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The antidiuretic effect of pneumadin requires a functional arginine vasopressin system
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ELSEVIER
Regulatory Peptides 57 (1995) 105-114
The antidiuretic effect of pneumadin requires a functional arginine vasopressin system J o h n D . W a t s o n a, D o n a l d B. J e n n i n g s c, I n d e r R . S a r d a b, S t e p h e n C. P a n g a, B a r b a r a L a w s o n °, D e n n i s A . W i g l e a, T . G e o f f r e y F l y n n b'* aDepartment of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada bDepartrnent of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada CDepartment of Physiology, Queen's University, Kingston, Ontario, K7L 3N6, Canada
Received 15 October 1994; revised version received 16 February 1995; accepted 20 February 1995
Abstract Pneumadin is an antidiuretic decapeptide, recently isolated from rat and human lung. Bolus intravenous injection of 5 nmol of pneumadin into water-loaded rats caused a rapid and significant antidiuresis and a reduction in Na ÷ and CIexcretion. Pneumadin administration did not alter mean arterial pressure, right atrial pressure, heart rate or haematocrit. Bolus intravenous injection of 20 nmol of pneumadin into non-water-loaded rats caused a significant increase in arginine vasopressin (AVP) within 10 min. Pneumadin administration also increased circulating atrial natriuretic peptide (ANP) but did not alter aldosterone or plasma renin activity levels. Injection of pneumadin into water-loaded Brattleboro rats, which genetically lack circulating AVP, did not change urine flow, confirming that the pneumadin induced antidiuresis is AVP dependent. Radioactive pneumadin was cleared from the circulation with a tl/2fl of 480.3 s. Radioactive pneumadin, isolated from plasma, eluted at an altered position on reverse phase HPLC, which indicated that the peptide was modified in vivo. This modification was also observed when synthetic pneumadin was incubated in rat plasma in vitro. Purification and sequencing of the modified synthetic peptide indicated that the modification is not a proteolytic cleavage. These results indicate that pneumadin injected into the rat caused an antidiuresis by altering circulating AVP levels.
Keywords: Antidiuresis; Atrial natriuretic peptide; AVP; Brattleboro rat
1. Introduction Diseases of the lung such as tuberculosis and small cell lung cancer (SCLC) are associated with abnor* Corresponding author. Fax: + 1 (613) 5452497. 0167-0115/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 0 2 4 - 0
mal fluid retention [ 1]. The retention of water and associated hyponatremia is known as the syndrome of inappropriate antidiuresis. This syndrome is often a result of abnormally high plasma levels of arginine vasopressin (AVP), caused by ectopic production from the tumour or by the hypothalamus [ 1]. To
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J.D. Watson et al, /Regulator),, Peptides 57 (1995) 105-114
date, it is unknown how a tumour of the lung could cause an increase in the hypothalamic release of AVP. Recently, a novel peptide called pneumadin was isolated from lung, which when injected into a water-loaded bioassay rat caused a significant antidiuresis [2]. The mechanism of action of this peptide has not been identified but it has been postulated that pneumadin acts by stimulating the release of AVP [2]. In order to further clarify this mechanism of action, synthetic pneumadin was injected intravenously into Sprague-Dawley rats, and several plasma hormones related to water and electrolyte homeostasis assayed. Further, Brattleboro rats that are genetically devoid of circulating AVP [3], were used to determine if the renal mechanism by which pneumadin caused an antidiuresis is dependent on the presence of AVP.
2. Materials and methods
Cardiovascular and renal responses to pneumadin were assayed following bolus injection of the peptide (YGEPKLDAGV-NH2, synthesized by the Core Facility for Protein/DNA Chemistry, Queen's University, Kingston, Ontario, Canada) in 100/~1 of 10 mM acetic acid into male Sprague-Dawley (Charles River Laboratories, St. Constant, Quebec, Canada) or homozygous Brattleboro (Harlan Sprague-Dawley, Indianapolis, IN, USA)rats. For control studies, rats were injected with an identical volume of I0 mM acetic acid. Rats were anaesthetized with sodium pentobarbitol (70 mg/kg), a tracheal tube was inserted, their penis tied off, and their carotid artery, femoral vein and urinary bladder cannulated. A catheter was also placed in the right cardiac atrium via the external jugular vein. Body temperature was monitored with a rectal temperature probe and maintained between 36.5-38.0°C. Animals were given a 1.1 ml infusion of saline over 20 min via the femoral vein and were water-loaded with a total of 8 ml/100 g body weight by stomach gavage. The gastric water-load was ad-
ministered in 2 equal volumes with a 30 min rest period in between. Upon observation of urine flow, data collection was begun for a 20 rain control period. The rat was then intravenously given 100 #1 of either the peptide or its vehicle and the catheter was flushed with 100 /21 of saline. During the experimental period, data were collected at 1 rain post-injection, then at 5 rain intervals for an additional 25 min. At the end of the experiment 50 #1 of blood was collected from the carotid artery for haematocrit determination. The animal was then sacrificed by decapitation. Blood was collected from the trunk into a pre-chilled tube containing EDTA (0.1 ml of a 15~o solution) and aprotinin (Trasylol, 200 KIU). Plasma was separated by centrifugation at 400 g for 20 rain at 4 ° C. Subsequently the atrial appendage and ventricular apex were rapidly excised and placed into liquid nitrogen; the kidneys were collected and weighed. Tissues and plasma were stored at -70 °C until processed. Urine electrolytes were determined using ion-specific electrodes (Baxter Paramax Autoanalyzer) by the Kingston General Hospital Clinical Chemistry Laboratory. Urine volume and electrolyte excretions were normalized by kidney weight. Data from each rat were analyzed as the change in each variable from time 0, the point immediately prior to injection of pneumadin or its vehicle. Three different protocols were conducted to determine physiological responses to pneumadin injection in rats with body weight varying between 125225 g. 2.1. Protocol 1 : pneumadin
To determine the changes in heart rate, mean arterial pressure, right atrial pressure, urine flow and electrolyte excretion, a bolus dose of 5 nmol ofpneumadin was given to 6 Sprague-Dawley rats. A control group of weight matched rats were given a bolus injection of vehicle to determine animal variability over the time-course of the experiment. The dose used was that which reduced urine flow in
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
water-loaded Sprague-Dawley rats by approximately
80%. 2.2. Protocol 2: time-course
To determine the effect of pneumadin on hormones involved in fluid homeostasis, a bolus dose of 20 nmol of pneumadin was given to 6 non-waterloaded Sprague-Dawley rats. The rats were anaesthetized as above and only their jugular vein cannulated. Pneumadin or vehicle was administered via the jugular cannula, 10 min after completion of the surgery. At 2, 5, 10, or 15 min following peptide injection, animals were sacrificed by decapitation and blood collected from the trunk as described above. Plasma was assayed for aldosterone, plasma renin activity (PRA), atrial natriuretic peptide (ANP) and arginine vasopressin (AVP) by radioimmunoassay [8,9]. A control group of 6 weight matched rats were injected with vehicle to determine animal variability during the experiment. The dose of pneumadin used was designed to maximize hormonal responses.
