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Interaction of Osmotic and Volemic Mechanisms in Secretion of Atrial and Ventricular Natriuretic Peptides in Eels
General and Comparative Endocrinology 107, 322–326 (1997) Article No. GC976928
Interaction of Osmotic and Volemic Mechanisms in Secretion of Atrial and Ventricular Natriuretic Peptides in Eels Hiroyuki Kaiya and Yoshio Takei Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Minamidai, Nakano, Tokyo 164, Japan Accepted April 17, 1997
In eels, plasma osmolality rather than blood volume is a major regulator of atrial and ventricular natriuretic peptide (ANP and VNP) secretion. The present study examined the effects of changes in blood volume on ANP and VNP secretion stimulated by an increase in plasma osmolality in freshwater-adapted eels. Basal plasma ANP and VNP levels were decreased by 2 ml of blood withdrawal (28% of total blood volume), but not changed by blood volume expansion with 2 ml of 0.9% NaCl solution containing 2% dextran. The blood loss suppressed the increased plasma ANP level caused by an injection of 2.5 ml/kg of 1.7 M NaCl solution at 60 min (120.5 6 31.0 fmol/ml, n 5 5) compared with controls without blood volume manipulation (586.6 6 43.6 fmol/ ml, n 5 5), but the plasma ANP level transiently increased in bled fish immediately after osmotic stimulus, probably due to the release of ANP stored in the cardiac tissues after the blood loss. Changes in plasma VNP were not so evident as those of ANP. In contrast, blood volume expansion augmented the increase in plasma ANP and VNP levels within 60 min after osmotic stimulus compared with controls. The recovery of plasma VNP level was quicker than that of plasma ANP. Increases in plasma Na, Cl concentrations, and osmolality were not different among hypovolemic, normovolemic, and hypervolemic eels after osmotic stimulation. It is concluded that volume itself is a minor regulator for ANP and VNP secretion compared with osmotic stimulus, but it plays a modulatory role in osmotically induced ANP and VNP secretion in eels. r 1997 Academic Press
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Atrial natriuretic peptide (ANP) is a cardiac hormone known to be involved in volume and blood pressure homeostasis (Brenner et al., 1990). In mammals, the diuretic/natriuretic actions of ANP decrease blood volume, and an increased blood volume stimulates ANP secretion (Ruskoaho, 1992). In fish, however, the actions of ANP may differ from those in mammals; ANP seems to extrude sodium but not water at least in seawater (SW)-adapted eels (Evans, 1990; Takei and Balment, 1993a,b). Recently, both plasma osmolality and blood volume were found to regulate ANP secretion, but the osmotic mechanism is more dominant in eels (Kaiya and Takei, 1996b). After transfer of eels from freshwater (FW) to SW, plasma ANP concentration showed a biphasic response; a rapid, transient increase followed by a delayed increase after 6 hr (Kaiya and Takei, 1996c). Since the transient restoration of plasma ANP concentration by 6 hr after transfer occurs despite a progressive increase in plasma osmolality, it is possible that the fluctuation is caused by a decreased blood volume. In fact, eels face a severe dehydration after SW exposure by obligatory losses of water through the skin and gill epithelia (Oide and Utida, 1968; Kirsh and Mayer-Gostan, 1973). Furthermore, an increased plasma ANP concentration after SW transfer did not occur when the volume of blood sampled was increased, probably due to hypovolemia (H. Kaiya and Y. Takei, unpublished observation). Therefore, blood volume may modulate osmotically induced ANP secretion although volume changes alone 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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ANP Secretion in Eel
affect ANP secretion only slightly (Kaiya and Takei, 1996b). The present study examined the interaction of osmotic and volemic mechanisms in the regulation of ANP and VNP secretion in FW-adapted eels.
MATERIALS AND METHODS Fish Immature, cultured Japanese eels, Anguilla japonica, of either sex, weighing 196.7 6 3.6 (n 5 21) were obtained from a commercial source. Fish were maintained in FW aquaria unfed for 1–2 weeks before use. Water in the aquaria was continuously filtered, aerated at 18°.
