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Hemodynamic effects of calcitonin in the normal rat
Peptides,Vol. 13, pp. 571-575, 1992
0196-9781/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.
Printed in the USA.
Hemodynamic Effects of Calcitonin in the Normal Rat ALFREDO
M. P E G U E R O - R I V E R A 1 A N D C L I N T O N
N. C O R D E R 2
Department of Clinical Pharmacology, Oklahoma Medical Research Foundation, Oklahoma City, OK R e c e i v e d 13 N o v e m b e r 1991 PEGUERO-RIVERA, A. M. AND C. N. CORDER. Hemodynamic effects ofcalcitonin in the normal rat. PEPTIDES 13(3) 571-575, 1992.--calcitonin (CT) was administered acutely (IV 4-8 U/kg) and chronically (SC 2 U/kg/day × 150 day) to normal male rats. Measurements included heart rate (HR), mean blood pressure (MBP), cardiac index (CI), peripheral vascular resistance (PVR), and stroke volume index (SVI). The MBP was higher in CT rats examined under pentobarbital anesthesia. Upon awakening from anesthesia, rats chronically on CT exhibited impaired recovery of CI and SVI. Hemodynamic effects were not seen in rats acutely treated with CT. Heart weight was unchanged in chronic treatment with CT. Therefore, CT had minimal hemodynamic effects in the normal male rat. Rat
Calcitonin
Cardiac output
Blood pressure
It appears that there are no controlled studies in which the hemodynamic effects of CT on cardiac output (CO), peripheral vascular resistance (PVR), and stroke volume, as measured by thermodilution catheter, have been determined. There is an indirect clinical evidence in a condition known as Paget's disease in which the CO is elevated due to incremental changes in blood flow in the bone following accelerated bone turnover. Calcitonin is utilized to treat this bone condition. The CO tended to decrease from 7.6 to 6.2 l/rain with chronic porcine CT (19), and from 5.5 to 4.9 1/min/m 2 with chronic salmon CT (l 1). Therefore, CT apparently has a hemodynamic effect in patients with Paget's disease. In order to further elucidate the effects of CT on normal heart function, we carried out the following study in which salmon CT was given acutely and chronically in normal male rats in which a thermodilution catheter was utilized for hempdynamic measurements.
CALCITONIN (CT, thyrocalcitonin) is commercially available as human CT and salmon CT. Calcitonin participates with parathyroid hormone in maintenance of blood calcium levels (3,8,34). In the kidney, CT either increased or decreased the excretion of phosphate and calcium (2). It may also decrease the nociceptive response (9,16,17). Calcitonin had variable effects on the cardiovascular system in rats when administered acutely [salmon CT (26,36,41), human CT (26,41)] or chronically [eel CT (40)], and in man, when given acutely [human CT (15,18), salmon (15), and porcine CT (13)], and in dogs [porcine (1)]. The CT effects were accompanied by the stimulation of adenyl cyclase, an enzyme involved in the generation of cyclic AMP (5,8,10,20,27,37). Other effects of CT that are suspected include blood pressure regulatory properties, as evidenced in the young spontaneously hypertensive rat in which hypertension and cardiac hypertrophy were associated with high plasma levels of CT (7,34). The literature reports evidence of CT-induced positive and negative inotropic effects, or no effect at all (10,13,20,27). Calcitonin affected the electrical activity of the myocardium, which was accompanied by diminution of Na+-K ÷ ATPase activity, alteration of the cyclic AMP system, and myocardial cell contraction (4-6,31,37). Also, there was evidence in the isolated atrium of the dog, but not in the papillary muscle, of a CTinduced negative inotropic effect (10). Additionally, intravenous infusion of epinephrine or isoproterenol that resulted in myocarditis and death in rats could be reversed by CT (14,38).
METHOD Male Wistar Kyoto rats (Charles River Company), weighing approximately 180 g, were separated into two different treatment groups. They were housed approximately four per cage at 2225°C, fed rat chow (Purina Mills Inc.) and tap water ad lib. The day/night cycle was 12/12 h, respectively. The animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Chronic subcutaneous injections were carded out with salmon calcitonin (Calcimar, Rorer Phar-
Present address: Department of Anesthesiology, University of Puerto Rico Medical School, Medical Science Campus, GPO Box 5067, San Juan, Puerto Rico, 00936. 2 Requests for reprints should be addressed to Dr. Clinton N. Corder, Department of Clinical Pharmacology, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104-5047.