107
phenol-chloroform method of Chomczynski and Sacchi [4] from tissues collected at the end of protocol 1. The RNA was dot blotted onto Biotrace charged nylon (BioRad) and probed for ANP and BNP message using standard procedures [5]. Specific cDNA probes were labelled using [ 0~-32p]dATP (ICN Biomedicals, Montreal, Quebec, Canada) and a nick translation kit (BRL/Gibco, Mississauga, Ontario, Canada) to a specific activity of (4-5). 108 dpm/#g. Autoradiographs were densitometrically scanned at 595 nm with an ELISA plate reader (Titretek). Signal intensity was corrected for variations in RNA amount by re-probing the dot blots with a 7S RNA specific probe [6,7]. 2.5. Radioimmunoassay
Plasma levels of ANP and AVP were determined by radioimmunoassay as previously described [8,9]. Plasma aldosterone and PRA were assayed using commercially available kits (Diagnostic Products Corporation, Los Angeles, CA, USA). 2.6. Measurement of pneumadin clearance
2.3. Protocol 3: Brattleboro rats
To determine if the renal response to pneumadin injection was due only to increases in circulating AVP, a bolus dose of 10 nmol of pneumadin was given to 11 Brattleboro rats. Animals were determined to be homozygous Brattleboro rats by monitoring water consumption for 24 hr prior to the protocol. A control group of 6 weight matched Brattleboro rats were given a bolus dose of vehicle to determine animal variability over the course of the experiment. The dose used was that which gave a >90~o decrease in urine flow in water-loaded Sprague-Dawley rats. 2.4. RNA preparation
Total cardiac atrial and ventricular RNA was extracted using the acid-guanidinium thiocyanate-
To determine the plasma clearance rate of pneumadin, Sprague-Dawley rats were anaesthetized with Inactin (100 mg/kg) and their jugular vein and carotid artery cannulated. Pneumadin was labelled with Na125I (ICN Biomedicals, Montreal, Quebec) using the chloramine T method [8] and purified by reverse phase HPLC. Each rat was injected through a jugular vein cannula with 1.107 cpm of radiolabelled peptide. The cannula was flushed with 100 gl of saline. Blood samples were taken via the carotid artery at 5, 10, 15, 20, 30, 60, 120 s and then every 5 min post-injection for 30 min. Aliquots (50 gl) were counted to determine radioactivity present in the blood. Data were analyzed using the computer program CHOISE [ 10]. To determine if radioactivity present in the blood represented iodinated peptide, animals were injected as above and 10 ml of blood was removed rapidly at 30 s, 5 min and
108
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
30 min, into a pre-chilled tube containing aprotinin and E D T A . A single animal was used for each time point. Blood was centrifuged and the plasma stored at - 7 0 o C until processed. Pneumadin was extracted by acetone precipitation of the plasma, followed by Sep-Pak purification. The acetonitrile was evaporated with a stream of N 2 and the extract was processed on a Waters #-BondPac Cls H P L C column. To determine if synthetic pneumadin was cleaved by circulating proteases when injected into the rat, 10 #g iodinated peptide was spiked with 10,000 cpm of radioiodinated peptide and incubated in rat plasma on ice for 30 min. The pneumadin was extracted and H P L C purified as before. The radioactive peak was submitted to the Core Facility For Protein/DNA Chemistry for amino acid sequencing.