Experimental Procedure After anesthesia by immersion in FW containing 0.3% (w/v) 3-aminobenzoic ethyl ester (MS222, Sigma, St. Louis, MO), a catheter (SP 10; OD, 0.61 mm, Natume, Japan) was inserted into the ventral aorta of FW eels as described previously (Kaiya and Takei, 1996a). After surgery, fish were transferred to an individual trough through which aerated water (18°) continuously circulated and allowed to recover for at least 20 hr before further study. To examine the effect of hypovolemia on osmotically induced ANP secretion, 2 ml of blood (approximately 28% of total blood volume; see Takei, 1988) was withdrawn through an aortic catheter 10 min before injection of a 1.7 M NaCl solution (2.5 ml/kg body wt) into five eels. Eels are still severely hypovolemic 15 min after 2 ml of blood withdrawal (Takei, 1988). Blood (200 µl) was sampled before controlled blood withdrawal and 0, 5, 15, 30, 60, and 180 min after injection of 1.7 M NaCl solution for measurement of plasma ANP and VNP concentrations and plasma solutes. Plasma obtained from before and 10 min after manipulation of blood volume was compared with plasma ANP, VNP, and Na concentrations to determine an acute effect of blood manipulation. After injection of hypertonic saline, the catheter (dead space ca. 10 µl) was flushed with 50 µl of 0.9% NaCl solution. The volume of collected blood was replaced with a 0.9% NaCl solution after each sampling. As a more physi-
ological stimulus, 0.85 M NaCl solution was injected after 1 ml blood withdrawal in another six eels. To examine the effect of hypervolemia, 2 ml of 0.9% NaCl solution containing 2% dextran (Sigma, Mr, 60,000– 90,000) was injected via an aortic catheter 10 min before injection of 1.7 M NaCl solution into another five eels. Dextran was added to adjust the colloidal osmotic pressure. The time course of blood sampling was the same as above. Five control fish received 1.7 M NaCl solution without manipulation of blood volume. Plasma dilution was taken into account for calculation of plasma hormone levels based on the method used by Nishida et al. (1988).
Determination of Plasma Parameters Blood was withdrawn into a chilled plastic syringe containing 10% 2K-EDTA solution (10 µl/ml blood). Forty microliters of blood was collected into a capillary tube to measure hematocrit. Plasma in the capillary tube was used for determination of osmolality with a vapor pressure osmometer (Model 5500, Wescor Inc., UT) and of Na concentration with an atomic absorption spectrophotometer (Model 180-50, Hitachi Ltd., Tokyo, Japan). Plasma Cl concentration was measured by Buchler–Cotlove chloridometer (Model 4-2500, Buchler Inc., NJ). The remaining blood was centrifuged at 11,000g for 5 min at 4°, and plasma was stored at 220° for later radioimmunoassay of ANP and VNP (Kaiya and Takei, 1996a).
Statistical Analysis The differences in plasma ANP, VNP, and Na concentrations before and after manipulation of blood volume were evaluated by Wilcoxon signed rank test. The differences between control and experimental groups were evaluated by two-way ANOVA (repeated measures), followed by Student’s t test or Cochran–Cox test to assess the difference at each time point. A P-value of less than 0.05 was considered significant. Values are expressed as means 6 SEM.
RESULTS Effect of Blood Volume Reduction In control fish without blood volume manipulation, plasma ANP concentration slightly but significantly
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increased immediately after injection of 1.7 M NaCl solution, and the peak increase was observed at 60 min (Fig. 1A). After 2 ml of blood was withdrawn, plasma ANP, VNP, and Na concentrations decreased significantly (Table 1). In the volume-depleted fish, plasma ANP concentration increased transiently 5 min after injection of 1.7 M NaCl solution. The increased level was higher than that of controls, but quickly returned to the initial level, so that the level became lower than that of controls at 60 min (Fig. 1A). The increase in plasma VNP concentration in control fish was faster and greater than that of ANP; the peak increase was observed 15 min after injection of hypertonic saline (Fig. 1D). In volume-depleted fish, the increase in plasma VNP concentration was less, but the difference was not significant compared with that of controls (Fig. 1D). The peak increase in plasma Na and Cl
Kaiya and Takei
TABLE 1 Changes in Plasma Atrial and Ventricular Natriuretic Peptide (ANP and VNP) Concentrations (fmol/ml) and Na Concentrations (mM) before and 10 min after Manipulation of Blood Volume in Freshwater Eels Group Hypovolemia (2 ml) ANP VNP Na Hypovolemia (1 ml) ANP VNP Na Hypervolemia (2 ml) ANP VNP Na Normovolemia ANP VNP Na
Before
After
69.1 6 10.5 (5) 131.1 6 14.8 141.1 6 1.5
44.7 6 1.8* 113.2 6 9.9* 136.9 6 2.8*
144.3 6 6.9 (6) 198.6 6 12.1 146.6 6 1.2
126.4 6 12.5 180.9 6 18.1 144.9 6 2.1
117.1 6 13.4 (5) 113.7 6 12.7 141.5 6 1.7
123.0 6 13.7 113.1 6 8.3 142.5 6 1.8
127.7 6 9.4 (5) 177.3 6 8.7 145.6 6 2.1
117.6 6 10.7 178.7 6 10.9 146.3 6 1.4
Note. Values are means 6 SEM. The number in parentheses indicates the number of animals used. * 0.01 , P , 50.05 (Wilcoxon signed rank test) compared with the value before manipulation of blood volume.
