On days 1, 3, 5 and 7 after surgery the rats were weighed and the systolic blood pressure was measured. Only the Sham rats on day seven of the protocol showed a significant gain in body weight (data not shown). None of the rats in the Sham-high salt, DOCA-high salt and DOCA-low salt groups gained significant body weight during the protocol. DOCA-high salt rats consumed significantly more fluid than the Sham, Sham-high salt and DOCA-low salt rats by day three and this continued through day seven (figure 1). Additionally, rats placed on high salt water alone increased intake significantly by day five and remained elevated through day seven (figure 1, bottom left and right, respectively). The Sham and DOCA-low salt rats did not vary significantly in their fluid consumption during the study.
DOCA-high salt rats showed a significant increase in blood pressure by day three [average systolic blood pressure (SBP) 124 ± 7.6 mm Hg, 110 ± 2.6 mm Hg and 112 ± 2.4 mm Hg, DOCA-high salt and Sham, respectively] (figure 2). The DOCA-salt rats reached hypertensive levels by day five (average SBP 147 ± 14.0). Sham-high salt rats had elevated blood pressures by day seven (average SBP 130 ± 9.3 mm Hg). Furthermore, by day seven the DOCA-low salt rats had significantly lower blood pressure than their Sham, DOCA-high salt and Sham-high salt counterparts (average SBP 114 ± 2.3 mm Hg, 142 ± 8.4 mm Hg, 130 ± 9.3 mm Hg and 101 ± 2.1 mm Hg, Sham, DOCA-high salt, Sham-high salt and DOCA-low salt, respectively). For further data please see Figure 9.
Contractile Studies on Day 1
Although there was no increase in systolic blood pressure on day one, arteries from DOCA-high salt rats contracted to the 5-HT2B receptor agonist BW723C86 (maximal contraction 52.5 ± 10.5 % of PE contraction). Arteries from DOCA-low salt, Sham-high salt and Sham rats did not respond to either BW723C86 or to the 5-HT1B receptor agonist CP93129 (figures 3A and 3B). There was no enhanced contraction to 5-HT observed in any of the arteries from the treatment groups (figure 3C). These data suggest that while 5-HT2B receptors are functional on day one, as indicated by contraction to BW723C86, this is not sufficient to result in an enhanced contraction to 5-HT. Additionally, the 5-HT1B receptor agonist CP93129 was unable to stimulate contraction, suggesting that the 5-HT1B receptor was not functionally coupled to contraction on day one.
Contractile Studies on Day 3
On day three, BW723C86 contracted arteries from DOCA-high salt and Sham-high salt rats (figure 4A). There was also a significant contraction observed to CP93129 in arteries from DOCA-high salt rats (figure 4B). Arteries from DOCA-low salt and Sham rats did not contract to BW723C86 or CP93129 (figures 4A and 4B). Contraction to 5-HT was enhanced, as defined by an increased maximal contraction, decreased threshold for contraction and/or an increased potency, in arteries from Sham-high salt rats (figure 4C). The arteries from the DOCA-high salt rats showed no change in the maximal contraction to 5-HT but demonstrated enhanced 5-HT potency (-log EC50 [M] values 6.02 ± 0.08, 5.74 ± 0.05 DOCA-high salt day 3 and Sham, respectively). Unexpectedly, arteries from DOCA-low salt rats showed an increased maximal response to 5-HT (maximal contraction 141.2 ± 11.8 % PE contraction). Since there was no response to CP93129 and very little response to BW723C86, this enhanced contraction to 5-HT was surprising. However, this enhancement of 5-HT-induced contraction in arteries from DOCA-low salt rats only occurred on day three. These findings suggest that the 5-HT1B receptor requires the presence of elevated levels of DOCA, salt and pressure to become functionally coupled to contraction. 5-HT2B receptors were able to mediate contraction in arteries from both DOCA-high salt and Sham-high salt rats, suggesting that the elevated levels of salt are required to observe functional changes in the 5-HT2B receptor.
