519
Journal of Physiology (1991), 443, pp. 519-531 With 4 figures Printed in Great Britain
THE EFFECT OF DISCRETE STIMULATION OF CAROTID BODY CHEMORECEPTORS ON ATRIAL NATRIURETIC PEPTIDE IN ANAESTHETIZED DOGS
BY M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM* From the Department of Physiology, The Worsley Medical and Dental Building, University of Leeds, Leeds LS2 9NQ
(Received 19 October 1990) SUMMARY
1. In seven chloralose-anaesthetized and artificially ventilated beagles, the carotid sinus regions were vascularly isolated and perfused with either arterial or mixed (arterial and venous) blood (PO2464+15 mmHg, mean + s.E.M.) to stimulate the chemoreceptors at constant flow and pressure. Cervical vagosympathetic trunks were ligated in all dogs, and gallamine triethiodide (2-0 mg kg-' h-', I.v.) was given in five dogs. Right atrial pressure was measured in all dogs, and left atrial pressure in four dogs. Mean aortic pressure was held constant (91-0 + 3-0 mmHg) by means of a reservoir connected to the animal via the common carotid and femoral arteries. Plasma atrial natriuretic peptide (ANP) was measured by radioimmunoassay and urinary sodium by flame photometry. 2. In seven dogs with mean carotid sinus pressure maintained at 96-0 + 4-3 mmHg, stimulation of the carotid chemoreceptors for 25 min produced significant increases in left atrial pressure of 41-2 + 3-3 % (n = 4; P < 0 005) from 5-4 + 0-6 cmH2O and of 30 9 + 4-5 % (n = 7; P < 0-002) in ANP from 31-6 + 2-1 pg ml-'. However, chemoreceptor stimulation produced significant decreases in urine flow rate of 26-1 + 1-9 % (n = 9; P < 0-001) from 0-29 + 0-03 ml min-1 (100 g kidney weight)-' and sodium excretion of 290+2 3 % (P < 0 001) from 8-5+ 17 /tmol min-' (100 g kidney weight)-' but right atrial pressure and heart rate did not change significantly. In three of the dogs, ,-adrenoceptor blockade by atenolol (2 mg kg-', i.v.) greatly reduced the effects of chemoreceptor stimulation on plasma levels of ANP. 3. The results show, for the first time, that discrete stimulation of the carotid chemoreceptors caused an increase in plasma ANP levels, probably due to the reflex increase in atrial pressure that results from an inhibition of the cardiac sympathetic nerves, and an increase in venous return from a reduction of peripheral vascular capacitance. INTRODUCTION
Acute generalized hypoxia and hypercapnia may cause an increase in circulating atrial natriuretic peptide (ANP) in man and in animals (Baertschi, Hausmaninger, Walsh, Mentzer, Wyatt & Pence, 1986; du Souich, Saunier, Harteman, Sautegeau, * To whom reprint requests should be sent. MS 8875
520
M. AL-OBAIDI,
E.
M. WHITAKER AND
F.
KARIM
Ong, Larose & Babini, 1987; Baertschi, Adams & Sullivan, 1988; Clozel, Saunier, Harteman, Allam & Fischli, 1989) and natriuresis and diuresis in man and dogs (Barker, Singer, Elkinton & Clark, 1957; Walker, 1982). However, it is not yet known whether carotid chemoreceptors are involved in the release of ANP and thereby influence sodium excretion and urine volume (Koller, Schopen, Keller, Lang & Vallotton, 1989). Besides stimulating chemoreceptors, generalized hypoxia and hypercapnia are likely to result in changes in blood chemistry that could have widespread effects on the cardiovascular system (Cross, Rieben, Barron & Salisbury, 1963; Folkow & Neil, 1971), and the central nervous system (Downing, Mitchell & Wallace, 1963). In addition, hypoxia and hypercapnia are associated with changes in a number of kidney hormones that could also influence ANP release (Anderson, Rose, Berns, Erikson & Arnold, 1980; Raff, Shinsako, Keil & Dallman, 1983; Wang, Sundet & Goetz, 1984), and thus complicate any interpretations of the mechanism of release of ANP. It is well established that atrial stretch is the main stimulus for ANP release (see Genest & Cantin, 1988), and carotid chemoreceptor stimulation produces a reflex negative inotropic effect, by inhibiting cardiac sympathetic nerves (Hainsworth, Karim & Sofola, 1979; Karim, Hainsworth, Sofola & Wood, 1980), and a reflex reduction in peripheral vascular capacitance by stimulating sympathetic nerves (Hainsworth, Karim, McGregor & Wood, 1983), which would lead to an increase in atrial pressure. Carotid chemoreceptor stimulation is, therefore, expected to cause a release of ANP, and contribute to the natriuretic response in the denervated kidney or at a high carotid sinus pressure in the intact kidney as demonstrated previously (Karim, Poucher & Summerill, 1987; Al-Obaidi, Karim, Majid & Goonewardene, 1988). So far as we know there have been no experiments to determine the effect of discrete stimulation of the carotid chemoreceptors on plasma ANP levels and its effect on sodium and water excretion. The present experiments were, therefore, carried out to determine the effects of discrete stimulation of the carotid chemoreceptors on plasma ANP concentration and sodium excretion in anaesthetized and artificially ventilated dogs. The aortic pressure (renal perfusion pressure) was adequately controlled, and the cervical vagosympathetic trunks were tied to eliminate the influence of receptors located in the aortic arch and cardiopulmonary regions. The carotid chemoreceptors were stimulated by perfusing them with moderately hypoxic (mixed arterial and venous) blood. A preliminary report of these experiments has been published (Al-Obaidi, Whitaker & Karim, 1990). METHODS
