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J Appl Physiol 98: 1013-1020, 2005. First published October 15, 2004; doi:10.1152/japplphysiol.01083.2004
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Effects of relaxin on systemic arterial hemodynamics and mechanical properties in conscious rats: sex dependency and dose response

Dan O. Debrah,1 Kirk P. Conrad,2 Lee A. Danielson,3 and Sanjeev G. Shroff1

1Department of Bioengineering, University of Pittsburgh, and 2Departments of Obstetrics, Gynecology and Reproductive Sciences, and of Cell Biology and Physiology, University of Pittsburgh School of Medicine and Magee-Womens Research Institute, Pittsburgh, Pennsylvania; and 3Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico

Submitted 29 September 2004 ; accepted in final form 10 October 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
We previously showed that chronic administration of recombinant human relaxin (rhRLX; 4 µg/h) to conscious female, nonpregnant rats to reach serum levels corresponding to early to midgestation (~20 ng/ml) increases cardiac output (CO) and global arterial compliance (AC) and decreases systemic vascular resistance (SVR), comparable to changes observed in midterm pregnancy. The goals of this study were to test whether chronic administration of rhRLX (4 µg/h) to conscious male rats will yield similar changes in CO and systemic arterial load and to determine whether higher infusion rates of rhRLX (50 µg/h) administered to nonpregnant female rats yielding serum concentrations corresponding to late pregnancy (~80 ng/ml) will further modify CO and SVR and global AC comparable to late gestation. CO and systemic arterial load, as quantified by SVR and AC, were obtained by using the same methods as in our previous studies. With respect to baseline, chronic rhRLX administration to male rats over 10 days at 4 µg/h increased both CO (20.5 ± 4.2%) and AC (19.4 ± 6.9%) and reduced SVR (12.7 ± 3.9%). These results were comparable to those elicited by the hormone in nonpregnant female rats. In contrast, neither acute (over 4 h) nor chronic (over 6 days) infusion of the higher dose of rhRLX administered to conscious female rats resulted in significant changes in CO, AC, or SVR from baseline. We conclude that 1) rhRLX increases CO and AC and reduces SVR irrespective of sex, and 2) the rhRLX dose response is biphasic insofar as significant alterations in CO and systemic arterial load fail to occur at high serum concentrations.

arteries; vasodilation; arterial compliance; arterial resistance

ONE OF THE EARLIEST AND MOST dramatic physiological changes to occur during normal pregnancy is a marked increase in cardiac output (CO) and reduction in both the steady and pulsatile components of systemic arterial load (23, 25). The steady arterial load is quantified by systemic vascular resistance (SVR), which is calculated as the quotient of the mean arterial pressure (MAP) and CO. The pulsatile arterial load arises from the branching nature of the vasculature, vessel geometry and wall viscoelasticity, and blood mechanical properties. Global arterial compliance (AC; ACarea) is one measure of pulsatile arterial load and is typically derived from the CO and diastolic decay of the aortic pressure waveform [P(t), where t is time]. The precise mediators of the reduction in arterial load observed during pregnancy are currently unknown. However, we have recently reported that the chronic administration of the ovarian hormone relaxin to nonpregnant female rats mimics pregnancy-associated changes in arterial load, i.e., a decrease in SVR and an increase in ACarea (4).

Relaxin is a peptide hormone of the insulin/relaxin family of structurally related hormones that is secreted by the corpus luteum of the ovary during pregnancy (26). Its impact on the renal circulation has been studied extensively in conscious rats (5, 8, 20). Analogous to pregnancy, chronic administration of relaxin to nonpregnant rats induces renal vasodilation and hyperfiltration, as well as reduces the myogenic reactivity of small renal arteries through a vasodilatory pathway involving vascular gelatinase activity, endothelin (ET), the endothelial ETB receptor, and nitric oxide (NO) (5, 7, 8, 15, 21). Administration of relaxin to male rats induces vasodilation of the renal circulation comparable to that reported for female rats (7). Furthermore, relaxin administration elicits a biphasic response in the renal circulation of conscious rats. That is, whereas administration of low doses of the hormone, which yield circulating levels of ~10–20 ng/ml, induces significant vasodilation, higher doses, which produce serum concentrations of ~80 ng/ml, are relatively inactive (6).