Contractile Studies on Day 5
Experiments performed on day five of the time course demonstrated a similar profile of results. Arteries from DOCA-high salt and Sham-high salt rats contracted to both BW723C86 (maximal contraction 54.3 ± 11.9 % and 46.3 ± 6.6 % of PE contraction, DOCA-high salt and Sham-high salt, respectively) and CP93129 (maximal contraction 35.0 ± 8.6 % and 27.8 ± 7.4 % of PE contraction, DOCA-high salt and Sham-high salt, respectively) (figures 5A and 5B). Arteries from Sham-high salt rats also displayed an increased maximal contraction to 5-HT (maximal contraction 148.5 ± 11.4 % and 106.2 ± 8.7 % of PE contraction, Sham-high salt and Sham, respectively), a decrease in the threshold of activation of contraction and an increase in potency, and as compared to Sham arteries (-log EC50 value [M] 6.06 ± 0.05 and 5.60 ± 0.07 Sham-high salt and Sham, respectively) (figure 5C). The arteries from DOCA-high salt rats also show a decrease in the threshold of activation of contraction (figure 5C). These results suggest that additional changes in the contractile mechanisms utilized by 5-HT, in addition to the 5-HT1B and 5-HT2B receptors being functionally coupled to contraction, are required in arteries from DOCA-high salt hypertensive rats to mediate hyperresponsiveness to 5-HT.
Contractile Studies on Day 7
On day seven, arteries from DOCA-high salt rats contracted to both BW723C86 (maximal contraction 57.4 ± 5.9 % of PE contraction) and CP93129 (maximal contraction 63.8 ± 7.8 % of PE contraction) (figures 6A and 6B). Arteries from Sham-high salt rats also contracted to both BW723C86 (maximal contraction 24.5 ± 9.4 % of PE contraction) and CP93129 (maximal contraction 19.6 ± 8.1 % of PE contraction) (figures 6A and 6B). Interestingly, although the arteries from Sham-high salt rats were exposed to both increased levels of salt and pressure, these arteries did not contract to the same magnitude in the presence of BW723C86 (maximal contraction 24.5 ± 9.4 % and 57.4 ± 5.9 % of PE contraction, Sham-high salt and DOCA-high salt, respectively) and CP93129 (maximal contraction 19.6 ± 8.1% and 63.8 ± 7.8 % of PE contraction, Sham-high salt and DOCA-high salt, respectively) as those that are taken from DOCA-high salt rats on day seven. Additionally, arteries from DOCA-high salt and Sham-high salt rats both show hyperresponsiveness to 5-HT (figure 6C). These arteries demonstrate a characteristic increased maximal contraction (maximal contraction 130.3 ± 19.5 % and 119.5 ± 5.1 % of PE contraction, DOCA-high salt and Sham-high salt, respectively), the decreased threshold for contraction and the increased potency (- log EC50 value [M] 6.41 ± 0.09, 6.13 ± 0.03 and 5.70 ± 0.07, DOCA-high salt, Sham-high salt and Sham, respectively). These data suggest that the combined presence of elevated levels of salt, DOCA and pressure all contribute to the functional changes in 5-HT1B and 5-HT2B receptors observed in the arteries from the DOCA-high salt hypertensive rats.
Protein Analysis Studies
To determine if upregulation of 5-HT1B and 5-HT2B receptor proteins was responsible for the acquired contractility, we performed western analysis of total aortic homogenates. The authors acknowledge that the limitations of this technique do not speak to localization of the receptor proteins within the cell, only to the total amount of the receptor protein present. There was no increase in 5-HT1B receptor protein levels in any of the treatment groups at any time point (figure 7), although there was a trend towards increasing levels of protein in the aortic homogenates from the DOCA-high salt rats. This was an unexpected finding as the arteries from both the DOCA-high salt and Sham-high salt rats contract to the 5-HT1B agonist CP93129 starting on day three of the time course. These data suggest that although the receptor protein is upregulated by 28 days of DOCA-salt treatment (13), upregulation is not required to enable this receptor to participate in contraction.
In contrast, the 5-HT2B receptor was upregulated significantly by day 3 of DOCA-high salt treatment (figure 8). However, 5-HT2B receptor protein levels were not increased in any of the other treatment groups at any time point. Interestingly, the level of receptor protein was not significantly increased on day 1, even though there was a significant contraction to the 5-HT2B receptor agonist BW723C86 on this day. There was also no significant increase in 5-HT2B receptor levels in the aortic homogenates from the Sham-high salt rats in which contraction to BW723C86 occurred on days 3, 5 and 7. These findings suggest that upregulation of the 5-HT2B receptor proteins is not absolutely required for this receptor to mediate agonist-induced contraction.