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3. Results
3.1. Physiological effects of pneumadin injection into Sprague-Dawley Rats Injection of 5 nmol of synthetic pneumadin into water-loaded Sprague-Dawley rats caused a significant decrease in urine production immediately upon peptide injection, with a continuing decrease in urine flow for the 25 min experimental period (Fig. 1). Urine output was decreased by greater than 80% in most animals. The urinary excretion of N a + and C1- were also decreased in pneumadin treated rats (Fig. 1). Changes in K ÷ excretion were not statistically significant. Heart rate, mean arterial pressure
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Fig. 1. Effect of pneumadin injection on urinary electrolytes. Urine samples collected in protocol 1 were analyzed for the concentration of(A) Na +, (B) K +, and (C) C1 . Data are the average for 5 animals and are presented as change from time 0 in mEq of ion excreted per 5 min per g kidney weight. Panel D in the change in urine flow from time 0 in #1 per 5 min per g kidney weight. An asterisk (*) indicates a significant difference between pneumadin-and vehicle-injected rats at P<0.05 as determined using an unpaired Student's t-test. and right atrial pressure were not affected by pneumadin administration (Fig. 2). Pneumadin had no affect upon haematocrit (Table 1) as compared with control animals. Radioimmunoassay of plasma samples collected at the end
J.D. Watson et al. / Regulatory" Peptides 57 (1995) 105-114
109
of protocol 1 (25 rain post-injection) showed a 1.5 fold increase in ANP, whereas aldosterone and AVP levels were unchanged from those of control animals (Table 1). Pneumadin treatment did not alter mRNA
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Fig. 2. Physiological effects of pneumadin administration. Data from protocol 1 are graphed as change from time 0, the preinjection value. (A) Mean arterial blood pressure, (B) heart rate, and (C) right atrial pressure. The data are the average of 5 animals. An asterisk (*) indicates a significant difference between control and pneumadin-treated animals at P < 0.05 as determined by an unpaired Student's t-test. Within study variance was determined using ANOVA.
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Table 1 Plasma ANP, AVP and aldosterone levels in water-loaded rats after pneumadin treatment Treated with
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Vehicle Pneumadin
684 + 43.6 725+50.1
ANP
27.4 + 2.5 51.5+5.7"
AVP
2.30 + 0.38 2.32+0.29
Hematocrit (%) 49.03 + 0.79 47.11+3.9
Results shown represent radioimmunoassay values obtained from rat plasma samples as described in protocol 1. Values are listed as mean + S.E.M. pg/ml; n = 5. The asterisk (*) denotes a significant difference vehicle- and pneumadin-treated animals at P < 0.05 as determined using an unpaired Student's t-test.
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Fig. 3. Plasma aldosterone, PRA, ANP, and AVP following pneumadin treatment of non-water-loaded rats. From top to bottom, data shown are: aldosterone, PRA, ANP, and AVP levels at 5, 10, and 15 rain following injection of vehicle (open bars) or 20 nmol of pneumadin (hatched bars). In the bottom panel (AVP) at 10 min post-injection, (A) represents 20 nmol pneumadin injection while (B) is 5 nmol of pneumadin injected. Each value represents the average of 6 animals + S.E.M. An asterisk (*) indicates a significant difference at P < 0 . 0 5 as determined by an unpaired Student's t-test.
J.D. Watson et al. / Regulator), Peptides 57 (1995) 105-114
110
levels for ANP or BNP in atrium or ventricle (data not shown).
3.2. Effects of pneumadin injection on ANP, A VP, aldosterone and plasma renin activity Injection of a 20 nmol bolus dose of pneumadin into anaesthetized, non-water-loaded rats (protocol 2) significantly increased blood levels of AVP as compared with vehicle injected animals at 10 min post-injection (Fig. 3, Group A) which returned to basal levels by 15 min. Injection of 5 nmol of pneu-
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3.3. Physiological effects of pneumadin injection into Brattleboro rats Injection of water-loaded Brattleboro rats with 10 nmol of synthetic pneumadin (protocol 3) did not cause any changes in heart rate, mean arterial pressure or urine flow as compared with vehicle injected animals (Fig. 4). While mean arterial pressure and heart rate of the Brattleboro rats were not significandy different from Sprague-Dawley (protocol 1) rats at the end of the control period, urine flow was significantly higher (Table 2).
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Fig. 4. Effect of p n e u m a d i n injection in Brattleboro rats. Brattleboro rats were treated as described in Fig. 1. Animals were injected with either 10 nmol of p n e u m a d i n (closed circles) or vehicle (open circles) at time 0. D a t a are the average values obtained from 6 animals (vehicle) or 11 animals (pneumadin)_+ S.E.M. D a t a shown are change in; (A) m e a n arterial pressure, (B) heart rate, and (C) urine flow.
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* Denotes the mean value for the Brattleboro rats is significantly higher than that for the Sprague-Dawley rats.
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
3.4. Pneurnadin clearance frorn the blood
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Fig. 5. Clearance of radioiodinated pneumadin from rat plasma. HPLC analysis of rat plasma extract collected at: (A) 30 s, (B) 5 min, and (C) 30 min followinginjection of 1.107 cpm of iodihated pneumadin. Results indicate rapid clearance of the iodinated peptide from the circulation. Panel D is the HPLC profile of iodinated synthetic pneumadin (peak II) or non-iodinated synthetic pneumadin (peak III). Pneumadin, when incubated in rat blood eluted at an altered position (panel A, B, C, peak I) as Compared with normal iodinated pneumadin (peak II). Iodinated pneumadin, when incubated in rat plasma on ice (panel E) also changed elution position from peak II to peak I, possiblywith an intermediate (peak IV). In all cases, peak V represents the injection artefact. Panel F is the clearance of radioactive pneumadin from the rat blood from one representative animal.
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Fig. 6. Modification of pneumadin in rat plasma. Incubation of synthetic pneumadin in rat plasma at 0 ° C for (A) 5 rain, (B) 10 min, (C) 15 min, and (D) 20 min or E) 20 min in water. In all panels peak I is the elution position for iodinated pneumadin, peak II is the position of modified pneumadin and peak IV is an injection artefact. Data represents epm of iodinated pneumadin in each fraction collected from the HPLC.
112
J.D. Watson et al. / Regulatoo' Peptides 57 (1995) 105-114
upon injection into the rat, indicating a rapid modification of the peptide. To further explore this possibility, pneumadin was incubated in rat plasma on ice for various time periods. This incubation revealed that iodinated pneumadin undergoes a shift in elution position, from 22~o to 145o acetonitrile, in a time-dependent fashion (Fig. 6). Furthermore, addition ofprotease inhibitors PMSF, aprotinin and leupeptin did not change the rate at which the positional change occurred, but caused an increase in peak heights (data not shown). The change in elution position did not occur when the peptide was incubated in water or phosphate buffer (Fig. 6E). The amino acid sequence of the modified peptide was determined to be 10 amino acids in length matching the sequence of the synthetic peptide.