In our previous investigation of the effects of relaxin on systemic hemodynamics and arterial properties (4), we used female rats and hormone infusion rates of 4 and 25 µg/h, which yielded serum concentrations of ~15 and ~36 ng/ml, respectively, corresponding to the levels reported in early to midterm pregnant rats (26). At this stage of pregnancy, CO is increased by ~25% and has not yet reached its maximum level of ~50% above nonpregnant values, which is typically observed during late gestation in rats (10, 27). At this late stage, circulating relaxin levels are further elevated: ~80 ng/ml. Thus there were two objectives of this study: first, to determine the effects of low-dose relaxin administration (4 µg/h) on systemic hemodynamics and arterial properties in male rats and to compare these effects with those previously observed in female rats (4); and, second, to determine whether a higher infusion rate of 50 µg/h when administered to female rats and yielding serum concentrations of ~80 ng/ml will result in a further increase in CO and decrease in systemic arterial load comparable to late pregnancy.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Long-Evans male and female rats of 12–14 wk were purchased from Harlan Sprague-Dawley (Frederick, MD). They were provided PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds, St. Louis, MO) and water ad libitum. The rats were maintained on a 12:12-h light-dark cycle. This investigation conforms with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Administration of recombinant human relaxin.   The recombinant human relaxin (rhRLX) (BAS, San Mateo, CA) was provided as a 5.0 mg/ml solution in a buffer (20 mM sodium acetate, pH 5.0). It was diluted as necessary in the same buffer. For the low-dose infusion protocol, two model 2002 osmotic minipumps (Durect, Cupertino, CA) were used to deliver the rhRLX for 10 days at the dose of 4 µg/h. This dose was designed to yield concentrations of circulating relaxin similar to those measured during early to midgestation in rats, i.e., 10–20 ng/ml (26). For the high-dose infusion protocol, one model 2ML2 osmotic minipump was used to deliver rhRLX at 50 µg/h for 6 days, which was expected to produce serum concentrations comparable to those recorded during late gestation (26) when further increases in CO and decreases in SVR are observed in this species (10, 27). Finally, in a third protocol, rhRLX was administered by intravenous bolus over 3 min (4.0 µg/0.3 ml) followed by a continuous intravenous infusion for 4 h at 4 µg/h.

Surgical preparation.   This surgical preparation has been described in detail previously (4). Briefly, rats were anesthetized with 60 mg/kg im ketamine and 21 mg/kg ip pentobarbital. They were then instrumented, using sterile technique, as follows: 1) a Tygon catheter was implanted in the right jugular vein with the tip lying at the junction of the anterior vena cava and right atrium; 2) a thermodilution microprobe (36 cm long, F-1.5; Columbus Instruments, Columbus, OH) was implanted in the abdominal aorta via the left femoral artery with the tip lying 1.0 cm below the left renal artery; and 3) a mouse pressure catheter (TA11PA-C20, F-1.2; Data Science International, St. Paul, MN) was implanted in the right carotid artery with the tip lying at the junction of the right carotid artery and aortic arch. For the acute administration of rhRLX, another Tygon catheter was implanted in the inferior vena cava via the left femoral vein such that the tip lay 1.5 cm below the left renal artery.

After instillation of 0.05 ml of a heparin solution into the venous catheters and plugging them with straight pins, rats were given ampicillin by drinking water for 2 days (100 mg/50 ml with 2 tablespoons of dextrose). Terbutrol was given subcutaneously for postoperative analgesia.

For chronic administration of low-dose rhRLX (4.0 µg/h rhRLX) in the male rats for 10 days, two Alzet model 2002 osmotic minipumps (Durect) were inserted subcutaneously in the back of the animal under isoflurane anesthesia. For chronic high-dose administration in the female rats for 6 days (80 µg/h), one Alzet model 2ML2 osmotic minipump was implanted. High-dose rhRLX was also administered to another group of female rats acutely by intravenous bolus over 3 min (4.0 µg/0.3 ml) followed by a continuous infusion for 4 h at 4 µg/h.

