The renin-angiotensin system is important in the regulation of blood pressure, body fluid homeostasis, and renal, neuronal, and endocrine functions associated with cardiovascular control in vertebrates (Inagami T, 1994). Renin, the rate-limiting enzyme of the renin-angiotensin system, hydrolyzes its specific protein substrate angiotensinogen to release the decapeptide angiotensin (Ang) I, which is further cleaved by a nonspecific dipeptidyl-carboxypeptidase angiotensin-converting enzyme (ACE) to produce the vasopressor octapeptide Ang II. Ang II functions by binding to Ang II type-1 (AT1) and type-2 (AT2) receptors.
A renin-like enzyme, an angiotensinogen-like protein, and ACE have been discovered in invertebrates such as leeches (Salzet and Stefano, 1997; Salzet et al., 1993; Vandenbulcke et al., 1997), locusts (Salzet et al., 2001; Schoofs et al., 1998; Veelaert et al., 1998), molluscs (Gonzalez et al., 1995; Laurent et al., 1997), and crabs (Delorenzi et al., 1996). Ang II-amide was isolated from leeches (Salzet et al., 1995), and immunoreactivity against anti-Ang I or anti-Ang II antibodies was demonstrated in leeches (Salzet et al., 2001), clam worms (Fewou and Dhainaut-Courtois, 1995), locusts (Schoofs et al., 1998), and crabs (Delorenzi et al., 1996; Frenkel et al., 2010). A 140-kDa protein with immunoreactivity to anti-AT1 receptor antibodies was purified from leech coelomocyte extract using an Ang II affinity column (Salzet and Verger-Bocquet, 2001). These findings suggest that invertebrates express essential components of the renin-angiotensin system.
There are some reports demonstrating the effects of Ang II on body fluid homeostasis in invertebrates. In the clam worm, a marine polychaete, Ang II attenuates body fluid loss under hyperosmotic and drying conditions and enhances body fluid gain under hypoosmotic conditions (Fewou and Dhainaut-Courtois, 1995; Satou et al., 2005a; Satou et al., 2005b). In slugs, Ang II facilitates water absorption from the foot (Makra and Prior, 1985). Accordingly, Ang II increases body fluid volume in these invertebrates as well as in vertebrates. In contrast to the physiological action of Ang II in the aforementioned invertebrates, Ang II accelerates body fluid loss under semidrying conditions in leeches (Salzet et al., 1995; Salzet et al., 1992), suggesting a negative regulation of body fluid volume by Ang II. However, it was concluded that the downregulating effect of Ang II in leeches was caused by their blood-feeding behavior. Therefore, in the present study, we tested if Ang II decreases body fluid volume in a freshwater oligochaete, Tubifex tubifex, which does not require blood-feeding.
MATERIALS AND METHODS
Ang I, Ang II (human type), [Sar1, Ala8]-Ang II (saralasin, human type), and [Sar1, Val5, Ala8]-Ang II (saralasin, bovine type) were purchased from the Peptide Institute (Osaka, Japan). Other chemical reagents were from Nacalai Tesque (Kyoto, Japan).
Freshwater sludge worms, T. tubifex (Annelida, Oligochaeta, Tubificidae) collected from a natural source were obtained from a local aquarium fish supplier in Gifu, Japan, and maintained at room temperature in artificial pond water (APW; Dietz and Alvarado, 1970) consisting of 0.5 mM NaCl, 0.05 mM KCl, 0.4 mM CaCl2, and 0.2 mM NaHCO3 until use. When the worms were used for the experiments, we carefully observed the morphology of individual animals and excluded worms which exhibited unexpected size and/or morphology.
Treatment of T. tubifex with Ang I or Ang II
T. tubifex worms (a total of approximately 100 mg) were exposed at room temperature to Ang I (from 0 to 1 × 10-8 M) or Ang II (from 0 to 1 × 10-7 M) dissolved in APW for 30 min. The total body weight of the worms was measured after briefly blotting on tissue paper to remove external liquid. The total body weight was expressed as a relative value to that observed at the 0-min time point of each treatment.