4. Discussion
It is evident from the present study that pneumadin injected into the circulation of the rat acts as a potent antidiuretic confirming the findings of Batra et al. [2]. Our original hypothesis was that pneumadin reduce the release and/or production of atrial natriuretic peptide which may contribute to the antidiuretic effect. Results of the present study do not support this hypothesis, since circulating ANP increased in response to pneumadin injection (Table I). This likely represents a feedback effect, possibly due to the action of increased AVP, which has been shown to cause increases in the release of A S P [11]. Intravenous injection of relatively high doses of pneumadin into non-water-loaded rats caused a statistically significant increase in circulating AVP, 10 min following injection (Fig. 3). The increase in circulating AVP in response to pneumadin infusion is consistent with results obtained by Batra et al. [2] who injected pneumadin directly into the third ventricle of the brain [2]. The lack of antidiuretic response to pneumadin injection seen in the Brattleboro rat model is a clear indication that the
pneumadin-induced antidiuresis is a result of increases in circulating AVP. The physiological responses to pneumadin injection are consistent with the actions of AVP on the kidney, these being retention of water, Na + and C1[ 12]. Other blood borne peptides, such as ANP [ 13 ], and endothelin [ 14], are known to modulate levels of circulating AVP by acting on specific receptors found on the circumventricular organs. It is possible that pneumadin also binds to specific receptors on the circumventricular organs to cause modulation of AVP levels. It is interesting that a 5 nmol bolus dose of pneumadin caused a statistically significant 1.5-fold increase in circulating ANP in a water-loaded rat (Table 1), while a 20 nmol pneumadin injection did not cause a statistically significant change in a nonwater-loaded rat (Fig. 3). This apparent contradiction may be due to the water-load which could amplify changes caused by pneumadin, by increasing atrial stretch as compared with a non-water-loaded rat. This supposition is supported by the trend of ANP increase (not statistically significant) in nonwater-loaded rats following pneumadin administration (Fig. 3). Iodinated pneumadin was rapidly cleared from the blood, with a typical biphasic clearance profile for circulating hormones (Fig. 5F). Since pneumadin does not precipitate efficiently in trichloroacetic acid, the clearance profile was determined directly from blood. The HPLC of radioactive peptide extracted from rat plasma (Fig. 5) indicate that the determined profile for pneumadin is accurate and did not merely represent clearance of 125I-tyrosine or other degradation products. The ti/2~ of 480 s is consistent with the clearance time for peptide hormones, such as ANP [15-17]. It is uncertain what role the novel modification ofpneumadin in rat circulation plays in the activity or clearance of the peptide. The actual change occured very rapidly with the unmodified form of the peptide undetectable as soon as 30 s following injection (Fig. 5A). It is clear, from the sequencing of the peptide incubated in plasma, that
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
the modification was not a proteolytic event. This event requires further study to determine what the modification is, and what affect it has on the activity of the peptide. In summary, this study showed that when pneumadin was injected into the rat, it was cleared from the circulation with pharmacokinetic parameters consistent with those observed for peptide hormones. Furthermore, the physiological effects of the peptide, i.e., retention of water, Na + and CI-, are undoubtedly due to increases in circulating AVP in response to pneumadin injection. The complete lack of response seen in the Brattleboro rat indicated that the antidiuresis caused by pneumadin injection was due only to release of AVP, and that pneumadin did not act directly on the kidney. Although the primary physiological function ofpneumadin has not yet been determined, it is possible that this peptide may play a role in pathophysiological states such as small cell lung cancer as some of these patients exhibit abnormal retention of fluid. In addition, more research is required to determine whether pneumadin is normally secreted into the circulation, and to determine the mechanism by which pneumadin increased circulating AVP.
Acknowledgements This work was supported by a research grant from the Medical Research Council of Canada (T.G.F. and S.C.P.).
Dedication This manuscript is dedicated to Dr. M. Ashwini Kumar of Vallabhbhai Patel Chest Institute, the University of Delhi, Delhi, India on the occasion of his retirement.
113
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[14] Wall, K.M. and Ferguson, A.V., Endothelin acts at the subfornical organ to influence the activity of putative vasopressin and oxytocin-secreting neurons, Brain Res., 586 (1992) 111-116. [15] Ohashi, M., Fujio, N., Nawata, H., Kato, K., Matsuo, H. and lbayashi, H,, Pharmicokinetics of synthetic alphahuman atrial natriuretic polypeptide in normal men: Effect of aging, Regul. Pept., 19 (1987) 265-272. [16] Gillies, A.H., Crozier, I.G., Nicholls, M.G., Espiner, E.A.
and Yandle, T.G., Effect of posture on clearance of atrial natriuretic peptide from plasma, J. Clin. Endocrinol. Metab., 65 (1987) 1095-1097. [ 17] Barclay, P.L., Bennett, J.A., Greengrass, P.M., Griffin, A., Samuels, G.M.R. and Shepperson, N.B., The pharmacokinetics of x25-Iatrial natriuretic factor in anaesthetized rats. Effects of neutral endopeptidase inhibition with candoxatrilate and of ANF-C receptor blockade, Biochem. Pharmacol., 44 (1992) 1013-1022.