concentrations and osmolality occurred 5 min after injection of 1.7 M NaCl solution and gradually declined thereafter (Figs. 1B, 1C, and 1E). Changes in these plasma solutes did not differ between volumedepleted and control fish. In eels subjected to milder blood loss (1 ml of blood withdrawal) and osmotic stimulus (0.85 M NaCl injection), the difference in plasma hormone levels was not significant between volume-depleted and control fish (data not shown).
Effect of Blood Volume Expansion
FIG. 1. Effect of blood volume reduction (28% of total blood volume) on the secretion of atrial and ventricular natriuretic peptides (ANP and VNP) after intra-arterial injection of a hypertonic 1.7 M NaCl solution (2.5 ml/kg) in freshwater eels. (A) Plasma ANP, (B) plasma Na, (C) plasma osmolality, (D) plasma VNP, (E) plasma Cl, and (F) hematocrit are expressed. (W) and (X) Volume-depleted and control eels, respectively. Values are means 6 SEM. An arrow indicates the point of blood volume manipulation. *P , 0.05 compared with controls.
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Plasma ANP and VNP concentrations did not change after injection of 2 ml of dextran–saline (Table 1). In volume-loaded fish, the increase in plasma ANP and VNP concentrations after injection of hypertonic saline was greater than that of controls (Figs. 2A and 2D). However, the increased VNP level returned to the control level more quickly than the ANP level. Changes in plasma Na and Cl concentrations and osmolality were similar in volume-loaded and control fish (Figs. 2B, 2C, and 2E).
ANP Secretion in Eel
FIG. 2. Effect of blood volume expansion (28% of total blood volume) on the secretion of ANP and VNP after intra-arterial injection of a hypertonic 1.7 M NaCl solution (2.5 ml/kg) in freshwater eels. (A) Plasma ANP, (B) plasma Na, (C) plasma osmolality, (D) plasma VNP, (E) plasma Cl, and (F) hematocrit are expressed. (W) and (X) Volume-loaded and control eels, respectively. Values are means 6 SEM. An arrow indicates the point of blood volume manipulation. *P , 0.05 compared with controls.
DISCUSSION The present study showed that the increased secretion of ANP and VNP by osmotic stimulus was modified by changes in blood volume in eels. Thus, although blood volume expansion alone is a minor stimulus for ANP secretion (Kaiya and Takei, 1996b), it is apparent that blood volume plays a modulatory role in osmotically induced ANP secretion in eels. Furthermore, the present detailed analysis of the time course showed that the peak response of ANP and VNP secretion occurs slightly later than that of plasma osmolality and that the response of VNP is more rapid than that of ANP. There have been a number of observations showing that plasma concentrations of ANP are altered by volume status in terrestrial animals, and the primary
325
regulator for ANP secretion is the change in atrial stretch (Ruskoaho, 1992). In eels, however, plasma ANP and VNP concentrations were not altered even by a 14% reduction in blood volume and by 28% blood volume expansion in this study. Similar blood volume manipulations in normohydrated terrestrial animals invariably alters plasma ANP concentration (Lang et al., 1985; Courneya et al., 1989; Gray et al., 1991). Since fish have a huge secondary vascular system (Olson, 1992), the system may buffer the changes in blood volume. Thus, the dominancy of blood volume as a regulator of ANP secretion seems to be smaller in eels than in terrestrial animals. This difference in ANP secretion may reflect the difference in the biological actions of ANP; ANP promotes sodium but not water loss in the eel (Takei and Balment, 1993b), but it promotes both sodium and water loss in terrestrial animals (Brenner et al., 1990). After blood withdrawal, the amount of ANP and VNP secretion immediately after osmotic stimulus was greater than that of controls without blood volume manipulation. Since ANP and VNP secretions were inhibited after blood loss as shown in this study, the immediate increase in plasma ANP and VNP may be caused by transient release of increased ANP and VNP stored in cardiac tissues caused by blood loss. When eels were transferred from FW to SW, plasma ANP and VNP concentrations increased just after transfer, then returned to initial