After completion of the measurement for the last time point, rats were anesthetized with 60 mg/kg iv pentobarbital. Blood was obtained from the abdominal aorta for measurements of plasma rhRLX levels. The position of the jugular catheter relative to the right atrium, the placement of the pressure catheter relative to the aortic arch, and the position of the thermocouple relative to the left renal artery were recorded.

Hemodynamics and systemic arterial mechanical properties.   The low- and high-dose rhRLX protocols entailed seven male and nine female rats, respectively. After two baseline measurements of systemic hemodynamics on days 5 and 7 after surgery, either low- or high-dose rhRLX was administered by osmotic minipump. Systemic hemodynamics were again assessed on days 3, 6, 8, and 10 after initiation of relaxin infusion for the low-dose male rats and days 3 and 6 for the high-dose female rats. Each measurement consisted of four to eight recordings of CO and blood pressure waveforms obtained when the rat was either sleeping or resting. Seven to ten minutes were allowed between recordings. These measurements were obtained between 9 AM and 3 PM.

For acute administration of high-dose rhRLX, five female rats were used. Baseline measurements of systemic hemodynamics were obtained followed by intravenous infusion of high-dose rhRLX for 4 h. Systemic hemodynamics were assessed continuously during the 4-h infusion.

We used the thermodilution technique (22) to measure CO. Instantaneous P(t) waveforms were recorded by using a blood pressure telemetry system (Data Sciences International) (19). The aortic pressure recorded by the pressure catheter implanted in the aortic arch was transmitted to an external receiver. Steady-state aortic pressure was digitized online by using a personal computer-based data-acquisition system with 16-bit resolution and 2,000-Hz sampling rate and stored as text files for offline analysis. Each measurement consisted of a 30-s sampling duration.

Analysis of the acquired data and calculation of ACarea were performed by using a custom computer program developed using Matlab software (MathWorks, Natick, MA). Briefly, individual beats were selected (3–15 cycles) from the 10 s of the aortic pressure recording, immediately preceding the measurement of CO. The ensemble was averaged as described by Burattini et al. (2) to yield a single representative beat for each trial. The MAP, peak systolic pressure, and end-diastolic pressure were calculated from this averaged beat. Pulse pressure (PP) was calculated as peak systolic pressure minus end-diastolic pressure. SVR was calculated by dividing the MAP by CO.

Two measures of ACarea were calculated. The first was calculated from the diastolic decay of the P(t) waveform using the area method (18):

where P1 and P2 are the pressures at the beginning and end of the diastolic decay curve, respectively, and Ad is the area under the P(t) waveform over this region. The second measure of ACarea was calculated as the stroke volume (SV)-to-PP ratio (SV/PP) (3). SV was defined as CO/heart rate (HR).

Serum measurements.   Serum osmolality was measured by using a freezing-point depression instrumentation osmometer (model 3MO; Advanced Instruments, Needham Heights, MA). The levels of rhRLX in serum were measured by a quantitative sandwich immunoassay, as previously described (15).

Statistical analysis.   Data are presented as means ± SE. For the sake of comparison, we used data from our previous study (4) wherein low and medium doses of rhRLX were administered to female rats. Two-factor repeated-measures ANOVA (29) was used to compare mean values between low-dose male and female rats at various time points. The same analysis was performed to compare mean values among low, medium, and high doses of rhRLX in female rats at various time points. One-factor repeated-measures ANOVA (29) was used to compare mean values at various time points during the short-term administration of high-dose rhRLX to baseline values. If significant main effects or interactions were observed, pairwise comparisons between groups were performed by using Fisher's least significant difference test. The Student's paired t-test was used to compare the composite mean values (defined later) during chronic infusion of rhRLX with baseline. P < 0.05 was taken to be significant. Finally, we used linear regression to analyze the relationships between the magnitudes of the change in each arterial property of individual rats in response to relaxin infusion and the baseline values of that property. Group differences in the linear regression parameters were examined using analysis of covariance, implemented as multiple linear regression with dummy variables (12).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Male rats administered low-dose rhRLX (4 µg/h).   The temporal patterns of several systemic hemodynamic variables, expressed as a percentage of baseline values, are illustrated in Fig. 1, and absolute values of these variables are presented in Table 1. For the purpose of comparison, the data from our previous study (4) examining the effects of rhRLX infusion at 4 µg/h in female rats are also presented in Fig. 1. Low-dose rhRLX significantly increased CO relative to baseline in male rats. There was a slight (~6%), but statistically significant, rise in HR in the relaxin-treated male rats (Fig. 1A). However, there was a greater rise in SV (Fig. 1B), indicating that the elevation in CO resulted primarily from an increase in SV and to a lesser degree from a rise in HR. MAP was not significantly changed during rhRLX infusion (Fig. 1D). At the final time point (i.e., day 10 after the onset of rhRLX infusion), there was no statistically significant difference between the effects of rhRLX administration on systemic hemodynamics in the male and female rats.