T. tubifex worms (a total of approximately 100 mg) were exposed to 1×10-8 M Ang I or Ang II dissolved in APW at room temperature. APW, which does not contain Ang, served as a control. The total body weight of the worms was measured and expressed as described above after treatment times of 0, 15, 30, and 60 min.
Treatment of T. tubifex with Ang II in the presence of varying concentrations of saralasin
T. tubifex worms (a total of approximately 100 mg) were exposed at room temperature to 1×10-8 M Ang II in combination with 0 to 1×10-8 M human- or bovine-type saralasin in APW for 30 min. The total body weight of the worms was measured and expressed as described above.
Data were expressed as mean ± SD of three independent experiments. Statistical comparison was performed by Welch’s t-test using Statcel (OMS, Japan). A p-value less than 0.05 was considered significant.
Effects of varying concentrations of Ang I and Ang II on body weight of T. tubifex
Ang II produced a decrease in body weight of T. tubifex in a concentration-dependent manner (Figure 1). Significant changes were observed at 1 × 10-12 to 1 × 10-7 M. The maximal and the half-maximal effect of Ang II were observed at about 1 × 10-8 and 2-5 × 10-11 M, respectively. Ang I did not alter the body weight.
Figure 1. Effects of Ang I and Ang II on the body weight of Tubifex tubifex. The worms were treated with indicated concentrations of Ang I (open circle) or Ang II (closed circle) in artificial pond water for 30 min. Data are expressed as mean ± SD of three independent experiments. Asterisks indicate a significant difference compared to the value obtained in the absence of Ang at *p < 0.05 or **p < 0.01.
Temporal changes in the body weight of T. tubifex treated with/without Ang II
In the control group exposed to APW, approximately 5% of body weight was lost after 60 min. Treatment with Ang II (1 × 10-8 M) enhanced the decrease in body weight in a time-dependent manner (Figure 2). Body weight markedly decreased to below 85% during the first 30 min of the treatment, and then, the decreased body weight was sustained in the following 30 min. Ang I-treated worms did not show any difference from the control.
Effect of saralasin on Ang II-induced change in body weight of T. tubifex
Both human- and bovine-type saralasins blunted the Ang II-induced body weight loss (Figure 3). Saralasins at 1 × 10-9 and 1 × 10-8 M recovered the body weight to the control level. The half-maximal inhibitory concentrations for human- and bovine-type saralasins were 2–3 × 10-10 and 4–5 × 10-11 M, respectively.
Figure 2. Temporal changes in the body weight of Tubifex tubifex treated with/without Ang II. The worms were treated with 1 × 10-8 M Ang I (open circle) or Ang II (closed circle) in artificial pond water (APW) up to 60 min. APW without Ang was used as a control (open triangle). Data are expressed as mean ± SD of three independent experiments. Asterisks indicate a significant difference compared to the value at the 0-min time point at *p < 0.05 or **p < 0.01.
Figure 3. Effect of saralasin on the Ang II-induced change in body weight of Tubifex tubifex. The worms were treated with 1×10-8 M Ang II in combination with 0 to 1×10-8 M human-type (closed circle) or bovine-type (open) saralasin in artificial pond water (APW) for 30 min. Data are expressed as mean ± SD of three independent experiments. Asterisks indicate a significant difference compared to the value obtained in the absence of saralasin at *p < 0.05 or **p < 0.01. The dotted line represents the mean value obtained from the worms treated with APW for 30 min in the absence of Ang and saralasin.
Ang II produced a time- and concentration-dependent decrease in body weight in T. tubifex, suggesting that Ang II downregulates the body fluid volume of the worms. This effect in T. tubifex resembles the diuretic effects of Ang II in the leech Theromyzon tessulatum (Salzet et al., 1995); however, it is not consistent with the upregulating effects on body weight in clam worms Nereis diversicolor and Perinereis sp. (Fewou and Dhainaut-Courtois, 1995; Satou et al., 2005a; Satou et al., 2005b), and in vertebrates. The differing effects of Ang II in invertebrates may have originated from evolutionary adaptations to environmental conditions. Considered together with the findings presented above, Ang II seems to upregulate the body fluid volume in invertebrates that undergo the passive outflow of body fluid water (e.g., marine and terrestrial organisms), but downregulate the body fluid volume in invertebrates that utilize the passive or active inflow of outside water to regulate body fluid volume (e.g., freshwater and blood-feeding organisms).
In the present study, the Kd value of Ang II for its receptor estimated from the concentration-dependence curve (Figure 1) was 2–5 × 10-11 M, which is a similar order to that reported in clam worms (Satou et al., 2005a) and mammals (Kobayashi and Takei, 1996). The minimum effective concentration of Ang II observed in this study was 1 × 10-12 M, supporting previous findings in clam worms (Satou et al., 2005a). In addition, the minimum effective concentration of Ang II in T. tubifex is similar to the human and rat plasma concentration of the peptide (Jacobsen and Poulsen, 1990; Matsui et al., 1999; Mizuno et al., 1992). Since these results suggest the existence of an Ang II receptor in T. tubifex, involvement of Ang II receptors in Ang II-induced body weight loss in T. tubifex was tested using Ang II receptor inhibitors. Antagonizing properties of pharmacological Ang II receptor blockers including losartan, candesartan, and olmesartan have not been evaluated in invertebrates. Thus, saralasins, Ang II analogs (Regoli et al., 1974; Timmermans et al., 1974), were employed in the study to avoid potential issues with species-dependent specificity of Ang II receptor blockers. The existence of Ang II receptors in T. tubifex is further supported by the observation that saralasin diminished the Ang II-induced effect (Figure 3). The receptor subtype; however, was not delineated in the current study because of the nonselectivity of the antagonist. The use of a specific AT2 receptor antagonist such as PD123319 will clarify the receptor subtype concerning the Ang II-induced effect if the inhibitor is crossreactive with putative AT2 receptors in the worm. The Ki values of human- and bovine-type saralasins for the receptor estimated from the inhibition curves (Figure 3) were 2–3 × 10-10 and 4–5 × 10-11 M, respectively. This suggests higher affinity of the Ang II receptor to bovine-type Ang II than human one. Although human-type Ang II was elucidated in leeches (Salzet et al., 1995), a more bovine-type-like Ang II is speculated in T. tubifex.
Ang I was ineffective even at 1 × 10-8 M, which is 10,000-fold higher than the lowest Ang II concentration that produced significant effects. As ACE is a nonspecific dipeptidyl-carboxypeptidase, this result suggests the possibility that Ang II acts cutaneously and is not taken up into the body fluid of the worm. This possibility is supported by the finding that Ang II affects frogs through skin exposure (Coviello and Brauckmann, 1973). In addition, ACE, which does not contain a C-terminal transmembrane domain, has been identified in leeches and other invertebrates (Rivière et al., 2004). The expression of only soluble ACE in invertebrates might also provide limited capability of local production of Ang II from exogenous Ang I in the present study.
In conclusion, Ang II downregulates body fluid volume of T. tubifex via the Ang II receptor, providing evidence that Ang II downregulates body fluid volume of non-blood-feeding invertebrates. Although further studies including identification of the components of the renin-angiotensin system will be required to elucidate the physiological significance of this hormonal system in course of evolution, the findings will help to evolutionarily evaluate functions of the renin-angiotensin system in mammals.
We thank Prof. Takashi Shimizu (Hokkaido University, Sapporo, Japan) for helpful information on tubificid taxonomy, biology, and maintenance.
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