Beagles (15-0-31-8 kg, of both sexes) were anaesthetized with thiopentone sodium (Intraval Sodium, May & Baker; 500 mg, i.v.) followed bya-chloralose (BDH Chemicals; 01 g kg-', i.v.). These anaesthetics were administered through a catheter that had been inserted into the left lateral saphenous vein under local anaesthesia (Lignocaine Hydrochloride, Phoenix Pharmaceuticals, 2 % w/v plain) and passed centrally through the vein so that the tip lay in the inferior venacava. Surgical anaesthesia was maintained by a continuous infusion of chloralose (approximately 0 3 mg kg-' min-'). The chloralose was dissolved in a sterile solution of isotonic saline (Steriflex, The Boots Company; 0 9 % w/v sodium chloride) to achieve a final concentration of 10 mg ml-,. Positive-pressure ventilation was started with 40 % oxygen in air, via an endotracheal tube, using a Starling 'Ideal' Pump at a rate of 18 strokes min-' and a stroke volume of approximately
CHEMORECEPTORS AND ATRIAL NATRIURETIC PEPTIDE
521
17 ml kg-'. End-tidal CO2 was measured continuously by means of a CO2 meter (Hartmann & Braun, type URAS 4). Blood samples for the measurement of plasma ANP, blood gases and pH were taken from a cannula placed in the left brachial artery. The pH of the arterial blood was maintained within normal limits by a continuous infusion of sodium bicarbonate solution at a rate of about 0-1 mmol kg-' min-' (Kappagoda, Linden & Snow, 1970). Pco0 was kept within normal limits by adjusting the stroke volume of the respiration pump. Po2 was maintained above 100 mmHg by altering the flow of oxygen in the inspired air-oxygen mixture. Deep body temperature was measured continuously by a telethermometer placed in the oesophagus, and temperature maintained at 36-8 + 0.04 'C. The aortic pressure (i.e. renal perfusion pressure) was measured via a cannula which was passed through the cardiac end of the left femoral artery so that its tip lay in the aorta close to the origin of the renal arteries (Fig. 1). Right atrial pressure was measured through a cannula inserted via the right external jugular vein. In four dogs, left atrial pressure was measured by means of a cannula inserted into the left atrial chamber via the left atrial appendage, through an incision made in the fourth left intercostal space. The chest was closed by suturing all the muscle layers and the skin, and pneumothorax was relieved and the intrathoracic negative pressure was restored as described by Pavlin & Cheney (1979). Pressures were recorded with Statham strain-gauge manometers (Model P23 ID) connected to the appropriate cannulae. The aortic pressure was held constant by connecting the right femoral artery to a pressure control device consisting of a Starling resistance, compressed air system and an arterial reservoir (Fig. 1). Blood was pumped from the left common carotid artery at about 15 ml min-' into the reservoir, which was primed with 20-40 ml dextran-saline (50 % dextran solution and 50 % sterile isotonic saline), and returned to the animal via a heat exchanger (37 °C) and a cannula connected to the right femoral artery. The temperature of the blood was measured continuously by a telethermometer. The regions of the carotid bifurcations were vascularly isolated. The distal ends of the common carotid arteries were cannulated, and both carotid bifurcations were perfused, using roller pumps, either with arterial blood taken from the cardiac end of the right common carotid artery, or moderately hypoxic (mixed, i.e. arterial and venous) blood taken from the right femoral vein and the cardiac end of the right common carotid artery. The carotid sinus pressure was measured by a strain-gauge manometer attached to the perfusion circuit as close to the carotid sinus regions as possible. Perfusion of the carotid sinus regions was made at a constant flow and pressure. The blood perfusing these regions passed through cannulae that were placed in the external carotid arteries and connected to the left external jugular vein via a wider bore cannula. The outflow circuit had a sample port such that blood samples could be taken to measure the pH, P.2 and P. of the blood perfusing the carotid bifurcation regions. Before connecting the perfusion circuit to the dog, heparin (250 units kg-', i.v.) was administered, followed by a continuous infusion into the carotid circuit at approximately 1 unit kg-' min-' to prevent platelet aggregation. In three dogs only the left ureter was exposed retroperitoneally and cannulated for urine collection. In four dogs the ureter was approached by a small suprapubic incision. In all experiments the vagosympathetic trunks were tied in the neck to eliminate the influence of receptors in the aortic arch and cardiopulmonary regions. In five dogs gallamine triethiodide (Flaxedil, May & Baker; 2-0 mg kg-' h-1) was given to prevent chest wall movements. Before administration of gallamine triethiodide, the chloralose infusion rate was increased to approximately 0-4 mg kg-' min-'. The level of anaesthesia was checked using the procedure described by Linden, Mary & Weatherill (1980). Urinary sodium was determined by flame photometry (Corning-Eel 400) and plasma ANP
by radioimmunoassay (RIA).