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  		Fig. 1. Temporal changes in systemic hemodynamics in response to low-dose (4 µg/h) recombinant human relaxin (rhRLX) administration in male and female rats. Heart rate (A), stroke volume (SV; B), cardiac output (C), and mean arterial pressure (D) data are presented as percentages of baseline. Values are means ± SE. *P < 0.05 vs. baseline [post hoc Fisher's least significant difference (LSD)]. Although our previous analysis (4) of SV response in female rats alone indicated a significant increase at day 3, the simultaneous analysis of male and female responses did not yield a significant interaction term. Therefore, significant increments in SV are shown only for days 6, 8, and 10.

 

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  		Table 1. Male rats administered low-dose rhRLX (4 µg/h)

 
The temporal effects of rhRLX infusion on systemic arterial properties in male rats, expressed as a percentage of baseline values, are depicted in Fig. 2. Once again, absolute values for these variables are presented in Table 1, and previous data from female rats (4) are also shown in Fig. 2 for comparison. SVR fell significantly relative to baseline (Fig. 2A), whereas both measures of AC (ACarea and SV/PP) were significantly increased (Fig. 2, B and C). At the final time point (i.e., day 10 after the onset of rhRLX infusion), the changes in arterial properties were not statistically different between male and female rats.



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  		Fig. 2. Temporal changes in systemic arterial properties in response to low-dose (4 µg/h) rhRLX administration in male and female rats. Systemic vascular resistance (A) and 2 measures of global arterial compliance (ACarea; B) and SV-to-pulse pressure (PP) ratio (SV/PP; C) data are presented as percentages of baseline. Values are means ± SE. *P < 0.05 vs. baseline (post hoc Fisher's LSD). Although our previous analysis (4) of ACarea and SV/PP responses in female rats alone indicated significant increases at days 3 and 6, the simultaneous analyses of male and female responses did not yield significant interaction terms. Therefore, significant increments in ACarea and SV/PP are shown only for days 8 and 10.

 
Although the final (peak) responses were similar, relaxin-induced changes in male rats tended to be delayed, especially for SVR (Fig. 2A) and ACarea (Fig. 2B). We expected the two-way ANOVA to yield a significant interaction effect. However, this was not the case because the difference between male and female responses was typically confined to the first time point following the initiation of rhRLX infusion (i.e., day 3), and we do not have sufficient power to detect this single time point difference. In some instances, the interaction effect did come close to being significant; e.g., interaction for HR and SVR had P values of 0.09 and 0.14, respectively.

We calculated a composite mean change from baseline for each variable by averaging values at all successive time points during the infusion of rhRLX that were characterized by a significant change from baseline and were not significantly different from each other (i.e., the plateau phase). This yielded overall increases in CO and ACarea of 20.5 ± 4.2 and 19.4 ± 6.9% from baseline, respectively, and an overall decrease in SVR of 12.7 ± 3.9% from baseline (all P < 0.05 vs. baseline). There was no statistical difference between these results in male rats and those previously reported for female rats (4). Serum rhRLX was 17.7 ± 1.1 ng/ml, a value similar to that previously observed in female rats administered the same rhRLX regimen, 14.0 ± 2.0 ng/ml.