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ELSEVIER
Regulatory Peptides 57 (1995) 105-114
The antidiuretic effect of pneumadin requires a functional arginine vasopressin system J o h n D . W a t s o n a, D o n a l d B. J e n n i n g s c, I n d e r R . S a r d a b, S t e p h e n C. P a n g a, B a r b a r a L a w s o n °, D e n n i s A . W i g l e a, T . G e o f f r e y F l y n n b'* aDepartment of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, K7L 3N6, Canada bDepartrnent of Biochemistry, Queen's University, Kingston, Ontario, K7L 3N6, Canada CDepartment of Physiology, Queen's University, Kingston, Ontario, K7L 3N6, Canada
Received 15 October 1994; revised version received 16 February 1995; accepted 20 February 1995
Abstract Pneumadin is an antidiuretic decapeptide, recently isolated from rat and human lung. Bolus intravenous injection of 5 nmol of pneumadin into water-loaded rats caused a rapid and significant antidiuresis and a reduction in Na ÷ and CIexcretion. Pneumadin administration did not alter mean arterial pressure, right atrial pressure, heart rate or haematocrit. Bolus intravenous injection of 20 nmol of pneumadin into non-water-loaded rats caused a significant increase in arginine vasopressin (AVP) within 10 min. Pneumadin administration also increased circulating atrial natriuretic peptide (ANP) but did not alter aldosterone or plasma renin activity levels. Injection of pneumadin into water-loaded Brattleboro rats, which genetically lack circulating AVP, did not change urine flow, confirming that the pneumadin induced antidiuresis is AVP dependent. Radioactive pneumadin was cleared from the circulation with a tl/2fl of 480.3 s. Radioactive pneumadin, isolated from plasma, eluted at an altered position on reverse phase HPLC, which indicated that the peptide was modified in vivo. This modification was also observed when synthetic pneumadin was incubated in rat plasma in vitro. Purification and sequencing of the modified synthetic peptide indicated that the modification is not a proteolytic cleavage. These results indicate that pneumadin injected into the rat caused an antidiuresis by altering circulating AVP levels.
Keywords: Antidiuresis; Atrial natriuretic peptide; AVP; Brattleboro rat
1. Introduction Diseases of the lung such as tuberculosis and small cell lung cancer (SCLC) are associated with abnor* Corresponding author. Fax: + 1 (613) 5452497. 0167-0115/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 5 ) 0 0 0 2 4 - 0
mal fluid retention [ 1]. The retention of water and associated hyponatremia is known as the syndrome of inappropriate antidiuresis. This syndrome is often a result of abnormally high plasma levels of arginine vasopressin (AVP), caused by ectopic production from the tumour or by the hypothalamus [ 1]. To
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J.D. Watson et al, /Regulator),, Peptides 57 (1995) 105-114
date, it is unknown how a tumour of the lung could cause an increase in the hypothalamic release of AVP. Recently, a novel peptide called pneumadin was isolated from lung, which when injected into a water-loaded bioassay rat caused a significant antidiuresis [2]. The mechanism of action of this peptide has not been identified but it has been postulated that pneumadin acts by stimulating the release of AVP [2]. In order to further clarify this mechanism of action, synthetic pneumadin was injected intravenously into Sprague-Dawley rats, and several plasma hormones related to water and electrolyte homeostasis assayed. Further, Brattleboro rats that are genetically devoid of circulating AVP [3], were used to determine if the renal mechanism by which pneumadin caused an antidiuresis is dependent on the presence of AVP.
2. Materials and methods
Cardiovascular and renal responses to pneumadin were assayed following bolus injection of the peptide (YGEPKLDAGV-NH2, synthesized by the Core Facility for Protein/DNA Chemistry, Queen's University, Kingston, Ontario, Canada) in 100/~1 of 10 mM acetic acid into male Sprague-Dawley (Charles River Laboratories, St. Constant, Quebec, Canada) or homozygous Brattleboro (Harlan Sprague-Dawley, Indianapolis, IN, USA)rats. For control studies, rats were injected with an identical volume of I0 mM acetic acid. Rats were anaesthetized with sodium pentobarbitol (70 mg/kg), a tracheal tube was inserted, their penis tied off, and their carotid artery, femoral vein and urinary bladder cannulated. A catheter was also placed in the right cardiac atrium via the external jugular vein. Body temperature was monitored with a rectal temperature probe and maintained between 36.5-38.0°C. Animals were given a 1.1 ml infusion of saline over 20 min via the femoral vein and were water-loaded with a total of 8 ml/100 g body weight by stomach gavage. The gastric water-load was ad-
ministered in 2 equal volumes with a 30 min rest period in between. Upon observation of urine flow, data collection was begun for a 20 rain control period. The rat was then intravenously given 100 #1 of either the peptide or its vehicle and the catheter was flushed with 100 /21 of saline. During the experimental period, data were collected at 1 rain post-injection, then at 5 rain intervals for an additional 25 min. At the end of the experiment 50 #1 of blood was collected from the carotid artery for haematocrit determination. The animal was then sacrificed by decapitation. Blood was collected from the trunk into a pre-chilled tube containing EDTA (0.1 ml of a 15~o solution) and aprotinin (Trasylol, 200 KIU). Plasma was separated by centrifugation at 400 g for 20 rain at 4 ° C. Subsequently the atrial appendage and ventricular apex were rapidly excised and placed into liquid nitrogen; the kidneys were collected and weighed. Tissues and plasma were stored at -70 °C until processed. Urine electrolytes were determined using ion-specific electrodes (Baxter Paramax Autoanalyzer) by the Kingston General Hospital Clinical Chemistry Laboratory. Urine volume and electrolyte excretions were normalized by kidney weight. Data from each rat were analyzed as the change in each variable from time 0, the point immediately prior to injection of pneumadin or its vehicle. Three different protocols were conducted to determine physiological responses to pneumadin injection in rats with body weight varying between 125225 g. 2.1. Protocol 1 : pneumadin
To determine the changes in heart rate, mean arterial pressure, right atrial pressure, urine flow and electrolyte excretion, a bolus dose of 5 nmol ofpneumadin was given to 6 Sprague-Dawley rats. A control group of weight matched rats were given a bolus injection of vehicle to determine animal variability over the time-course of the experiment. The dose used was that which reduced urine flow in
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
water-loaded Sprague-Dawley rats by approximately
80%. 2.2. Protocol 2: time-course
To determine the effect of pneumadin on hormones involved in fluid homeostasis, a bolus dose of 20 nmol of pneumadin was given to 6 non-waterloaded Sprague-Dawley rats. The rats were anaesthetized as above and only their jugular vein cannulated. Pneumadin or vehicle was administered via the jugular cannula, 10 min after completion of the surgery. At 2, 5, 10, or 15 min following peptide injection, animals were sacrificed by decapitation and blood collected from the trunk as described above. Plasma was assayed for aldosterone, plasma renin activity (PRA), atrial natriuretic peptide (ANP) and arginine vasopressin (AVP) by radioimmunoassay [8,9]. A control group of 6 weight matched rats were injected with vehicle to determine animal variability during the experiment. The dose of pneumadin used was designed to maximize hormonal responses.
107
phenol-chloroform method of Chomczynski and Sacchi [4] from tissues collected at the end of protocol 1. The RNA was dot blotted onto Biotrace charged nylon (BioRad) and probed for ANP and BNP message using standard procedures [5]. Specific cDNA probes were labelled using [ 0~-32p]dATP (ICN Biomedicals, Montreal, Quebec, Canada) and a nick translation kit (BRL/Gibco, Mississauga, Ontario, Canada) to a specific activity of (4-5). 108 dpm/#g. Autoradiographs were densitometrically scanned at 595 nm with an ELISA plate reader (Titretek). Signal intensity was corrected for variations in RNA amount by re-probing the dot blots with a 7S RNA specific probe [6,7]. 2.5. Radioimmunoassay
Plasma levels of ANP and AVP were determined by radioimmunoassay as previously described [8,9]. Plasma aldosterone and PRA were assayed using commercially available kits (Diagnostic Products Corporation, Los Angeles, CA, USA). 2.6. Measurement of pneumadin clearance
2.3. Protocol 3: Brattleboro rats
To determine if the renal response to pneumadin injection was due only to increases in circulating AVP, a bolus dose of 10 nmol of pneumadin was given to 11 Brattleboro rats. Animals were determined to be homozygous Brattleboro rats by monitoring water consumption for 24 hr prior to the protocol. A control group of 6 weight matched Brattleboro rats were given a bolus dose of vehicle to determine animal variability over the course of the experiment. The dose used was that which gave a >90~o decrease in urine flow in water-loaded Sprague-Dawley rats. 2.4. RNA preparation
Total cardiac atrial and ventricular RNA was extracted using the acid-guanidinium thiocyanate-
To determine the plasma clearance rate of pneumadin, Sprague-Dawley rats were anaesthetized with Inactin (100 mg/kg) and their jugular vein and carotid artery cannulated. Pneumadin was labelled with Na125I (ICN Biomedicals, Montreal, Quebec) using the chloramine T method [8] and purified by reverse phase HPLC. Each rat was injected through a jugular vein cannula with 1.107 cpm of radiolabelled peptide. The cannula was flushed with 100 gl of saline. Blood samples were taken via the carotid artery at 5, 10, 15, 20, 30, 60, 120 s and then every 5 min post-injection for 30 min. Aliquots (50 gl) were counted to determine radioactivity present in the blood. Data were analyzed using the computer program CHOISE [ 10]. To determine if radioactivity present in the blood represented iodinated peptide, animals were injected as above and 10 ml of blood was removed rapidly at 30 s, 5 min and
108
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
30 min, into a pre-chilled tube containing aprotinin and E D T A . A single animal was used for each time point. Blood was centrifuged and the plasma stored at - 7 0 o C until processed. Pneumadin was extracted by acetone precipitation of the plasma, followed by Sep-Pak purification. The acetonitrile was evaporated with a stream of N 2 and the extract was processed on a Waters #-BondPac Cls H P L C column. To determine if synthetic pneumadin was cleaved by circulating proteases when injected into the rat, 10 #g iodinated peptide was spiked with 10,000 cpm of radioiodinated peptide and incubated in rat plasma on ice for 30 min. The pneumadin was extracted and H P L C purified as before. The radioactive peak was submitted to the Core Facility For Protein/DNA Chemistry for amino acid sequencing.
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3. Results
3.1. Physiological effects of pneumadin injection into Sprague-Dawley Rats Injection of 5 nmol of synthetic pneumadin into water-loaded Sprague-Dawley rats caused a significant decrease in urine production immediately upon peptide injection, with a continuing decrease in urine flow for the 25 min experimental period (Fig. 1). Urine output was decreased by greater than 80% in most animals. The urinary excretion of N a + and C1- were also decreased in pneumadin treated rats (Fig. 1). Changes in K ÷ excretion were not statistically significant. Heart rate, mean arterial pressure
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Fig. 1. Effect of pneumadin injection on urinary electrolytes. Urine samples collected in protocol 1 were analyzed for the concentration of(A) Na +, (B) K +, and (C) C1 . Data are the average for 5 animals and are presented as change from time 0 in mEq of ion excreted per 5 min per g kidney weight. Panel D in the change in urine flow from time 0 in #1 per 5 min per g kidney weight. An asterisk (*) indicates a significant difference between pneumadin-and vehicle-injected rats at P<0.05 as determined using an unpaired Student's t-test. and right atrial pressure were not affected by pneumadin administration (Fig. 2). Pneumadin had no affect upon haematocrit (Table 1) as compared with control animals. Radioimmunoassay of plasma samples collected at the end
J.D. Watson et al. / Regulatory" Peptides 57 (1995) 105-114
109
of protocol 1 (25 rain post-injection) showed a 1.5 fold increase in ANP, whereas aldosterone and AVP levels were unchanged from those of control animals (Table 1). Pneumadin treatment did not alter mRNA
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Fig. 2. Physiological effects of pneumadin administration. Data from protocol 1 are graphed as change from time 0, the preinjection value. (A) Mean arterial blood pressure, (B) heart rate, and (C) right atrial pressure. The data are the average of 5 animals. An asterisk (*) indicates a significant difference between control and pneumadin-treated animals at P < 0.05 as determined by an unpaired Student's t-test. Within study variance was determined using ANOVA.
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Table 1 Plasma ANP, AVP and aldosterone levels in water-loaded rats after pneumadin treatment Treated with
Aldo
Vehicle Pneumadin
684 + 43.6 725+50.1
ANP
27.4 + 2.5 51.5+5.7"
AVP
2.30 + 0.38 2.32+0.29
Hematocrit (%) 49.03 + 0.79 47.11+3.9
Results shown represent radioimmunoassay values obtained from rat plasma samples as described in protocol 1. Values are listed as mean + S.E.M. pg/ml; n = 5. The asterisk (*) denotes a significant difference vehicle- and pneumadin-treated animals at P < 0.05 as determined using an unpaired Student's t-test.
5 5
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15
MINUTES POSTINJECTION
Fig. 3. Plasma aldosterone, PRA, ANP, and AVP following pneumadin treatment of non-water-loaded rats. From top to bottom, data shown are: aldosterone, PRA, ANP, and AVP levels at 5, 10, and 15 rain following injection of vehicle (open bars) or 20 nmol of pneumadin (hatched bars). In the bottom panel (AVP) at 10 min post-injection, (A) represents 20 nmol pneumadin injection while (B) is 5 nmol of pneumadin injected. Each value represents the average of 6 animals + S.E.M. An asterisk (*) indicates a significant difference at P < 0 . 0 5 as determined by an unpaired Student's t-test.
J.D. Watson et al. / Regulator), Peptides 57 (1995) 105-114
110
levels for ANP or BNP in atrium or ventricle (data not shown).
3.2. Effects of pneumadin injection on ANP, A VP, aldosterone and plasma renin activity Injection of a 20 nmol bolus dose of pneumadin into anaesthetized, non-water-loaded rats (protocol 2) significantly increased blood levels of AVP as compared with vehicle injected animals at 10 min post-injection (Fig. 3, Group A) which returned to basal levels by 15 min. Injection of 5 nmol of pneu-
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madin did not significantly alter AVP levels at 10 min (Fig. 3, Group B). Aldosterone, ANP and PRA were unaffected by injection of 20 nmol of pneumadin (Fig. 3).
3.3. Physiological effects of pneumadin injection into Brattleboro rats Injection of water-loaded Brattleboro rats with 10 nmol of synthetic pneumadin (protocol 3) did not cause any changes in heart rate, mean arterial pressure or urine flow as compared with vehicle injected animals (Fig. 4). While mean arterial pressure and heart rate of the Brattleboro rats were not significandy different from Sprague-Dawley (protocol 1) rats at the end of the control period, urine flow was significantly higher (Table 2).
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Fig. 4. Effect of p n e u m a d i n injection in Brattleboro rats. Brattleboro rats were treated as described in Fig. 1. Animals were injected with either 10 nmol of p n e u m a d i n (closed circles) or vehicle (open circles) at time 0. D a t a are the average values obtained from 6 animals (vehicle) or 11 animals (pneumadin)_+ S.E.M. D a t a shown are change in; (A) m e a n arterial pressure, (B) heart rate, and (C) urine flow.
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* Denotes the mean value for the Brattleboro rats is significantly higher than that for the Sprague-Dawley rats.
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
3.4. Pneurnadin clearance frorn the blood
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Fig. 5. Clearance of radioiodinated pneumadin from rat plasma. HPLC analysis of rat plasma extract collected at: (A) 30 s, (B) 5 min, and (C) 30 min followinginjection of 1.107 cpm of iodihated pneumadin. Results indicate rapid clearance of the iodinated peptide from the circulation. Panel D is the HPLC profile of iodinated synthetic pneumadin (peak II) or non-iodinated synthetic pneumadin (peak III). Pneumadin, when incubated in rat blood eluted at an altered position (panel A, B, C, peak I) as Compared with normal iodinated pneumadin (peak II). Iodinated pneumadin, when incubated in rat plasma on ice (panel E) also changed elution position from peak II to peak I, possiblywith an intermediate (peak IV). In all cases, peak V represents the injection artefact. Panel F is the clearance of radioactive pneumadin from the rat blood from one representative animal.
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Fig. 6. Modification of pneumadin in rat plasma. Incubation of synthetic pneumadin in rat plasma at 0 ° C for (A) 5 rain, (B) 10 min, (C) 15 min, and (D) 20 min or E) 20 min in water. In all panels peak I is the elution position for iodinated pneumadin, peak II is the position of modified pneumadin and peak IV is an injection artefact. Data represents epm of iodinated pneumadin in each fraction collected from the HPLC.