Experimental protocol Approximately 45-50 min after the perfusion circuit had been connected to the dog, a number of procedures were carried out. Arterial blood gases and pH were adjusted to the normal range, and the aortic and carotid sinus pressures were set at a desired level within normal limits. When all measured variables were steady, urine collection was started for two consecutive 10 min periods. If the urine volumes of these two collections were very different, further collections were made until two consecutive volumes were similar, indicating a steady state of kidney function. At the fifth minute of each period an arterial blood sample (4-5 ml) was taken for the determination of plasma ANP, and the blood sample was immediately replaced by a mixture of warm dextran-saline
M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM
522
Blood sample
SC
I,
Damping chamber
P2
vein
Common carotid arteries
SG3
SG4
RA RVLV
Starling OT
resistance
i
Urine
Water
__ MMI
Compressed air
/
11 . _ /
_
\G2 F
Femoral vein
HE2 Arterial reservoirHE Fig. 1. Diagram of the preparation. At a constant carotid sinus pressure, the vascularly isolated carotid sinus (CS) regions were perfused at a constant flow either with arterial blood from the right common carotid artery, or mixed blood from the right femoral vein and the right common carotid artery to stimulate the carotid body (CB) chemoreceptors. The blood was pumped through a damping chamber and a heat exchanger (HE1) using roller pumps (P1 and P2). Carotid sinus pressure was recorded by a strain gauge (SG1) and set at a desired level by regulating the outflow resistance using a screw clamp (SC); blood from the carotid sinuses was returned to the left external jugular vein. Blood was collected from a sample port for the measurement of blood gases and pH of the carotid perfusate.
CHEMORECEPTORS AND ATRIAL NATRIURET1C PEPTIDE
523
solution. The carotid chemoreceptors were then stimulated by changing their perfusion from arterial to mixed blood (PO 46-4+1-5 mmHg). The aortic and carotid sinus pressures were corrected to the control levels. After a 5 min collection period, during which the volume of urine of the estimated dead space of the kidney and ureteral cannula had passed, two further consecutive urine collections were made. Urine collection was continued for a further two 10 min periods after changing the carotid perfusion back to arterial blood. The average of the values obtained during the initial two 10 min control periods, and the second 10 min period of arterial perfusion after withdrawal of carotid chemoreceptor stimulation, were taken as control and recovery values respectively. The averages of these values were compared with those obtained during the two 10 min periods of mixed blood perfusion, and the difference between these two values was considered as the response to carotid chemoreceptor stimulation. In three dogs the above protocol was repeated after administration of atenolol. Radioimmunoassay of atrial natriuretic peptide The method used for extraction of ANP from plasma samples and subsequent RIA was a modification of the method described by Tan, Tosmalen, Theelen, Kloppenborg, Benraad & Benraad (1987). Blood samples were collected into chilled plastic tubes containing potassium EDTA, and centrifuged at 3000 r.p.m. and 4 °C for 20 min in pre-chilled centrifuge. The plasma was dispensed into aliquotes and stored at -80 'C. Sep-Pak C18 cartridges (Waters, Division of Millipore, Cheshire, UK) were used to extract ANP from plasma samples. The cartridges were activated by washing with 5 ml 4% acetic acid in 86% ethanol, 5 ml methanol, 5 ml water, and finally 5 ml 4% acetic acid (Tan et al. 1987). Prior to extraction, plasma samples (1 ml) were acidified with 3 ml 4 % acetic acid, and the acidified plasma was applied to individual cartridges. After washing the cartridges twice with 3 ml water, the ANP was eluted with 4 ml 4 % acetic acid in 86 % ethanol (Tan et al. 1987). The eluants were evaporated to dryness under a stream of air at 37 'C and the dried extract reconstituted in 500 ,ul RIA buffer (19 mM-monobasic and 81 mMdibasic sodium phosphate (pH 7 4), 005 M-sodium chloride, 0-1 % bovine serum albumin, 0-1 % Triton X-100 and 0-01 % sodium azide). RIA ANP standard (a-human atrial natriuretic peptide (a-hANP); Peninsula Laboratories, UK) was diluted in assay buffer to concentrations ranging from 1 to 100 pg 100 ul-1. Lyophilized rabbit anti-a-ANP (Peninsula Laboratories) was diluted 1:100 with 0-1 % Triton X-100. Primary antiserum (100 ,l) was mixed with 100 ,ul (3-[1251]iodotyrosyl 28) a-hANP (Amersham, Buckinghamshire, UK) containing approximately 15000 c.p.m., and either 100 ul of reconstituted plasma extract or 100 ,ul of the standard solutions. The reagents were incubated for 24 h at 4 'C. The primary antiserum was precipitated by adding 100,u each of pre-titered goat anti-rabbit IgG serum and normal rabbit serum (Peninsula Laboratories) to each tube. After a further 2 h incubation at room temperature the reaction was stopped by the addition of 500 ,ul assay buffer. The tubes were centrifuged at 3000 r.p.m for 20 min, and the supernatant Aortic pressure (renal perfusion pressure) was measured by a strain gauge (SG2) attached to a cannula placed in the abdominal aorta via the left femoral artery and held constant by means of a pressure control device consisting of a Starling resistance and compressed air system, which was connected to an arterial reservoir. Blood was pumped from the left common carotid artery using a roller pump (P3) into the reservoir, which was primed with 20-40 ml dextran-saline mixture and returned to the animal via a heat exchanger (HE2) and a cannula connected to the right femoral artery. Temperature of the blood was measured continuously by a telethermometer (T). A desired pressure was set initially in the system by a pressure bottle (PB) connected to a mercury manometer (MM). When the systemic blood pressure exceeded the set level, the pressure was transmitted to the air chamber (AC) and caused an enhancement of air flow through the Starling resistance and the outflow tube (OT) placed in a bottle containing water, and thus the pressure in the system returned to the initial level. Conversely, when the pressure fell, this outflow of air decreased bringing the pressure back to the set level. In four dogs, left atrial (LA) pressure was recorded by a strain gauge (SG3) connected to a cannula inserted into the left atrial chamber via the left atrial appendage, through an incision made in the fourth left intercostal space. The chest was closed and pneumothorax was relieved. In all dogs, right atrial (RA) pressure was recorded by a strain gauge (SG4) connected to a cannula inserted via the right external jugular vein. LV, left ventricle; RV, right ventricle. Arrows alongside the circuit indicate direction of the flow of blood.
M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM
524
was aspirated immediately. The precipitate was counted in an automatic y-counter (LKB-Wallac Clinigamma 1272; counting efficiency: 80%). The extraction method gave a recovery of 880 + 30% (mean + S.D; n = 8) when iodinated a-hANP was added to plasma. Concentrations of ANP were not, therefore, corrected for extraction losses. The sensitivity of the assay was 12-5 pg ml-', and the intra- and inter-assay coefficients of variation were 5 and 9 % respectively. The RIA was further validated by studying the effect of blood volume expansion on the plasma concentration of ANP. In the first five dogs, a 10 % volume expansion with dextran-saline mixture was performed over a period of 5 min, and urine collection was started for 10 min. At the end of the urine collection period another 10 % volume expansion was done as before to produce a total of 20% expansion; 10 min urine collection was then started. In the remaining dog, only a 20% blood volume expansion was performed as above (Benjamin, Metzler & Peterson, 1987; Fig. 2).
Statistical analysis Student's t test for paired observations was used to determine the statistical significance of the differences in values. When test values were compared with pre-test (control) and recovery values within the same experiment, a one-way analysis of variance was applied. Differences between groups were considered significant at P < 0-05. All values quoted are the mean + standard error of the mean. RESULTS
General variables During the experimental procedure which commenced 5-6 h after the induction of anaesthesia (the time taken for completion of surgical procedures and to obtain steady-state levels of all variables), the mean values of arterial pH, Po., Pco2 and
E
120-.
10.
150--
E 0
100-
6-
E 0,
0
60-
*
Z
2
30-
01-
50-
~160 2 120X0
150
--
1
*
E n
1.2-
EC o
E
40-
04
~~00. Control VE Control VE Control. VE *) and 20 % Fig. 2. The summarized results of the responses to 10 % (n = 5, * (n = 6, A----A) blood volume expansion (VE). Each point shows the mean values with their standard error bars obtained during control and VE periods. MAoP, mean aortic pressure; MRAP, mean right atrial pressure; ANP, atrial natriuretic peptide; UNa, urinary sodium concentration; UNaV, urinary sodium excretion; V, urine volume. Note that the increase in ANP was associated with parallel increases in MAoP, MRAP, UNa V, and V. All the values obtained during control periods were statistically compared with those during VE periods using t test for paired observations. Where no symbol is present, P > 0-05; otherwise, tP < 001, *P < 0001. 0
CHEMORECEPTORS AND ATRIAL NATRIURETIC PEPTIDE
P02 of carotid perfusate (mmHg)
Control
Stimulation
117
49
525
Recovery 121
PET,CO2 (mmHg) 40150
CSP (mmHg) 50
150 _~~~~~~~~~~~ AoP (mmHg)
J 50 HR (beats min-1) RAP 10 (cmH20) 0
LAP
15_
(cmH20)
0
194
176
188 vI
_
30 s
ANP (pg ml1)
31.2
53.8
30-9
V(ml min-1)
0-20
0.13
0.18
10.3
7.8
9.6
UNaV(Iumol min-1)
Fig. 3. Records and values from one experiment showing the responses to moderate stimulation of the carotid chemoreceptors, with a mixture of arterial and venous blood. Note that chemoreceptor stimulation produced an increase in left atrial pressure (LAP), which was associated with an increase in plasma ANP concentration. AoP, aortic pressure; CSP, carotid sinus pressure; RAP, right atrial pressure; HR, heart rate; PET, C02' end-tidal pressure of CO2. Other abbreviations are the same as in Fig. 2. Kidney weight = 48-3 g.
+ 5-7 mmHg, 37±0+ 07 mmHg and 36-8 + 004 °C, temperature were 7-41 + 003, 119±0 respectively. The values of pH, Po2 and Pco2 of the mixed blood perfusing the carotid sinus regions were 7-38 + 002, 46-4 + 1-5 mmHg and 38-4 + 1-5 mmHg, respectively.
M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM
526
Effects of blood volume expansion Blood volume expansion was associated with increases in mean aortic pressure, right atrial pressure, urine flow rate and sodium excretion (Fig. 2). Ten per cent blood volume expansion increased plasma ANP from 37-7 + 1P5 to 7041 + 3-2 pg ml-' 125 T
125
100
Q-Z
100 + 75 t
75 w-
50.
Ic
300 .
I=,
200- .
4 .
3-I
,
a
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-0
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1
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60 -*
Journal of Physiology (1991), 443, pp. 519-531 With 4 figures Printed in Great Britain
THE EFFECT OF DISCRETE STIMULATION OF CAROTID BODY CHEMORECEPTORS ON ATRIAL NATRIURETIC PEPTIDE IN ANAESTHETIZED DOGS
BY M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM* From the Department of Physiology, The Worsley Medical and Dental Building, University of Leeds, Leeds LS2 9NQ
(Received 19 October 1990) SUMMARY
1. In seven chloralose-anaesthetized and artificially ventilated beagles, the carotid sinus regions were vascularly isolated and perfused with either arterial or mixed (arterial and venous) blood (PO2464+15 mmHg, mean + s.E.M.) to stimulate the chemoreceptors at constant flow and pressure. Cervical vagosympathetic trunks were ligated in all dogs, and gallamine triethiodide (2-0 mg kg-' h-', I.v.) was given in five dogs. Right atrial pressure was measured in all dogs, and left atrial pressure in four dogs. Mean aortic pressure was held constant (91-0 + 3-0 mmHg) by means of a reservoir connected to the animal via the common carotid and femoral arteries. Plasma atrial natriuretic peptide (ANP) was measured by radioimmunoassay and urinary sodium by flame photometry. 2. In seven dogs with mean carotid sinus pressure maintained at 96-0 + 4-3 mmHg, stimulation of the carotid chemoreceptors for 25 min produced significant increases in left atrial pressure of 41-2 + 3-3 % (n = 4; P < 0 005) from 5-4 + 0-6 cmH2O and of 30 9 + 4-5 % (n = 7; P < 0-002) in ANP from 31-6 + 2-1 pg ml-'. However, chemoreceptor stimulation produced significant decreases in urine flow rate of 26-1 + 1-9 % (n = 9; P < 0-001) from 0-29 + 0-03 ml min-1 (100 g kidney weight)-' and sodium excretion of 290+2 3 % (P < 0 001) from 8-5+ 17 /tmol min-' (100 g kidney weight)-' but right atrial pressure and heart rate did not change significantly. In three of the dogs, ,-adrenoceptor blockade by atenolol (2 mg kg-', i.v.) greatly reduced the effects of chemoreceptor stimulation on plasma levels of ANP. 3. The results show, for the first time, that discrete stimulation of the carotid chemoreceptors caused an increase in plasma ANP levels, probably due to the reflex increase in atrial pressure that results from an inhibition of the cardiac sympathetic nerves, and an increase in venous return from a reduction of peripheral vascular capacitance. INTRODUCTION
Acute generalized hypoxia and hypercapnia may cause an increase in circulating atrial natriuretic peptide (ANP) in man and in animals (Baertschi, Hausmaninger, Walsh, Mentzer, Wyatt & Pence, 1986; du Souich, Saunier, Harteman, Sautegeau, * To whom reprint requests should be sent. MS 8875
520
M. AL-OBAIDI,
E.
M. WHITAKER AND
F.
KARIM
Ong, Larose & Babini, 1987; Baertschi, Adams & Sullivan, 1988; Clozel, Saunier, Harteman, Allam & Fischli, 1989) and natriuresis and diuresis in man and dogs (Barker, Singer, Elkinton & Clark, 1957; Walker, 1982). However, it is not yet known whether carotid chemoreceptors are involved in the release of ANP and thereby influence sodium excretion and urine volume (Koller, Schopen, Keller, Lang & Vallotton, 1989). Besides stimulating chemoreceptors, generalized hypoxia and hypercapnia are likely to result in changes in blood chemistry that could have widespread effects on the cardiovascular system (Cross, Rieben, Barron & Salisbury, 1963; Folkow & Neil, 1971), and the central nervous system (Downing, Mitchell & Wallace, 1963). In addition, hypoxia and hypercapnia are associated with changes in a number of kidney hormones that could also influence ANP release (Anderson, Rose, Berns, Erikson & Arnold, 1980; Raff, Shinsako, Keil & Dallman, 1983; Wang, Sundet & Goetz, 1984), and thus complicate any interpretations of the mechanism of release of ANP. It is well established that atrial stretch is the main stimulus for ANP release (see Genest & Cantin, 1988), and carotid chemoreceptor stimulation produces a reflex negative inotropic effect, by inhibiting cardiac sympathetic nerves (Hainsworth, Karim & Sofola, 1979; Karim, Hainsworth, Sofola & Wood, 1980), and a reflex reduction in peripheral vascular capacitance by stimulating sympathetic nerves (Hainsworth, Karim, McGregor & Wood, 1983), which would lead to an increase in atrial pressure. Carotid chemoreceptor stimulation is, therefore, expected to cause a release of ANP, and contribute to the natriuretic response in the denervated kidney or at a high carotid sinus pressure in the intact kidney as demonstrated previously (Karim, Poucher & Summerill, 1987; Al-Obaidi, Karim, Majid & Goonewardene, 1988). So far as we know there have been no experiments to determine the effect of discrete stimulation of the carotid chemoreceptors on plasma ANP levels and its effect on sodium and water excretion. The present experiments were, therefore, carried out to determine the effects of discrete stimulation of the carotid chemoreceptors on plasma ANP concentration and sodium excretion in anaesthetized and artificially ventilated dogs. The aortic pressure (renal perfusion pressure) was adequately controlled, and the cervical vagosympathetic trunks were tied to eliminate the influence of receptors located in the aortic arch and cardiopulmonary regions. The carotid chemoreceptors were stimulated by perfusing them with moderately hypoxic (mixed arterial and venous) blood. A preliminary report of these experiments has been published (Al-Obaidi, Whitaker & Karim, 1990). METHODS
Beagles (15-0-31-8 kg, of both sexes) were anaesthetized with thiopentone sodium (Intraval Sodium, May & Baker; 500 mg, i.v.) followed bya-chloralose (BDH Chemicals; 01 g kg-', i.v.). These anaesthetics were administered through a catheter that had been inserted into the left lateral saphenous vein under local anaesthesia (Lignocaine Hydrochloride, Phoenix Pharmaceuticals, 2 % w/v plain) and passed centrally through the vein so that the tip lay in the inferior venacava. Surgical anaesthesia was maintained by a continuous infusion of chloralose (approximately 0 3 mg kg-' min-'). The chloralose was dissolved in a sterile solution of isotonic saline (Steriflex, The Boots Company; 0 9 % w/v sodium chloride) to achieve a final concentration of 10 mg ml-,. Positive-pressure ventilation was started with 40 % oxygen in air, via an endotracheal tube, using a Starling 'Ideal' Pump at a rate of 18 strokes min-' and a stroke volume of approximately
CHEMORECEPTORS AND ATRIAL NATRIURETIC PEPTIDE
521
17 ml kg-'. End-tidal CO2 was measured continuously by means of a CO2 meter (Hartmann & Braun, type URAS 4). Blood samples for the measurement of plasma ANP, blood gases and pH were taken from a cannula placed in the left brachial artery. The pH of the arterial blood was maintained within normal limits by a continuous infusion of sodium bicarbonate solution at a rate of about 0-1 mmol kg-' min-' (Kappagoda, Linden & Snow, 1970). Pco0 was kept within normal limits by adjusting the stroke volume of the respiration pump. Po2 was maintained above 100 mmHg by altering the flow of oxygen in the inspired air-oxygen mixture. Deep body temperature was measured continuously by a telethermometer placed in the oesophagus, and temperature maintained at 36-8 + 0.04 'C. The aortic pressure (i.e. renal perfusion pressure) was measured via a cannula which was passed through the cardiac end of the left femoral artery so that its tip lay in the aorta close to the origin of the renal arteries (Fig. 1). Right atrial pressure was measured through a cannula inserted via the right external jugular vein. In four dogs, left atrial pressure was measured by means of a cannula inserted into the left atrial chamber via the left atrial appendage, through an incision made in the fourth left intercostal space. The chest was closed by suturing all the muscle layers and the skin, and pneumothorax was relieved and the intrathoracic negative pressure was restored as described by Pavlin & Cheney (1979). Pressures were recorded with Statham strain-gauge manometers (Model P23 ID) connected to the appropriate cannulae. The aortic pressure was held constant by connecting the right femoral artery to a pressure control device consisting of a Starling resistance, compressed air system and an arterial reservoir (Fig. 1). Blood was pumped from the left common carotid artery at about 15 ml min-' into the reservoir, which was primed with 20-40 ml dextran-saline (50 % dextran solution and 50 % sterile isotonic saline), and returned to the animal via a heat exchanger (37 °C) and a cannula connected to the right femoral artery. The temperature of the blood was measured continuously by a telethermometer. The regions of the carotid bifurcations were vascularly isolated. The distal ends of the common carotid arteries were cannulated, and both carotid bifurcations were perfused, using roller pumps, either with arterial blood taken from the cardiac end of the right common carotid artery, or moderately hypoxic (mixed, i.e. arterial and venous) blood taken from the right femoral vein and the cardiac end of the right common carotid artery. The carotid sinus pressure was measured by a strain-gauge manometer attached to the perfusion circuit as close to the carotid sinus regions as possible. Perfusion of the carotid sinus regions was made at a constant flow and pressure. The blood perfusing these regions passed through cannulae that were placed in the external carotid arteries and connected to the left external jugular vein via a wider bore cannula. The outflow circuit had a sample port such that blood samples could be taken to measure the pH, P.2 and P. of the blood perfusing the carotid bifurcation regions. Before connecting the perfusion circuit to the dog, heparin (250 units kg-', i.v.) was administered, followed by a continuous infusion into the carotid circuit at approximately 1 unit kg-' min-' to prevent platelet aggregation. In three dogs only the left ureter was exposed retroperitoneally and cannulated for urine collection. In four dogs the ureter was approached by a small suprapubic incision. In all experiments the vagosympathetic trunks were tied in the neck to eliminate the influence of receptors in the aortic arch and cardiopulmonary regions. In five dogs gallamine triethiodide (Flaxedil, May & Baker; 2-0 mg kg-' h-1) was given to prevent chest wall movements. Before administration of gallamine triethiodide, the chloralose infusion rate was increased to approximately 0-4 mg kg-' min-'. The level of anaesthesia was checked using the procedure described by Linden, Mary & Weatherill (1980). Urinary sodium was determined by flame photometry (Corning-Eel 400) and plasma ANP
by radioimmunoassay (RIA).
Experimental protocol Approximately 45-50 min after the perfusion circuit had been connected to the dog, a number of procedures were carried out. Arterial blood gases and pH were adjusted to the normal range, and the aortic and carotid sinus pressures were set at a desired level within normal limits. When all measured variables were steady, urine collection was started for two consecutive 10 min periods. If the urine volumes of these two collections were very different, further collections were made until two consecutive volumes were similar, indicating a steady state of kidney function. At the fifth minute of each period an arterial blood sample (4-5 ml) was taken for the determination of plasma ANP, and the blood sample was immediately replaced by a mixture of warm dextran-saline
M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM
522
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HE2 Arterial reservoirHE Fig. 1. Diagram of the preparation. At a constant carotid sinus pressure, the vascularly isolated carotid sinus (CS) regions were perfused at a constant flow either with arterial blood from the right common carotid artery, or mixed blood from the right femoral vein and the right common carotid artery to stimulate the carotid body (CB) chemoreceptors. The blood was pumped through a damping chamber and a heat exchanger (HE1) using roller pumps (P1 and P2). Carotid sinus pressure was recorded by a strain gauge (SG1) and set at a desired level by regulating the outflow resistance using a screw clamp (SC); blood from the carotid sinuses was returned to the left external jugular vein. Blood was collected from a sample port for the measurement of blood gases and pH of the carotid perfusate.
CHEMORECEPTORS AND ATRIAL NATRIURET1C PEPTIDE
523
solution. The carotid chemoreceptors were then stimulated by changing their perfusion from arterial to mixed blood (PO 46-4+1-5 mmHg). The aortic and carotid sinus pressures were corrected to the control levels. After a 5 min collection period, during which the volume of urine of the estimated dead space of the kidney and ureteral cannula had passed, two further consecutive urine collections were made. Urine collection was continued for a further two 10 min periods after changing the carotid perfusion back to arterial blood. The average of the values obtained during the initial two 10 min control periods, and the second 10 min period of arterial perfusion after withdrawal of carotid chemoreceptor stimulation, were taken as control and recovery values respectively. The averages of these values were compared with those obtained during the two 10 min periods of mixed blood perfusion, and the difference between these two values was considered as the response to carotid chemoreceptor stimulation. In three dogs the above protocol was repeated after administration of atenolol. Radioimmunoassay of atrial natriuretic peptide The method used for extraction of ANP from plasma samples and subsequent RIA was a modification of the method described by Tan, Tosmalen, Theelen, Kloppenborg, Benraad & Benraad (1987). Blood samples were collected into chilled plastic tubes containing potassium EDTA, and centrifuged at 3000 r.p.m. and 4 °C for 20 min in pre-chilled centrifuge. The plasma was dispensed into aliquotes and stored at -80 'C. Sep-Pak C18 cartridges (Waters, Division of Millipore, Cheshire, UK) were used to extract ANP from plasma samples. The cartridges were activated by washing with 5 ml 4% acetic acid in 86% ethanol, 5 ml methanol, 5 ml water, and finally 5 ml 4% acetic acid (Tan et al. 1987). Prior to extraction, plasma samples (1 ml) were acidified with 3 ml 4 % acetic acid, and the acidified plasma was applied to individual cartridges. After washing the cartridges twice with 3 ml water, the ANP was eluted with 4 ml 4 % acetic acid in 86 % ethanol (Tan et al. 1987). The eluants were evaporated to dryness under a stream of air at 37 'C and the dried extract reconstituted in 500 ,ul RIA buffer (19 mM-monobasic and 81 mMdibasic sodium phosphate (pH 7 4), 005 M-sodium chloride, 0-1 % bovine serum albumin, 0-1 % Triton X-100 and 0-01 % sodium azide). RIA ANP standard (a-human atrial natriuretic peptide (a-hANP); Peninsula Laboratories, UK) was diluted in assay buffer to concentrations ranging from 1 to 100 pg 100 ul-1. Lyophilized rabbit anti-a-ANP (Peninsula Laboratories) was diluted 1:100 with 0-1 % Triton X-100. Primary antiserum (100 ,l) was mixed with 100 ,ul (3-[1251]iodotyrosyl 28) a-hANP (Amersham, Buckinghamshire, UK) containing approximately 15000 c.p.m., and either 100 ul of reconstituted plasma extract or 100 ,ul of the standard solutions. The reagents were incubated for 24 h at 4 'C. The primary antiserum was precipitated by adding 100,u each of pre-titered goat anti-rabbit IgG serum and normal rabbit serum (Peninsula Laboratories) to each tube. After a further 2 h incubation at room temperature the reaction was stopped by the addition of 500 ,ul assay buffer. The tubes were centrifuged at 3000 r.p.m for 20 min, and the supernatant Aortic pressure (renal perfusion pressure) was measured by a strain gauge (SG2) attached to a cannula placed in the abdominal aorta via the left femoral artery and held constant by means of a pressure control device consisting of a Starling resistance and compressed air system, which was connected to an arterial reservoir. Blood was pumped from the left common carotid artery using a roller pump (P3) into the reservoir, which was primed with 20-40 ml dextran-saline mixture and returned to the animal via a heat exchanger (HE2) and a cannula connected to the right femoral artery. Temperature of the blood was measured continuously by a telethermometer (T). A desired pressure was set initially in the system by a pressure bottle (PB) connected to a mercury manometer (MM). When the systemic blood pressure exceeded the set level, the pressure was transmitted to the air chamber (AC) and caused an enhancement of air flow through the Starling resistance and the outflow tube (OT) placed in a bottle containing water, and thus the pressure in the system returned to the initial level. Conversely, when the pressure fell, this outflow of air decreased bringing the pressure back to the set level. In four dogs, left atrial (LA) pressure was recorded by a strain gauge (SG3) connected to a cannula inserted into the left atrial chamber via the left atrial appendage, through an incision made in the fourth left intercostal space. The chest was closed and pneumothorax was relieved. In all dogs, right atrial (RA) pressure was recorded by a strain gauge (SG4) connected to a cannula inserted via the right external jugular vein. LV, left ventricle; RV, right ventricle. Arrows alongside the circuit indicate direction of the flow of blood.
M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM
524
was aspirated immediately. The precipitate was counted in an automatic y-counter (LKB-Wallac Clinigamma 1272; counting efficiency: 80%). The extraction method gave a recovery of 880 + 30% (mean + S.D; n = 8) when iodinated a-hANP was added to plasma. Concentrations of ANP were not, therefore, corrected for extraction losses. The sensitivity of the assay was 12-5 pg ml-', and the intra- and inter-assay coefficients of variation were 5 and 9 % respectively. The RIA was further validated by studying the effect of blood volume expansion on the plasma concentration of ANP. In the first five dogs, a 10 % volume expansion with dextran-saline mixture was performed over a period of 5 min, and urine collection was started for 10 min. At the end of the urine collection period another 10 % volume expansion was done as before to produce a total of 20% expansion; 10 min urine collection was then started. In the remaining dog, only a 20% blood volume expansion was performed as above (Benjamin, Metzler & Peterson, 1987; Fig. 2).
Statistical analysis Student's t test for paired observations was used to determine the statistical significance of the differences in values. When test values were compared with pre-test (control) and recovery values within the same experiment, a one-way analysis of variance was applied. Differences between groups were considered significant at P < 0-05. All values quoted are the mean + standard error of the mean. RESULTS
General variables During the experimental procedure which commenced 5-6 h after the induction of anaesthesia (the time taken for completion of surgical procedures and to obtain steady-state levels of all variables), the mean values of arterial pH, Po., Pco2 and
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CHEMORECEPTORS AND ATRIAL NATRIURETIC PEPTIDE
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Fig. 3. Records and values from one experiment showing the responses to moderate stimulation of the carotid chemoreceptors, with a mixture of arterial and venous blood. Note that chemoreceptor stimulation produced an increase in left atrial pressure (LAP), which was associated with an increase in plasma ANP concentration. AoP, aortic pressure; CSP, carotid sinus pressure; RAP, right atrial pressure; HR, heart rate; PET, C02' end-tidal pressure of CO2. Other abbreviations are the same as in Fig. 2. Kidney weight = 48-3 g.
+ 5-7 mmHg, 37±0+ 07 mmHg and 36-8 + 004 °C, temperature were 7-41 + 003, 119±0 respectively. The values of pH, Po2 and Pco2 of the mixed blood perfusing the carotid sinus regions were 7-38 + 002, 46-4 + 1-5 mmHg and 38-4 + 1-5 mmHg, respectively.
M. AL-OBAIDI, E. M. WHITAKER AND F. KARIM
526
Effects of blood volume expansion Blood volume expansion was associated with increases in mean aortic pressure, right atrial pressure, urine flow rate and sodium excretion (Fig. 2). Ten per cent blood volume expansion increased plasma ANP from 37-7 + 1P5 to 7041 + 3-2 pg ml-' 125 T
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