Female rats administered high-dose rhRLX (50 µg/h).   Absolute values of systemic hemodynamics and arterial properties are listed in Table 2, and their temporal patterns following the initiation of rhRLX infusion are depicted in Figs. 3 and 4. For the purpose of comparison, data from our previous study (4) examining the effects of low- (serum concentration = 14 ± 2 ng/ml) and medium-dose (serum concentration = 36 ± 3 ng/ml) rhRLX infusion in female rats are also shown in Figs. 3 and 4. Low- and medium-dose rhRLX infusion significantly increased CO, mainly by increasing SV. Both doses also significantly reduced SVR and increased AC (4). These alterations were all observed by the earliest time point studied: 3 days after the onset of rhRLX administration. Serum rhRLX concentration for the high-dose infusion in the present study was 71.5 ± 1.6 ng/ml. However, there was no change from baseline in any of the systemic hemodynamics or arterial properties (Figs. 3 and 4). Thus the effects of rhRLX on systemic hemodynamics and arterial properties are apparently biphasic.


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  		Table 2. Female rats administered high-dose rhRLX (50 µg/h)

 


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  		Fig. 3. Temporal changes in systemic hemodynamics in response to 3 doses of rhRLX administration in female rats: low (4 µg/h), medium (25 µg/h), and high (50 µg/h). Heart rate (A), SV (B), cardiac output (C), and mean arterial pressure (D) data are presented as percentages of baseline. Values are means ± SE. *P < 0.05 vs. baseline (post hoc Fisher's LSD).

 


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  		Fig. 4. Temporal changes in systemic hemodynamics in response to 3 doses of rhRLX administration in female rats: low (4 µg/h), medium (25 µg/h), and high (50 µg/h). Systemic vascular resistance (A) and 2 measures of ACarea (B) and SV/PP (C) data are presented as percentages of baseline. *P < 0.05 vs. baseline (post hoc Fisher's LSD).

 
Next, we wanted to determine whether there would be significant alterations in systemic hemodynamics and arterial properties in response to high-dose rhRLX treatment at a time point earlier than 3 days after the onset of rhRLX administration. Accordingly, an additional five female conscious rats were treated with acute intravenous infusion of rhRLX over 4 h. Serum rhRLX concentration was 64.1 ± 1.0 ng/ml. The temporal effects of short-term, high-dose rhRLX infusion on systemic hemodynamics and arterial properties in female rats, expressed as a percentage of baseline values, are depicted in Figs. 5 and 6. HR was significantly increased (~13%) at both the 2- and 4-h time points (Fig. 5A). This increase in HR was offset by a decrease (although statistically insignificant) in SV (Fig. 5B), resulting in no significant change in CO (Fig. 5C). There was a small (~8%), albeit statistically significant, increase in MAP (Fig. 5D). There were no statistically significant changes from baseline in any of the systemic arterial properties (Fig. 6).



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  		Fig. 5. Temporal changes in systemic hemodynamics in response to short-term high-dose rhRLX administration in female rats. Heart rate (A), SV (B), cardiac output (C), and mean arterial pressure (D) data are presented as percentages of baseline. Values are means ± SE. *P < 0.05 vs. baseline (post hoc Fisher's LSD).

 


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  		Fig. 6. Temporal changes in systemic arterial properties in response to short-term high-dose rhRLX administration in female rats. Systemic vascular resistance (A) and 2 measures of ACarea (B) and SV/PP (C) data are presented as percentages of baseline. Values are means ± SE.

 
Additional analysis.   We noticed an interesting trend in the data, suggesting that the magnitude of the change in arterial properties of individual rats (male or female) in response to infusion of low-dose rhRLX was dependent on the baseline value of that particular property. To validate this trend, we analyzed the relationship between baseline values of SVR, ACarea, and SV/PP and their respective, composite percent changes from baseline during rhRLX infusion. Linear regression analysis revealed that the effect of rhRLX infusion (i.e., the percent change from baseline) on SVR (Fig. 7A) and AC, as measured by both ACarea (Fig. 7B) and SV/PP (Fig. 7C), were all highly dependent on their baseline values. Specifically, rats with low AC at baseline were characterized by a greater increase in AC in response to relaxin treatment. Similarly, rats that had high SVR at baseline responded to relaxin with a greater decrease in SVR. Further analysis (analysis of covariance) indicated that these linear relationships were not different between male and female rats.



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  		Fig. 7. Relationships between composite percentage changes from baseline and baseline values for systemic vascular resistance (A) and 2 measures of ACarea (B) and SV/PP (C) in male and female rats administered low-dose rhRLX (4 µg/h). These relationships were sex independent. The solid line in each panel corresponds to the plot of the relationship obtained by linear regression (male and female rats combined).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
We previously reported that chronic administration of rhRLX to nonpregnant female rats that produced serum levels corresponding to early/midterm pregnancy significantly increased SV and CO and reduced steady and pulsatile arterial loads (4). In related work, we demonstrated that the influence of relaxin on the renal circulation was independent of sex and was biphasic (6, 7). The major findings of the present study were as follows: 1) chronic administration of low-dose rhRLX to conscious male rats resulted in an increase in CO and a reduction in steady and pulsatile arterial loads comparable to female rats; and 2) the effect of relaxin on systemic hemodynamics and arterial properties was biphasic, with higher doses yielding minimal and statistically insignificant changes from baseline.

Methodological considerations: Time controls.   We have previously established the stability of CO, SVR, and AC (ACarea and SV/PP) over a 17-day period beginning 5 days after surgery in chronically instrumented conscious rats (4). In addition, the vehicle for rhRLX was without any effect when administered alone for at least a 10-day period.

Methodological considerations: Serum levels of rhRLX.   In the protocols involving chronic administration of rhRLX by osmotic minipump, we obtained blood for serum rhRLX determinations at the end of the last experiment. Because we need 1.0 ml of blood to determine serum rhRLX in duplicate, we did not want to stress the rats with multiple blood drawings during the 6- to 10-day protocols. Nevertheless, we have previously conducted rhRLX infusions of shorter duration and measured rhRLX levels at the end (6). For example, after 48 h of rhRLX delivery at 40 µg/h, serum concentration was 80.2 ± 10.5 ng/ml. In the present study, the serum level of rhRLX was 71.5 ± 1.6 ng/ml after 10 days at 50 µg/h. Following 12 and 24 h of rhRLX administration at 4.0 µg/h, serum rhRLX was 14.0 ± 2.4 and 16.8 ± 2.7 ng/ml, respectively. In the present study, the level was 17.7 ± 1.1 ng/ml after 10 days at 4.0 µg/h. Thus circulating relaxin levels are relatively stable and constant over time.

Potential mechanisms of action.   Although the precise mechanisms for the relaxin-induced reduction in steady and pulsatile arterial loads are currently unknown, we propose three possible pathways. First, the hormone has been shown to exert renal vasodilation by increasing vascular gelatinase activity that, in turn, processes big ET to ET1–32 (9, 15). Subsequently, ET1–32 stimulates the production of endothelial NO via the endothelial ETB receptor subtype. This vasodilatory pathway may also be activated in other organ circulations during relaxin infusion, thereby contributing to the reduction in SVR and increase in AC. Second, vascular gelatinase and other matrix metalloproteinases can remodel extracellular matrix too, thereby contributing to the increase in ACarea (along with the decrease in arterial tone). Third, there is also the possibility that relaxin induces the formation of new blood vessels and/or increases branching of the vasculature (5, 28), which can contribute to the reduction in SVR and/or the increase in AC. Interestingly, this process of angiogenesis may also require the participation of vascular gelatinase activity (24), the endothelial ETB receptor, and NO (11). Increased skin capillary density has been reported in normal pregnancy (13). However, it is not known whether relaxin is responsible for this pregnancy-associated angiogenesis.

Male vs. female rats.   Analogous to our laboratory's previous work on relaxin in the renal circulation (7), this hormone elicited similar effects on systemic hemodynamics and arterial properties in both male and female rats, even though relaxin is traditionally considered to be a female hormone and is not believed to circulate in male rats (26). The renal effects of relaxin were also noted in ovariectomized rats, suggesting that female sex steroids play little or no intermediary role (8). Other studies have demonstrated that relaxin increases coronary blood flow in male rats and guinea pigs (1) and exerts chronotropic effects on the heart in male rats (16). Although not statistically significant, our results tend to show a delay in the response to relaxin treatment in males compared with females. This issue needs to be explored in future investigations. Potential mechanisms for this seemingly delayed response in male rats are not immediately evident. The three potential pathways for relaxin-induced changes in systemic arterial properties discussed above are likely to exhibit different temporal patterns, with the reduction in tone being the fastest and angiogenesis being the slowest. It is possible, therefore, that there is a difference in the relative contributions of these three pathways between male and female rats.

