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Christchurch Cardioendocrine Research Group, Christchurch School of Medicine and Health Sciences, Christchurch 8001, New Zealand
Address all correspondence and requests for reprints to: Dr. C. J. Charles, Christchurch Cardioendocrine Research Group, Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch 8001, New Zealand. E-mail: chris.charles{at}chmeds.ac.nz.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AM and CGRP interact with different combinations of a type II G protein-coupled receptor known as calcitonin receptor-like receptor (CRLR) and three receptor activity-modifying proteins (RAMPs) (3). CGRP preferentially activates CRLR/RAMP1, whereas AM preferentially stimulates CRLR/RAMP2 and CRLR/RAMP3. AM-2 dose-dependently stimulates cAMP production in cells coexpressing CRLR and RAMPs but is reported to be a nonselective agonist for CRLR/RAMP receptor complexes (2). Tissue distribution of mRNA and immunostaining shows that AM-2 is widely expressed including high expression in pituitary, submaxillary gland, kidney, and stomach and lower expression through the gastrointestinal tract, lung, thymus, and ovary and is present in the endothelial cells of the vasculature of the heart and kidney (1, 2, 4).
In anesthetized mice, AM-2 exhibits dose-dependent and long-lasting hypotensive effects (more potent than AM) with an associated rise in heart rate (1). AM-2 has also been shown to reduce arterial pressure and increase heart rate in conscious Sprague Dawley and spontaneously hypertensive rats with a similar potency to AM (2). AM-2-induced reductions in arterial pressure are associated with not only increased heart rate but also increased renal sympathetic nerve activity (RSNA), effects that were attenuated but not abolished by sinoaortic denervation (5). AM-2 also acts within the central nervous system to elevate blood pressure and heart rate and alter stress hormone secretion (6, 7). Intrarenal infusions of AM-2 have been shown to increase renal blood flow, urine flow, and sodium excretion in anesthetized rats (8).
However, beyond these limited hemodynamic measurements in rodents, as mentioned above, there is little information available regarding other biological actions of systemically administered AM-2. Accordingly, we have studied the integrated hemodynamic, hormonal, and renal actions of iv AM-2 in normal conscious sheep.
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Materials and Methods |
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Each animal was studied on two occasions at least 2 d apart receiving vehicle (hemaccel) control and AM-2 according to a balanced, random-order design. AM-2 was infused iv at two incremental doses of 10 ng/kg·min for 90 min followed immediately by 100 ng/kg·min for 90 min. Human AM-2 (intermedin) was purchased from Bachem (Bubendorf, Switzerland).
Arterial pressure and RAP recordings using an online data acquisition system (Dataflow; Crystal Biotech, Hopkinton, MA) commenced 30 min before infusions and continued 90 min post infusions. 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 (CTPR) was calculated as mean arterial pressure (MAP) divided by cardiac output.
Venous blood was drawn at preset intervals throughout the experiment. Blood was taken into chilled EDTA tubes and centrifuged and the plasma stored at –80 C before assay for aldosterone (9), cortisol (10), plasma renin activity (PRA) (11), atrial natriuretic peptide (ANP) (12), brain natriuretic peptide (BNP) (13), endothelin (14), catecholamines (15), and cAMP (commercially available kit; Biotrak, Amersham, Little Chalfont, UK). Additional venous blood was drawn at 0, 90, 180, and 270 min into heparin tubes for analysis of plasma sodium and potassium by standard flame photometry and plasma creatinine by the standard Jaffe method.
Urine was collected for a 90-min baseline period immediately before infusions and then at 90-min periods for the duration of the study. Volume was measured before assay for sodium, potassium (standard flame photometry), and creatinine (standard Jaffe method) excretion rates.
Statistics
Results are expressed as mean ± SEM. Two-way ANOVA with time as a repeated measure was used to determine time and treatment differences between AM-2 and control arms of the study. Statistical significance was assumed at P < 0.05. Where significant differences were identified by ANOVA, a priori Fisher’s protected least square difference (LSD) tests were used to identify individual time points significantly different from time-matched data.
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Results |
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AM-2 infusion reduced MAP (P < 0.001), with pressures significantly lower than time-matched control during the high dose (Fig. 1
). MAP was lowered by approximately 14 mm Hg by the end of infusion and had returned to control levels by 60 min post infusion. Heart rate rose significantly (P < 0.001) and dose-dependently with a modest increment of approximately 10 beats/min by the end of the low dose and a much greater rise in response to the high dose of 40 beats/min (Fig. 1). As with MAP, heart rate had returned to control levels by 60 min post infusion. Cardiac output increased by approximately 4 liters/min (50%) in response to AM-2 (P < 0.001), with modest but significant increases in response even to the lower dose (Fig. 1). Cardiac output remained elevated above control levels at 90 min post infusion. Thus, CTPR was dose-dependently reduced in response to AM-2 (P < 0.001). As seen in Fig. 2, blood pressure-lowering effects of AM-2 were greater for diastolic (DAP) than systolic arterial pressure (SAP). Trends for SAP to fall did not achieve statistical significance (P = 0.15), whereas DAP (P < 0.001) fell to a greater extent than MAP (approximately 18 vs. 14 mm Hg, respectively). There was an overall significant treatment x time interaction observed for RAP (P = 0.026). However, RAP was not significantly different from time-matched control during infusions but was significantly higher on the AM-2 day than control levels during the post-infusion recording period (Fig. 2). Similarly, hematocrit was significantly lower on the AM-2 day than time-matched control during the post-infusion period to give an overall significant difference (P = 0.001; Fig. 2).
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). PRA tended to increase with low-dose AM-2 and was significantly elevated above time-matched control during the high dose, with levels returning to control at 90 min post infusion. Rises in PRA with AM-2 were paralleled by 2- to 3-fold increases in plasma aldosterone (P < 0.001). Aldosterone increased dose-dependently with modest but significant increments during low-dose AM-2 and greater rises observed during the high dose (Fig. 3). In contrast, plasma cortisol levels tended to be lower during the AM-2 infusions (not significant; Fig. 3). Plasma cAMP levels increased in response to AM-2 (P < 0.001) being significantly above time-matched control throughout the high dose and returning to control levels by 90 min post infusion (Fig. 3). Both plasma ANP (P = 0.039) and BNP (P = 0.036) levels were higher than time-matched control on the AM-2 study day, particularly during the post-infusion period where levels continued to rise (Fig. 4). There were no significant effects of AM-2 on plasma endothelin-1, epinephrine, or norepinephrine concentrations (Fig. 4). Infusion of AM-2 had no significant effects on plasma sodium, potassium, or creatinine levels (Table 1).
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. Urine sodium excretion tended to be reduced during high-dose AM-2 infusion, although these changes did not achieve statistical significance. AM-2 had no significant effect on urine volume, potassium, or creatinine excretion.
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Discussion |
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Hemodynamic actions for AM-2 were reported in both initial reports of the peptide (1, 2). In anesthetized mice, AM-2 exhibits dose-dependent, long-lasting hypotensive effects, more potent than AM, with an associated rise in heart rate (1). AM-2 has also been shown to reduce arterial pressure and increase heart rate in both conscious Sprague Dawley and spontaneously hypertensive rats with a similar potency to AM (2). The present study confirms and extends these findings in a large-animal model and is the first to report effects of systemic AM-2 on other hemodynamic indices. Hemodynamic effects of AM have been reported by a number of authors. These actions include lowering of arterial pressure associated with increases in cardiac output and falls in peripheral resistance (16, 17, 18). Thus, the hemodynamic actions for AM-2 observed in the present study are similar to those previously reported for AM. Given such similarities, it is likely that underlying mechanisms are similar for the two peptides. AM’s effect on cardiac output likely results from alterations in cardiac preload and afterload, baroreflex-induced augmentation of efferent cardiac sympathetic activity, and/or a direct positive inotropic action (18, 19). However, others have demonstrated no evidence for AM acting as a positive inotroph (20, 21). Indeed, AM-2 has recently been reported to decrease cardiac function (reduced dP/dt) in vivo in rats and in isolated perfused rat hearts (22). Clearly, additional studies are required to assess whether AM-2 exhibits inotropic effects under other experimental conditions. The consistent observation that iv AM reduces peripheral resistance and that intraarterial administration of the hormone increases local blood flow (23, 24) suggests a direct effect of AM on arterial tone. Of note, AM-2’s hypotensive actions were greater on DAP than SAP as previously shown for AM (16). AM-2 has also been demonstrated to have vasodilator actions in vitro on coronary, carotid, and supramesenteric artery rings that are equipotent with AM (25) and relaxes preconstricted aortic rings in a dose-related manner (22). RAP was not significantly reduced in the present study but did rebound post infusion to be higher than time-matched control. Of interest, effects of AM on filling pressures are more pronounced in experimental heart failure than normal sheep (16, 26). It remains to be determined whether AM-2 is equally efficacious in heart failure. The rise in RAP post infusion coincided with a significant fall in hematocrit, suggesting that there was a shift of fluid from the extravascular space into plasma, causing a rise in atrial pressures that may, in part, underlie the concurrent rise in ANP/BNP. The reasons for such rebound changes in RAP occurring post infusion are unclear. Likewise, the mechanism underlying probable fluid shifts require further study.
Taken together, there is convincing evidence that both AM and AM-2 are effective vasodilators, which may be the primary mechanism underlying the hypotensive actions of these peptides. The observed increases in heart rate and cardiac output may be primarily a reflex response (reduced vagal tone and increased sympathetic activity) to the fall in arterial pressure, with a reduction in afterload also having some effect. This is consistent with the reported effect of AM-2 to increase both heart rate and RSNA in rats (5). Of note in that study, AM-2-induced rises in both these indices were greater than those observed with pressure-matched nitroprusside administration, similar to previous reports for AM (27). Furthermore, sinoaortic denervation attenuated but did not abolish rises in heart rate and RSNA rise, suggesting rises in heart rate and RSNA were not all baroreflex mediated (5). In the present study, the increase in heart rate and cardiac output preceded the decrease in blood pressure. This supports the hypothesis that AM-2 may also have direct actions on the heart. Likewise, rises in plasma aldosterone, discussed in more detail below, appear to have preceded the fall in blood pressure and increase in PRA, raising the possibility that AM-2 may also have direct actions at the adrenal. Clearly, additional studies, including administration of appropriate AM-2 antagonists, are required examining the possibility of direct cardiac and adrenal actions of AM-2 and to characterize the underlying mechanisms.
AM-2 administered within the central nervous system has been shown to alter stress hormone secretion (7). This includes stimulation of prolactin, ACTH, oxytocin, and arginine vasopressin and inhibition of GH. However, there have been no reports of the effects of systemic administration of AM-2 on hormone secretion. Results from the present study demonstrate for the first time that, like AM, infusion of AM-2 results in stimulation of PRA. In addition to reducing arterial pressure (and thereby stimulating the afferent renal arteriolar baroreceptor) and augmenting (or at least not inhibiting) delivery of chloride and sodium to the macula densa, it is likely that AM acts directly on juxtaglomerular granular cells to augment renin release (28). Whether AM-2 also demonstrates such direct actions on the kidney remains to be determined. There are conflicting reports regarding in vivo studies of the action of AM on aldosterone (for review see Ref.29), but accumulating evidence supports a role for AM in the regulation of aldosterone secretion, presumably by a direct inhibitory action on aldosterone secretion from the adrenal glomerulosa (30). In contrast, results from the present study show no evidence for such a role for AM-2. Rather, AM-2 infusions resulted in dose-dependent increases in aldosterone consistent with the observed rises in PRA. Differential effects on aldosterone suggest that a unique AM-2 receptor may yet be discovered. Whether AM-2 inhibits aldosterone under other experimental conditions, or in cardiovascular disease, remains to be determined. Plasma cortisol results in the present study provide no evidence of systemic AM-2 significantly affecting the hypothalamo-pituitary-adrenal axis, again indicating that aldosterone responses are angiotensin II rather than ACTH mediated.
There are no reports of the effects of AM-2 on natriuretic peptide secretion. Results from the present study clearly suggest that AM-2 can enhance circulating levels of ANP and BNP in vivo. Previous reports of AM’s effects on the natriuretic peptides have been inconsistent. An in vitro study has demonstrated suppression of ANP mRNA expression by AM in neonatal rat cardiocytes (31). In contrast, in vivo data from our laboratory suggests AM may stimulate plasma ANP and BNP levels. In an ovine pacing model of heart failure, we have consistently shown that plasma natriuretic peptide levels remain elevated during AM infusion despite precipitous falls in filling pressures (26, 32). Furthermore, in those studies, plasma ANP and BNP levels increase significantly post AM infusion concurrent with filling pressures returning to baseline levels. AM also enhances the ANP response to acute volume loading in normal sheep (33). The reason for the predominantly delayed accentuation of natriuretic peptides by AM-2 is not clear. It may be difficult to postulate a direct stimulatory action of AM-2 itself on natriuretic peptide secretion because, presumably, levels of AM-2 would have been falling at a time when ANP and BNP levels were most augmented (post infusion). Of note, RAP (and presumably left atrial pressure) was higher post AM-2 infusion compared with control. Thus, it is likely that at least some of the rise in natriuretic peptide levels post infusion was in response to raised atrial pressures. However, it should be noted that both ANP and BNP levels were significantly raised at some time points throughout the infusions of AM-2 when RAP was matched to control data. It is also possible that AM-2 may promote natriuretic peptide secretion indirectly via stimulation of angiotensin II, a known secretagogue for natriuretic peptides (34), which, although not measured in the present study, presumably rose in parallel with PRA. Taken together, it is clear that AM and AM-2 can enhance circulating levels of natriuretic peptides under a variety of experimental conditions, but whether this is by direct or indirect pathways remains to be determined.
AM-2 significantly increased circulating levels of cAMP in the present study. This is consistent with AM-2’s action to stimulate cAMP from cells expressing CRLR/RAMP complexes (2). Roh et al. (2) showed that the rank order of potency for cAMP production was CGRP > AM-2 = AM for CRLR/RAMP1; AM > AM-2 = CGRP for CRLR/RAMP2; and AM > AM-2 > CGRP for CRLR/RAMP3. Thus, AM-2 exhibits a receptor activation profile distinct from that of CGRP or AM, suggesting that AM-2 could be important for specific or unique CRLR/RAMP-mediated physiological responses. Of note, plasma cAMP levels were only raised in response to the high-dose infusion of AM-2, whereas heart rate, cardiac output, CTPR, aldosterone, and natriuretic peptide changes were observed also during the low dose. This suggests that either cAMP concentrations were elevated sufficiently at the target tissue level to induce these responses or that these AM-2 actions may be mediated by non-cAMP mechanisms yet to be determined. Alternative signal transduction pathways have been reported for AM, including intracellular calcium mobilization and activation of inositol phosphate, nitric oxide, and prostaglandins. Because the amino acid sequence of ovine AM-2 is yet to be determined, we infused the human form in the present studies. There is a high degree of homology for AM-2 between species sequences thus far identified (1, 2). However, because bioactivity may be species specific, it will ultimately be important to determine the biological actions of the hormone in the species of origin. Experimental design and doses of AM-2 employed in the present study were identical to those used in our previous study of AM in normal conscious sheep (16). This allows some comparison of relative efficacy/potency of AM and AM-2 between the two studies. We have already highlighted the different effects of AM-2 and AM on plasma aldosterone levels. All other indices measured showed qualitatively similar changes. However, AM-2 appears to exhibit more potent effects on hemodynamics than AM. Of note, AM-2 induced significant changes in heart rate, cardiac output, and CTPR during the low-dose infusion, changes that did not achieve significance for AM (16). Additional studies employing wider dose-response data are required to fully elucidate such differences.
To our knowledge, there is only one report of plasma levels of AM-2, that being in normal rats where AM-2 circulates at approximately 40 pmol/liter (6). Clearly, additional studies are required documenting endogenous levels of AM-2 in large experimental animals and in man. Given that AM has a putative role in the pathophysiology and treatment of cardiovascular disease (for reviews see Refs.35 and 36), it will be important to examine AM-2 levels in human health and cardiovascular diseases such as hypertension and heart failure. Such studies, combined with examination of biological responses to AM-2 antagonists, are required to clarify the physiological and/or pathophysiological significance of the present results.
This study showed no significant renal effects of AM-2 in normal conscious sheep. Urine sodium excretion tended to be lower during the high dose, but this did not achieve statistical difference and occurred at a time when arterial pressure (and hence presumably renal perfusion pressure) was reduced. Takei et al. (1) demonstrated that urine flow and sodium excretion was reduced by AM-2 in mice, also during significant concurrent hypotension. The only other study to report renal effects of AM-2 to date showed that intrarenal infusion of the peptide in anesthetized rats, at a dose that had minimal systemic hemodynamic effects, increased renal blood flow, urinary flow, and sodium excretion without affecting glomerular filtration (8). For AM, natriuretic and diuretic actions were demonstrated in an ovine model of heart failure (26) but not in normal conscious sheep (16). It remains to be determined whether AM-2 promotes renal excretion of volume and electrolytes under other experimental conditions.
In conclusion, iv infusions of AM-2 administered to normal conscious sheep induced significant hemodynamic actions including reduced MAP and CTPR and increased heart rate and cardiac output. Concurrently, AM-2 activated plasma cAMP, PRA, aldosterone, and the natriuretic peptides. With the exception of actions on aldosterone, these actions are similar to those previously reported for AM. Thus, AM-2 may be another important regulator of volume and pressure homeostasis and may play a role in the pathophysiology of heart disease.
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Acknowledgments |
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Footnotes |
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Disclosures: C.J.C., M.T.R., and A.M.R. have nothing to declare.
First Published Online January 12, 2006
Abbreviations: AM, Adrenomedullin; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CGRP, calcitonin gene-related peptide; CRLR, calcitonin receptor-like receptor; CTPR, calculated total peripheral resistance; DAP, diastolic arterial pressure; LSD, least square difference; MAP, mean arterial pressure; PRA, plasma renin activity; RAMP, receptor activity-modifying protein; RAP, right atrial pressure; RSNA, renal sympathetic nerve activity; SAP, systolic arterial pressure.
Received November 4, 2005.
Accepted for publication January 4, 2006.
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) or vehicle control (
) in eight sheep. Values shown are mean ± SEM. Significant differences were observed for MAP (P < 0.001), heart rate (P < 0.001), cardiac output (P < 0.001), and CTPR (P < 0.001). Individual time points significantly different from time-matched data (Fisher’s protected LSD from two-way ANOVA) are indicated as follows: *, P < 0.05; 
, P < 0.01.