112
J.D. Watson et al. / Regulatoo' Peptides 57 (1995) 105-114
upon injection into the rat, indicating a rapid modification of the peptide. To further explore this possibility, pneumadin was incubated in rat plasma on ice for various time periods. This incubation revealed that iodinated pneumadin undergoes a shift in elution position, from 22~o to 145o acetonitrile, in a time-dependent fashion (Fig. 6). Furthermore, addition ofprotease inhibitors PMSF, aprotinin and leupeptin did not change the rate at which the positional change occurred, but caused an increase in peak heights (data not shown). The change in elution position did not occur when the peptide was incubated in water or phosphate buffer (Fig. 6E). The amino acid sequence of the modified peptide was determined to be 10 amino acids in length matching the sequence of the synthetic peptide.
4. Discussion
It is evident from the present study that pneumadin injected into the circulation of the rat acts as a potent antidiuretic confirming the findings of Batra et al. [2]. Our original hypothesis was that pneumadin reduce the release and/or production of atrial natriuretic peptide which may contribute to the antidiuretic effect. Results of the present study do not support this hypothesis, since circulating ANP increased in response to pneumadin injection (Table I). This likely represents a feedback effect, possibly due to the action of increased AVP, which has been shown to cause increases in the release of A S P [11]. Intravenous injection of relatively high doses of pneumadin into non-water-loaded rats caused a statistically significant increase in circulating AVP, 10 min following injection (Fig. 3). The increase in circulating AVP in response to pneumadin infusion is consistent with results obtained by Batra et al. [2] who injected pneumadin directly into the third ventricle of the brain [2]. The lack of antidiuretic response to pneumadin injection seen in the Brattleboro rat model is a clear indication that the
pneumadin-induced antidiuresis is a result of increases in circulating AVP. The physiological responses to pneumadin injection are consistent with the actions of AVP on the kidney, these being retention of water, Na + and C1[ 12]. Other blood borne peptides, such as ANP [ 13 ], and endothelin [ 14], are known to modulate levels of circulating AVP by acting on specific receptors found on the circumventricular organs. It is possible that pneumadin also binds to specific receptors on the circumventricular organs to cause modulation of AVP levels. It is interesting that a 5 nmol bolus dose of pneumadin caused a statistically significant 1.5-fold increase in circulating ANP in a water-loaded rat (Table 1), while a 20 nmol pneumadin injection did not cause a statistically significant change in a nonwater-loaded rat (Fig. 3). This apparent contradiction may be due to the water-load which could amplify changes caused by pneumadin, by increasing atrial stretch as compared with a non-water-loaded rat. This supposition is supported by the trend of ANP increase (not statistically significant) in nonwater-loaded rats following pneumadin administration (Fig. 3). Iodinated pneumadin was rapidly cleared from the blood, with a typical biphasic clearance profile for circulating hormones (Fig. 5F). Since pneumadin does not precipitate efficiently in trichloroacetic acid, the clearance profile was determined directly from blood. The HPLC of radioactive peptide extracted from rat plasma (Fig. 5) indicate that the determined profile for pneumadin is accurate and did not merely represent clearance of 125I-tyrosine or other degradation products. The ti/2~ of 480 s is consistent with the clearance time for peptide hormones, such as ANP [15-17]. It is uncertain what role the novel modification ofpneumadin in rat circulation plays in the activity or clearance of the peptide. The actual change occured very rapidly with the unmodified form of the peptide undetectable as soon as 30 s following injection (Fig. 5A). It is clear, from the sequencing of the peptide incubated in plasma, that
J.D. Watson et al. / Regulatory Peptides 57 (1995) 105-114
the modification was not a proteolytic event. This event requires further study to determine what the modification is, and what affect it has on the activity of the peptide. In summary, this study showed that when pneumadin was injected into the rat, it was cleared from the circulation with pharmacokinetic parameters consistent with those observed for peptide hormones. Furthermore, the physiological effects of the peptide, i.e., retention of water, Na + and CI-, are undoubtedly due to increases in circulating AVP in response to pneumadin injection. The complete lack of response seen in the Brattleboro rat indicated that the antidiuresis caused by pneumadin injection was due only to release of AVP, and that pneumadin did not act directly on the kidney. Although the primary physiological function ofpneumadin has not yet been determined, it is possible that this peptide may play a role in pathophysiological states such as small cell lung cancer as some of these patients exhibit abnormal retention of fluid. In addition, more research is required to determine whether pneumadin is normally secreted into the circulation, and to determine the mechanism by which pneumadin increased circulating AVP.
Acknowledgements This work was supported by a research grant from the Medical Research Council of Canada (T.G.F. and S.C.P.).
Dedication This manuscript is dedicated to Dr. M. Ashwini Kumar of Vallabhbhai Patel Chest Institute, the University of Delhi, Delhi, India on the occasion of his retirement.
113
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[14] Wall, K.M. and Ferguson, A.V., Endothelin acts at the subfornical organ to influence the activity of putative vasopressin and oxytocin-secreting neurons, Brain Res., 586 (1992) 111-116. [15] Ohashi, M., Fujio, N., Nawata, H., Kato, K., Matsuo, H. and lbayashi, H,, Pharmicokinetics of synthetic alphahuman atrial natriuretic polypeptide in normal men: Effect of aging, Regul. Pept., 19 (1987) 265-272. [16] Gillies, A.H., Crozier, I.G., Nicholls, M.G., Espiner, E.A.
and Yandle, T.G., Effect of posture on clearance of atrial natriuretic peptide from plasma, J. Clin. Endocrinol. Metab., 65 (1987) 1095-1097. [ 17] Barclay, P.L., Bennett, J.A., Greengrass, P.M., Griffin, A., Samuels, G.M.R. and Shepperson, N.B., The pharmacokinetics of x25-Iatrial natriuretic factor in anaesthetized rats. Effects of neutral endopeptidase inhibition with candoxatrilate and of ANF-C receptor blockade, Biochem. Pharmacol., 44 (1992) 1013-1022.