571
572 maceuticals, Lot# CR-4002C-201039/-89), 2 U per kg body weight. Dosing was selected according to known chronic metabolic effects on rats (12,28). The solution was diluted with 0.9% saline daily and freshly injected. For the subsequent intravenous study outline in Table 2, CT was diluted to 8 U/ml and 16 U/ ml with normal saline and the appropriate volume was injected intravenously; controls received equal volumes of normal saline. Rats were anesthetized with sodium pentobarbital given IP at 40 mg/kg and restrained supine on an operating board; 10 mg/kg of sodium pentobarbital was given supplementally before the end of surgery based upon the corneal reflex and body movement. Under an aseptic technique, three incisions were made: ventral neck (longitudinal, right side, 2 cm), dorsal neck (sagittal, middle side, l cm) and in the right groin area (oblique, parallel to the femoral vessels, 2 cm). Subsequent ligatures and sutures were done with 3-0 silk. A 15 cm polyethylene tubing (i.d. 0.86 mm, o.d. 1.27 mm, 70 tzl, PE90, Clay Adams) and a 15 cm thermistor (1.5 F, bead 0.5 ram, specially manufactured by Clen Leiber, Edwards Lab, San Diego, CA) were passed subcutaneously from the dorsal to the ventral incision and a 17.5 cm PE90 tubing was passed from the groin incision to the dorsal neck incision. The thermistor placement was through a 0.35 × 7 cm plastic sleeve to protect the thermistor bead, after which the sleeve was removed. By blunt dissection of the ventral neck area, the right external jugular vein and right carotid artery were isolated with ligatures in the proximal and distal sides. The PE90 tubing was passed 3.5 cm intravenously into the right superior vena cava, and the thermistor was introduced intraarterially with the tip 3-3.5 cm into the aortic arch. Ligatures were placed around the catheter and thermistor, and the incision was closed. The iliac artery was cannulized with a 4 cm PE50 (i.d. 0.58 mm, o.d. 0.66 mm) tubing to an intraarterial distance of 1.5 cm placing it in the abdominal aorta. It was secured with a ligature and connected to the PE90 tube coming out subcutaneously from the dorsal neck, and the incision was sutured. The two PE90 catheters and thermistor exiting the dorsal neck were sutured with closing of the dorsal incision. The catheters were filled with 0.9% saline containing heparin at 12.5 U/ml and the ends plugged with metal wire. The thermistor was connected to a thermodilution cardiac output computer (Edwards 9520) and the calculated curve by the computer was depicted in a polygraph (Grass Model 7). The iliac artery catheter was connected in a PE90-PE50-PE90 interphase to a strain gauge transducer (P23XL Viggo-Spectramed) for mean blood pressure and heart rate measurements. The venous catheter was used for injections of 200 #l (500 #1 Hamilton syringe) of 0.9% saline at 22-25°C for cardiac output measurements. Measurements were recorded in triplicate and the mean values were taken for each time point: the coefficient of variation of sequential determination of cardiac index was 6.9%. The catheter was flushed with 0.9% saline containing 12.5 U heparin/ml between triplicate measurements. The catheters and the thermistor were protected by wrapping the rat's neck, chest, and back with surgical tape, in such a way that there was no interference to respiration. The rats were unrestrained and the four extremities free for movement. Blood temperature was monitored by the thermistor probe and maintained between 36-38°C, with a heat lamp of 60 watts. The surgical preparation time was about 1 h. The initial hemodynamic measurements were made while the rats were still under pentobarbital anesthesia. They were then moved to a regular unrestricted cage of 61 X 46 × 41 cm to regain consciousness, which took approximately 3 h. Although there would still be residual effects from the incomplete elimination (half-life 15-48 h) of barbiturates from the animal, and stress of the surgical procedures,