Low vs. high dose.   Analogous to our previous work on the influence of relaxin in the renal circulation and on osmoregulation (6), the effects of relaxin on systemic hemodynamics and arterial properties were biphasic. In direct opposition to our hypothesis, higher infusion rates of relaxin administered for 6 days that yielded serum concentrations corresponding to late pregnancy in the rat, when CO is highest and SVR lowest (10, 27), had no statistically significant effects on systemic hemodynamics and arterial properties. This finding suggests that relaxin alone is unlikely to be responsible for the alterations in systemic hemodynamics and arterial properties during the later stages of pregnancy. Alternatively, during late gestation, increasing serum concentrations of relaxin may require other pregnancy factors to further augment and maintain the increase in CO and decrease in arterial load. High-dose relaxin infusion alone, in the absence of these other factors, therefore, is incapable of reproducing these changes. Further analogous to our findings in the renal circulation in which high-dose infusion of rhRLX over a 4- to 6-h period was ineffectual (6), comparable administration also failed to increase systemic hemodynamics and ACarea. Thus there was not an initial positive effect of high-dose rhRLX after short-term infusion.

The LGR7 relaxin receptor, a leucine-rich, G-protein-coupled receptor, was recently discovered (14). To our knowledge, very little is known about the regulation of this receptor. Nevertheless, high-dose relaxin infusion alone may lead to classical downregulation of the putative relaxin receptor in the vasculature. Alternatively, we speculate that high-dose relaxin infusion alone may lead to inactivation of its receptor through proteolytic cleavage at a Gly-Leu bond in the extracellular domain by gelatinases or other matrix metalloproteinases that relaxin stimulates (15). Yet another possibility is that high levels of circulating relaxin leads to excessive production of ET1–32 via proteolytic cleavage of big ET at a Gly-Leu bond to the active peptide, ET1–32 (9, 15). Too much ET1–32 may then interact not only with the endothelial ETB receptor to produce NO but also with the vascular smooth muscle ETA and ETB receptors, thereby neutralizing the vasodilatory action of the endothelial ETB receptor.

Relationship to baseline values.   The present results demonstrate that the degree of reduction in arterial load during rhRLX infusion is dependent on the magnitude of arterial load before relaxin infusion. Irrespective of sex, rats that exhibited the greatest response to relaxin treatment were typically those that had high SVR and low AC at baseline. An intriguing explanation for this phenomenon that we are currently exploring is that relaxin is also a vascular-derived, locally acting compliance and relaxing factor. In those animals with low SVR and high AC at baseline, this local system may be more active, thereby mitigating the action of circulating hormone. Alternatively, the phenomenon may be related to the general principle that vasodilators are typically more effective when baseline SVR or MAP is high. Whether this axiom also holds true for AC, however, is unknown (i.e., factors that increase compliance are more effective when baseline compliance is low).

Summary.   Our previous work has shown that exogenous relaxin administration reproduces the pregnancy-associated alterations in systemic hemodynamics and arterial mechanical properties in nonpregnant female rats, thereby suggesting that the hormone may be responsible for triggering these alterations during pregnancy. Results of the current study indicate that these effects of relaxin are sex independent and that the dose response is biphasic. Next, we plan to determine whether elimination or neutralization of circulating relaxin in pregnant rats will prevent these changes in systemic hemodynamics and arterial properties as it did for the changes in the renal circulation (20).


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This project was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-67937 and McGinnis Chair Endowment funds. D. O. Debrah was the recipient of a Minority Undergraduate Student Research Supplement (to RO1 HL-67937) and a Beckman Undergraduate Research Award (2003–04).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Caroline Evans for technical assistance. We are grateful to Dr. Elaine Unemori of Connetics (Palo Alto, CA) and BAS Medical (San Mateo, CA) for providing the rhRLX and the antibodies for the rhRLX ELISA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. P. Conrad, Magee-Womens Research Institute, 204 Craft Ave., Pittsburgh, PA 15213 (E-mail: rsikpc{at}mwri.magee.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
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 GRANTS
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 REFERENCES
 

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