values, to further increase after 6 hr (Kaiya and Takei, 1996c). This biphasic response of natriuretic peptide secretion may be accounted for by the transient reduction of blood volume after transfer to SW. Eels exposed to SW face a severe dehydration by losses of water through the skin and gill epithelia (Oide and Utida, 1968; Kirsh and Mayer-Gostan, 1973) and blood volume in fact decreases for a few hours after SW transfer as determine by dye dilution (Y. Takei, unpublished data). The reason for the temporary diminution of ANP and VNP secretion and relatively smaller increase in plasma ANP and VNP levels despite a sufficient osmotic stimulus for its secretion after transfer to SW may reflect blood volume reduction, as shown in this study. The present in vivo study of eels suggests a close interaction between osmotic and volume mechanisms in the regulation of ANP secretion. In vitro studies using mammalian cardiac tissues suggest that influx of
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extracellular Ca through Ca channels plays an important role in stretch-induced ANP secretion (Laine et al., 1994a,b), while a Na–Ca exchanger is involved in osmotically induced ANP secretion (Schiebinger et al., 1995). Thus, cellular mechanisms for ANP secretion between stretch-induced and osmotically induced ANP secretion seem to be functionally separated, although the Ca ion is key in both mechanisms. To further analyze the interaction of osmotic and volume mechanisms at the cellular level, it is necessary to do in vitro studies using eel cardiac tissues or cardiocytes. The present detailed analysis of the time course showed that a peak increase in plasma osmolality was more rapid than those of plasma ANP and VNP levels. This result supports the idea that ANP and VNP are secreted in response to increased osmolality. Preliminary data suggest that the metabolic clearance rate of VNP is three times greater than that of ANP (H. Kaiya and Y. Takei, unpublished data). It may be, therefore, that the more rapid peak in plasma VNP results from the faster clearance of VNP than ANP.
ACKNOWLEDGMENTS We thank Sumotomo Bioscience Co. Ltd. for the use of equipment and facilities for iodination of eel ANP and VNP. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (06454020) and the Fisheries Agency of Japan.
REFERENCES Brenner, B. M., Ballerman, B. J., Gunning, M. E., and Zeidel, M. L. (1990). Diverse biological actions of atrial natriuretic peptide. Physiol. Rev. 70, 665–699. Courneya, C. A., Wilson, N., and Ledsome, R. J. (1989). Plasma vasopressin and atrial natriuretic factor in response to blood volume changes in the anaesthetized rabbit. Can J. Physiol. Pharmacol. 67, 344–352. Evans, D. H. (1990). An emerging role for a cardiac peptide hormone in fish osmoregulation. Annu. Rev. Physiol. 52, 43–60. Gray, D. A., Schutz, H., and Gerstberger, R. (1991). Plasma atrial
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natriuretic factor responses to blood volume changes in the Pekin duck. Endocrinology 128, 1655–1660. Kaiya, H., and Takei, Y. (1996a). Atrial and ventricular natriuretic peptide concentrations in plasma of freshwater- and seawateradapted eels. Gen. Comp. Endocrinol. 102, 183–190. Kaiya, H., and Takei, Y. (1996b). Osmotic and volaemic regulation of atrial and ventricular natriuretic peptide secretion in conscious eels. J. Endocrinol. 149, 441–447. Kaiya, H., and Takei, Y. (1996c). Changes in plasma atrial and ventricular natriuretic peptide concentration after transfer of eels from freshwater and seawater or vice versa. Gen. Comp. Endocrinol. 104, 337–345. Kirsh, R., and Mayer-Gostan, N. (1973). Kinetics of water and chloride exchanges during adaptation of the European eel to sea water. J. Exp. Biol. 58, 105–121. Laine, M., Weckstrom, M., Vuolteenaho, O., and Arjamaa, O. (1994a). Effect of ryanodine on atrial natriuretic peptide secretion by contracting and quiescent rat atrium. Eur. J. Physiol. 426, 276–283. Laine, M., Arjamaa, O., Vuolteenaho, O., Ruskoaho, H., and Weckstrom, M. (1994b). Block of stretch-activated atrial natriuretic peptide secretion by gadolinium in isolated rat atrium. J. Physiol. 