PEGUERO-RIVERA AND CORDER the rats were awake, walked, and had withdrawal reflexes to confrontation. Awake (acute recovery) measurements were then made with the rats unrestrained. These procedures, with modifications, were previously reported (21,24,25,30,32). The following hemodynamic measurements were made and/ or calculated: heart rate (bpm) (HR): number of arterial pulsations in six seconds at 25 mm/s paper speed times ten; mean blood pressure (mmHg) (MBP): an electronic mean of t/2 amplitude of the high frequency response at 0. l (Operational Manual, Grass Model 7 Polygraph); cardiac output (ml/min) (CO): automatic calculation dial (Edwards 9520 Computer) of the time integral of the dilution curve; also depicted graphically by the Grass polygraph for qualitative inspection; cardiac index (ml/ min/100 g) (CI): [cardiac output (ml/min) × 1000] + [body weight (kg/20) × 25] (25 is the factor of conversion of the injectate volume to the ideal volume 5 cc/0.02 cc at cardiac constant of 0.306 and temperature 22-25°C); stroke volume index (#lfloeat/100 g) (SVI): (CI × 1000) + HR; and peripheral vascular resistance (mmHg/ml/min/100 g) (PVR): MBP/CI. At the end of the study, animals were heparinized with heparin IV 150 U/kg, then sacrificed with sodium pentobarbital 100 mg/kg, IV. The catheters and thermistor were removed after their placement was confirmed. The heart was excised, rinsed with saline, and blotted. The wet heart was immediately weighed. All the data was analyzed using the Student t-test (Table 1) or analysis of variance for multiple comparisons (Table 2). The null hypothesis was rejected at p < 0.05, unless otherwise stated. RESULTS Results of the chronic subcutaneous administration of salmon CT are shown in Table t. The body weights increased to 547 g in control, and heart to body weight ratio was 2.52 × 10-31 The CT-treated rats had a significantly decreased heart to body weight ratio, suggesting a relatively decreased heart size, even though the heart weights were not different. The control HR was about 404 bpm and MBP was 113 mmHg. This was accompanied by a CI of 30.6 ml/min/100 g and SVI of 75.2 #lfoeat/100 g. The PVR was calculated at 3.81 mmHg/ml/min/100 g. These were the values on the control rats approximately 30 min after the end of surgery and still under sodium pentobarbital anesthesia at 40 mg/kg of body weight with a supplemental dose of l0 rag/ kg for maintenance. Shown in the anesthetized column are the corresponding values for the CT-treated rats, The results in this parallel treatment group indicated that CT rats, just after the surgical procedure, had the same HR as in controls, but the MBP may have been slightly elevated at 123 mmHg (17 = 0.09). Other parameters (CI, PVR, and SVI) were no different from the parallel control. In the awake columns (Table 1), the measurements are shown approximately 3 h after the animals awoke. These animals were unrestrained in their cage with no previous conditioning. There was no difference between control and CT treatment on HR, MBP, CI, and SVI. However, there was an insignificant elevation of PVR in the CT-treated group, 3.96 vs. 3.58 mmHg/ml/min/ 100 g (p = 0.06). Therefore, in the group chronically treated with salmon CT, there is some evidence of increase in the PVR and possible increase in MBP, but in neither case was the change significant. Pentobarbital anesthesia decreased CI and SVI by 12% and 7%, respectively, in the control animals (anesthetized versus awake columns, Table l). The HR was diminished about 5% by pentobarbital in both control and CT rats, but was only significant in control rats. This was contrary to the expected va-
CALCITONIN AND CARDIAC OUTPUT
573
TABLE 1 EFFECT OF CHRONICALLY INJECTED SALMON CALCITONIN ON HEMODYNAMICS IN THE RAT Anesthetized Measurement
Control [9l
Heart weight (g) Body weight (g) Heart/Body Weight × 10-3 HR (bpm) MBP (mmHg) CI (ml/min/100 g) PVR (mmHg/ml/min/100 g) SVI (#l/beat/100 g)
1.37 547 2.52 404 113 30.6 3.81 75.2
___0.05 + 24 + 0.09 + 7 +__3 ___2.1 ___0.26 +__4.7
Awake
Calcitonin [7]
Control [9]
Calcitonin [7]
1.40 + 0.06 577 ___23 2.44 +__0.12" 392 + 17 123 --- 6:~ 30.0 + 2.4 4.21 + 0.28 77.4 + 6.2
---423 + 10t 122 _+ 6 34.8 + 2.3f 3.58 + 0.22 81.2 + 4.9I"
--409 + 15 126 +__5 31.9 + 1.1 3.96 + 0.09§ 77.4 + 1.9
Number of subjects in brackets. Values are mean +- standard error. CT treatment was for 150 days at 2 U salmon calcitonin/kg weight SC. Anesthesia was with pentobarbital 40 mg/kg + 10 mg/kg. Anesthesia observations were 30 min after the surgery was completed. Awake observations about 3 h later with animals conscious. Heart rate (HR), mean blood pressure (MBP), cardiac index (CI), peripheral vascular resistance (PVR), stroke volume index (SVI). *t*§ Control vs. calcitonin within anesthetic state (i.e., anesthetized or awake): * p < 0.01, § p < 0.066, ~tp < 0.09. Anesthetized vs. awake within group (i.e., control or calcitonin): 'f p < 0.05.
golytic effect o f pentobarbital. Therefore, the effect o f pentobarbital anesthesia is m o r e suppressive o n h e m o d y n a m i c s in Cq" rats t h a n in control rats; i.e., the control rats recover CI a n d SVI at a b o u t 3 h postanesthesia, whereas CT-treated rats r e m a i n suppressed. T h e effect of acutely a d m i n i s t e r e d s a l m o n C T is s h o w n in Table 2. In this case, there were control rats t h a t h a d never received C T a n d were awake approximately 4 h after surgery. Calcitonin at 4 U / k g was given at 0 t i m e a n d t h e n 5 a n d 15 m i n u t e s values were recorded; C T 8 U / k g was repeated d u r i n g this total o f 3 5 - m i n observation period. A parallel control group o f rats received saline injections. T h e r e were n o statistical differences between CT- a n d saline-treated groups with respect to HR, MBP, CI, PVR, a n d SVI. In s u m m a r y , the effects o f C T a d m i n i s t e r e d chronically are evident, b u t it depends u p o n w h e t h e r o n e is looking at the effect u n d e r pentobarbital anesthesia or in the postsurgical awake animal.
DISCUSSION The aortic thermodilution m e t h o d used in this study is similar to that which has b e e n utilized a n d p r o v e n reliable for meas u r e m e n t o f CI in conscious a n d anesthetized rats (21,24,25,30). T h e CI values range from 21 to 58 m l / m i n / 1 0 0 g b.wt. depending o n strain, grade, a n d type o f stress, a n d anesthetic t e c h n i q u e (32). For the Wistar strain 40 (30), 28 (24), a n d 36 (39) m l / m i n / 100 g values have been reported a n d are similar to o u r values o f 35. T h e accuracy a n d reproducibility o f this m e t h o d to detect h e m o d y n a m i c changes o n pharmacological or physiological challenges in the rat model have m a d e it reliable (35,39). T h e h e m o d y n a m i c effects o f s o d i u m p e n t o b a r b i t a l n o t e d in this study are similar to previous results. Pentobarbital was administered IP at 50 m g / k g (23) or IV at 30 m g / k g a n d meas u r e m e n t s were d o n e 2 days after t h o r a c o t o m y (22); IP 40 m g / kg with m a i n t e n a n c e o f IP 20 m g / k g followed by c o n t i n u o u s IV infusion of 6 m g / k g / h (35); a n d in this study 40 m g / k g IV with m a i n t e n a n c e o f 10 mg/kg. T h e M B P was reported as u n c h a n g e d
TABLE 2 EFFECT OF ACUTELY INJECTED SALMON CALCITONIN ON HEMODYNAMICS IN THE AWAKE RAT Group
Time (min)
HR (bpm)
MBP (mmHg)
CI (ml/min/lO0 g)
PVR (mmHg/ml/min/lO0 g)
SVI (#lfoeat/lO0 g)
Control (n = 4)
0 5 15 20 35
418 436 431 440 435
+ 14 + 20 + 17 + 12 +__11
114 116 120 119 123
+ 3 + 2 + 3 + 1 ___3
34.8 35.5 36.0 36.0 39.0
+__1.5 + 1.2 + 2.4 + 1.9 ___1.7
3.32 3.28 3.39 3.33 3.15
___0.19 ___0.13 ___0.30 ___0.19 +__0.10
83.4 84.5 84.0 81.8 86.0
_+ 4.9 + 5.2 + 8.1 _ 6.2 ___4.9
Calcitonin (n = 4)
0 5 15 20 35
428 435 435 430 430
+ 21 ___ 18 +__21 ___ 16 + 18
122 124 125 126 130
+ 11 +__12 ___12 + 12 + 13
35.5 34.3 36.8 37.8 40.0
+ 4.8 _+ 5.3 _+ 5.2 + 2.8 ___3.4
3.52 3.69 3.45 3.06 3.24
___0.25 ___0.20 + 0.15 ___0.28 + 0.10
82.5 78.0 83.3 87.3 92.0
+_ 8.9 + 9.8 + 8.8 +__4.2 ___4.7
Values are mean _+ standard error. