothesized that, in addition to rPAMP, rAM2 and rAM may also have such an additional activity. To assess this hypothesis, we compared the HR and RSNA responses to rAM2 and rAM with those of SNP. As shown in Fig. 6, the HR and RSNA responses to rAM2 and rAM were significantly greater than the HR and RSNA responses to SNP, although the magnitude of BP reduction did not differ among the three groups. These findings indicate augmentation of the baroreflex function by rAM2 or rAM. Similar findings that intravenous injection of AM augments the reflex increase in HR and RSNA have been reported (24). The reason for the magnitude of the response of HR and RSNA to rAM2 is not clear. As shown in Fig. 7, sinoaortic denervation reduced, but did not diminish, the increases in HR and RSNA by rAM2. These results indicate that the rAM2-induced increases in HR and RSNA were caused by reflex responses to the hypotensive effect and additional mechanisms. Administration of AM2/IMD into the lateral cerebroventricle of rats elevated BP, and this effect was inhibited by phentolamine (27). Furthermore, the authors demonstrated that AM2/IMD was present in brain (27). These findings suggest that AM2/IMD may have a physiological role in the regulation of autonomic nerve activity via the central nervous system. In terms of the effect on the heart, a direct action of rAM2 on the heart is possible, because Taylor et al. (27). also reported that AM2/IMD was found in heart. AM has been detected in the heart (3, 25) and has a direct action on the heart (10, 22). Thus AM2/IMD, as well as AM, may exert a direct positive inotropic effect. Thus it can be assumed that AM2 augments the baroreflex function, although how circulating AM2 affects the central nervous system remains to be clarified.
Intravenous infusion of rAM2 increased RBF, even though BP was reduced, indicating that rAM2 strongly dilates the renal blood vessels. In addition, the rAM2-induced renal vasodilation was long lasting. This evidence is highly characteristic for this peptide. As described above, the tissue distribution of AM2/IMD is disproportionate to and distinct from that of AM, and the results of gene expression and peptide immunoreactivity analyses showed abundant expression and peptide localization in the kidneys (26, 27). Thus the strong response of the kidneys to AM2 may be explained by their very high AM2 levels. The mechanisms of vasodilation induced by AM2/IMD have not been defined. Roh et al. (23) reported that BP-lowering effects of IMD could be blocked by the CGRP receptor antagonist CGRP-(8–37) and partially blocked by AM-(22–52). Furthermore, Taylor et al. (27) demonstrated that blockade of nitric oxide production did not alter the effect of AM2/IMD on BP, although the BP-lowering effects of AM and CGRP were partially mediated via nitric oxide production. Taken together, it is likely that the vasodilatory actions may be mediated by the CGRP receptor CRLR/RAMP1.
In summary, intravenous infusion of rAM2 in conscious rats resulted in a significant decrease in BP and increase in HR, RSNA, and RBF. The effects of rAM2 on BP, HR, and RSNA were diminished within 30 min after cessation of the peptide infusion, whereas the RBF response was long lasting. Taken together, these findings suggest that rAM2 is a potent vasodilator peptide and may play an important role in the regulation of cardiovascular and sympathetic outflow.
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FOOTNOTES |
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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.
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) and rAM (
) on MBP, HR, RSNA, and RBF. *P < 0.05 vs. control.