480, 553–561. Lang, R. E., Thoelken, H., Ganten, D., Luft, F. C., Ruskoaho, H., and Unger, T. H. (1985). Atrial natriuretic factor-a circulating hormone stimulated by volume loading. Nature 314, 264–266. Nishida, Y., Miyata, A., Morita, H., Uemura, N., Kangawa, K., Matsuo, H., and Hosomi, H. (1988). Effect of rapid sodium load on circulating atrial natriuretic polypeptide. Am. J. Physiol. 254, F540–F546. Oide, M., and Utida, S. (1968). Changes in intestinal absorption and renal extraction of water during adaptation to sea water in the Japanese eel. Mar. Biol. 1, 172–176. Olson, K. R. (1992). Blood and extracellular fluid volume regulation: Role of renin-angiotensin system, kallilrein-kinin system, and atrial natriuretic peptides. In ‘‘Fish Physiology, Vol. XII, part B, The Cardiovascular System’’ (W. S. Hoar, D. J. Randall, and A. P. Farrell, Eds.), pp. 136–232. Academic Press, San Diego. Ruskoaho, H. (1992). Atrial natriuretic peptide: Synthesis, release and metabolism. Pharmacol. Rev. 44, 479–601. Schiebinger, R. J., Craig, M. J., Yangfan, L., and Cragoe, E. J. Jr. (1995). Mechanism of hyperosmolality stimulation of ANP secretion: its dependency on calcium and sodium. Am. J. Physiol. 268, E476– E483. Takei, Y. (1988). Changes in blood volume after alteration of hydromineral balance in conscious eels, Anguilla japonica. Comp. Biochem. Physiol. 94A, 293–297. Takei, Y., and Balment, R. J. (1993a). Biochemistry and physiology of a family of eel natriuretic peptides. Fish Physiol. Biochem. 11, 1–6. Takei, Y., and Balment, R. J. (1993b). Natriuretic factors in nonmammalian vertebrates. In ‘‘New Insights in Vertebrate Kidney Function’’ (J. A. Brown, R. J. Balment, and J. C. Rankin, Eds.), pp. 351–385. Cambridge Univ. Press, Cambridge.
Interaction of Osmotic and Volemic Mechanisms in Secretion of Atrial and Ventricular Natriuretic Peptides in Eels Hiroyuki Kaiya and Yoshio Takei Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Minamidai, Nakano, Tokyo 164, Japan Accepted April 17, 1997
In eels, plasma osmolality rather than blood volume is a major regulator of atrial and ventricular natriuretic peptide (ANP and VNP) secretion. The present study examined the effects of changes in blood volume on ANP and VNP secretion stimulated by an increase in plasma osmolality in freshwater-adapted eels. Basal plasma ANP and VNP levels were decreased by 2 ml of blood withdrawal (28% of total blood volume), but not changed by blood volume expansion with 2 ml of 0.9% NaCl solution containing 2% dextran. The blood loss suppressed the increased plasma ANP level caused by an injection of 2.5 ml/kg of 1.7 M NaCl solution at 60 min (120.5 6 31.0 fmol/ml, n 5 5) compared with controls without blood volume manipulation (586.6 6 43.6 fmol/ ml, n 5 5), but the plasma ANP level transiently increased in bled fish immediately after osmotic stimulus, probably due to the release of ANP stored in the cardiac tissues after the blood loss. Changes in plasma VNP were not so evident as those of ANP. In contrast, blood volume expansion augmented the increase in plasma ANP and VNP levels within 60 min after osmotic stimulus compared with controls. The recovery of plasma VNP level was quicker than that of plasma ANP. Increases in plasma Na, Cl concentrations, and osmolality were not different among hypovolemic, normovolemic, and hypervolemic eels after osmotic stimulation. It is concluded that volume itself is a minor regulator for ANP and VNP secretion compared with osmotic stimulus, but it plays a modulatory role in osmotically induced ANP and VNP secretion in eels. r 1997 Academic Press
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Atrial natriuretic peptide (ANP) is a cardiac hormone known to be involved in volume and blood pressure homeostasis (Brenner et al., 1990). In mammals, the diuretic/natriuretic actions of ANP decrease blood volume, and an increased blood volume stimulates ANP secretion (Ruskoaho, 1992). In fish, however, the actions of ANP may differ from those in mammals; ANP seems to extrude sodium but not water at least in seawater (SW)-adapted eels (Evans, 1990; Takei and Balment, 1993a,b). Recently, both plasma osmolality and blood volume were found to regulate ANP secretion, but the osmotic mechanism is more dominant in eels (Kaiya and Takei, 1996b). After transfer of eels from freshwater (FW) to SW, plasma ANP concentration showed a biphasic response; a rapid, transient increase followed by a delayed increase after 6 hr (Kaiya and Takei, 1996c). Since the transient restoration of plasma ANP concentration by 6 hr after transfer occurs despite a progressive increase in plasma osmolality, it is possible that the fluctuation is caused by a decreased blood volume. In fact, eels face a severe dehydration after SW exposure by obligatory losses of water through the skin and gill epithelia (Oide and Utida, 1968; Kirsh and Mayer-Gostan, 1973). Furthermore, an increased plasma ANP concentration after SW transfer did not occur when the volume of blood sampled was increased, probably due to hypovolemia (H. Kaiya and Y. Takei, unpublished observation). Therefore, blood volume may modulate osmotically induced ANP secretion although volume changes alone 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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ANP Secretion in Eel
affect ANP secretion only slightly (Kaiya and Takei, 1996b). The present study examined the interaction of osmotic and volemic mechanisms in the regulation of ANP and VNP secretion in FW-adapted eels.
MATERIALS AND METHODS Fish Immature, cultured Japanese eels, Anguilla japonica, of either sex, weighing 196.7 6 3.6 (n 5 21) were obtained from a commercial source. Fish were maintained in FW aquaria unfed for 1–2 weeks before use. Water in the aquaria was continuously filtered, aerated at 18°.
Experimental Procedure After anesthesia by immersion in FW containing 0.3% (w/v) 3-aminobenzoic ethyl ester (MS222, Sigma, St. Louis, MO), a catheter (SP 10; OD, 0.61 mm, Natume, Japan) was inserted into the ventral aorta of FW eels as described previously (Kaiya and Takei, 1996a). After surgery, fish were transferred to an individual trough through which aerated water (18°) continuously circulated and allowed to recover for at least 20 hr before further study. To examine the effect of hypovolemia on osmotically induced ANP secretion, 2 ml of blood (approximately 28% of total blood volume; see Takei, 1988) was withdrawn through an aortic catheter 10 min before injection of a 1.7 M NaCl solution (2.5 ml/kg body wt) into five eels. Eels are still severely hypovolemic 15 min after 2 ml of blood withdrawal (Takei, 1988). Blood (200 µl) was sampled before controlled blood withdrawal and 0, 5, 15, 30, 60, and 180 min after injection of 1.7 M NaCl solution for measurement of plasma ANP and VNP concentrations and plasma solutes. Plasma obtained from before and 10 min after manipulation of blood volume was compared with plasma ANP, VNP, and Na concentrations to determine an acute effect of blood manipulation. After injection of hypertonic saline, the catheter (dead space ca. 10 µl) was flushed with 50 µl of 0.9% NaCl solution. The volume of collected blood was replaced with a 0.9% NaCl solution after each sampling. As a more physi-
ological stimulus, 0.85 M NaCl solution was injected after 1 ml blood withdrawal in another six eels. To examine the effect of hypervolemia, 2 ml of 0.9% NaCl solution containing 2% dextran (Sigma, Mr, 60,000– 90,000) was injected via an aortic catheter 10 min before injection of 1.7 M NaCl solution into another five eels. Dextran was added to adjust the colloidal osmotic pressure. The time course of blood sampling was the same as above. Five control fish received 1.7 M NaCl solution without manipulation of blood volume. Plasma dilution was taken into account for calculation of plasma hormone levels based on the method used by Nishida et al. (1988).
Determination of Plasma Parameters Blood was withdrawn into a chilled plastic syringe containing 10% 2K-EDTA solution (10 µl/ml blood). Forty microliters of blood was collected into a capillary tube to measure hematocrit. Plasma in the capillary tube was used for determination of osmolality with a vapor pressure osmometer (Model 5500, Wescor Inc., UT) and of Na concentration with an atomic absorption spectrophotometer (Model 180-50, Hitachi Ltd., Tokyo, Japan). Plasma Cl concentration was measured by Buchler–Cotlove chloridometer (Model 4-2500, Buchler Inc., NJ). The remaining blood was centrifuged at 11,000g for 5 min at 4°, and plasma was stored at 220° for later radioimmunoassay of ANP and VNP (Kaiya and Takei, 1996a).
Statistical Analysis The differences in plasma ANP, VNP, and Na concentrations before and after manipulation of blood volume were evaluated by Wilcoxon signed rank test. The differences between control and experimental groups were evaluated by two-way ANOVA (repeated measures), followed by Student’s t test or Cochran–Cox test to assess the difference at each time point. A P-value of less than 0.05 was considered significant. Values are expressed as means 6 SEM.
RESULTS Effect of Blood Volume Reduction In control fish without blood volume manipulation, plasma ANP concentration slightly but significantly
Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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increased immediately after injection of 1.7 M NaCl solution, and the peak increase was observed at 60 min (Fig. 1A). After 2 ml of blood was withdrawn, plasma ANP, VNP, and Na concentrations decreased significantly (Table 1). In the volume-depleted fish, plasma ANP concentration increased transiently 5 min after injection of 1.7 M NaCl solution. The increased level was higher than that of controls, but quickly returned to the initial level, so that the level became lower than that of controls at 60 min (Fig. 1A). The increase in plasma VNP concentration in control fish was faster and greater than that of ANP; the peak increase was observed 15 min after injection of hypertonic saline (Fig. 1D). In volume-depleted fish, the increase in plasma VNP concentration was less, but the difference was not significant compared with that of controls (Fig. 1D). The peak increase in plasma Na and Cl
Kaiya and Takei
TABLE 1 Changes in Plasma Atrial and Ventricular Natriuretic Peptide (ANP and VNP) Concentrations (fmol/ml) and Na Concentrations (mM) before and 10 min after Manipulation of Blood Volume in Freshwater Eels Group Hypovolemia (2 ml) ANP VNP Na Hypovolemia (1 ml) ANP VNP Na Hypervolemia (2 ml) ANP VNP Na Normovolemia ANP VNP Na
Before
After
69.1 6 10.5 (5) 131.1 6 14.8 141.1 6 1.5
44.7 6 1.8* 113.2 6 9.9* 136.9 6 2.8*
144.3 6 6.9 (6) 198.6 6 12.1 146.6 6 1.2
126.4 6 12.5 180.9 6 18.1 144.9 6 2.1
117.1 6 13.4 (5) 113.7 6 12.7 141.5 6 1.7
123.0 6 13.7 113.1 6 8.3 142.5 6 1.8
127.7 6 9.4 (5) 177.3 6 8.7 145.6 6 2.1
117.6 6 10.7 178.7 6 10.9 146.3 6 1.4
Note. Values are means 6 SEM. The number in parentheses indicates the number of animals used. * 0.01 , P , 50.05 (Wilcoxon signed rank test) compared with the value before manipulation of blood volume.
concentrations and osmolality occurred 5 min after injection of 1.7 M NaCl solution and gradually declined thereafter (Figs. 1B, 1C, and 1E). Changes in these plasma solutes did not differ between volumedepleted and control fish. In eels subjected to milder blood loss (1 ml of blood withdrawal) and osmotic stimulus (0.85 M NaCl injection), the difference in plasma hormone levels was not significant between volume-depleted and control fish (data not shown).
Effect of Blood Volume Expansion
FIG. 1. Effect of blood volume reduction (28% of total blood volume) on the secretion of atrial and ventricular natriuretic peptides (ANP and VNP) after intra-arterial injection of a hypertonic 1.7 M NaCl solution (2.5 ml/kg) in freshwater eels. (A) Plasma ANP, (B) plasma Na, (C) plasma osmolality, (D) plasma VNP, (E) plasma Cl, and (F) hematocrit are expressed. (W) and (X) Volume-depleted and control eels, respectively. Values are means 6 SEM. An arrow indicates the point of blood volume manipulation. *P , 0.05 compared with controls.
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Plasma ANP and VNP concentrations did not change after injection of 2 ml of dextran–saline (Table 1). In volume-loaded fish, the increase in plasma ANP and VNP concentrations after injection of hypertonic saline was greater than that of controls (Figs. 2A and 2D). However, the increased VNP level returned to the control level more quickly than the ANP level. Changes in plasma Na and Cl concentrations and osmolality were similar in volume-loaded and control fish (Figs. 2B, 2C, and 2E).
ANP Secretion in Eel
FIG. 2. Effect of blood volume expansion (28% of total blood volume) on the secretion of ANP and VNP after intra-arterial injection of a hypertonic 1.7 M NaCl solution (2.5 ml/kg) in freshwater eels. (A) Plasma ANP, (B) plasma Na, (C) plasma osmolality, (D) plasma VNP, (E) plasma Cl, and (F) hematocrit are expressed. (W) and (X) Volume-loaded and control eels, respectively. Values are means 6 SEM. An arrow indicates the point of blood volume manipulation. *P , 0.05 compared with controls.
DISCUSSION The present study showed that the increased secretion of ANP and VNP by osmotic stimulus was modified by changes in blood volume in eels. Thus, although blood volume expansion alone is a minor stimulus for ANP secretion (Kaiya and Takei, 1996b), it is apparent that blood volume plays a modulatory role in osmotically induced ANP secretion in eels. Furthermore, the present detailed analysis of the time course showed that the peak response of ANP and VNP secretion occurs slightly later than that of plasma osmolality and that the response of VNP is more rapid than that of ANP. There have been a number of observations showing that plasma concentrations of ANP are altered by volume status in terrestrial animals, and the primary
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regulator for ANP secretion is the change in atrial stretch (Ruskoaho, 1992). In eels, however, plasma ANP and VNP concentrations were not altered even by a 14% reduction in blood volume and by 28% blood volume expansion in this study. Similar blood volume manipulations in normohydrated terrestrial animals invariably alters plasma ANP concentration (Lang et al., 1985; Courneya et al., 1989; Gray et al., 1991). Since fish have a huge secondary vascular system (Olson, 1992), the system may buffer the changes in blood volume. Thus, the dominancy of blood volume as a regulator of ANP secretion seems to be smaller in eels than in terrestrial animals. This difference in ANP secretion may reflect the difference in the biological actions of ANP; ANP promotes sodium but not water loss in the eel (Takei and Balment, 1993b), but it promotes both sodium and water loss in terrestrial animals (Brenner et al., 1990). After blood withdrawal, the amount of ANP and VNP secretion immediately after osmotic stimulus was greater than that of controls without blood volume manipulation. Since ANP and VNP secretions were inhibited after blood loss as shown in this study, the immediate increase in plasma ANP and VNP may be caused by transient release of increased ANP and VNP stored in cardiac tissues caused by blood loss. When eels were transferred from FW to SW, plasma ANP and VNP concentrations increased just after transfer, then returned to initial values, to further increase after 6 hr (Kaiya and Takei, 1996c). This biphasic response of natriuretic peptide secretion may be accounted for by the transient reduction of blood volume after transfer to SW. Eels exposed to SW face a severe dehydration by losses of water through the skin and gill epithelia (Oide and Utida, 1968; Kirsh and Mayer-Gostan, 1973) and blood volume in fact decreases for a few hours after SW transfer as determine by dye dilution (Y. Takei, unpublished data). The reason for the temporary diminution of ANP and VNP secretion and relatively smaller increase in plasma ANP and VNP levels despite a sufficient osmotic stimulus for its secretion after transfer to SW may reflect blood volume reduction, as shown in this study. The present in vivo study of eels suggests a close interaction between osmotic and volume mechanisms in the regulation of ANP secretion. In vitro studies using mammalian cardiac tissues suggest that influx of
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extracellular Ca through Ca channels plays an important role in stretch-induced ANP secretion (Laine et al., 1994a,b), while a Na–Ca exchanger is involved in osmotically induced ANP secretion (Schiebinger et al., 1995). Thus, cellular mechanisms for ANP secretion between stretch-induced and osmotically induced ANP secretion seem to be functionally separated, although the Ca ion is key in both mechanisms. To further analyze the interaction of osmotic and volume mechanisms at the cellular level, it is necessary to do in vitro studies using eel cardiac tissues or cardiocytes. The present detailed analysis of the time course showed that a peak increase in plasma osmolality was more rapid than those of plasma ANP and VNP levels. This result supports the idea that ANP and VNP are secreted in response to increased osmolality. Preliminary data suggest that the metabolic clearance rate of VNP is three times greater than that of ANP (H. Kaiya and Y. Takei, unpublished data). It may be, therefore, that the more rapid peak in plasma VNP results from the faster clearance of VNP than ANP.
ACKNOWLEDGMENTS We thank Sumotomo Bioscience Co. Ltd. for the use of equipment and facilities for iodination of eel ANP and VNP. This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (06454020) and the Fisheries Agency of Japan.
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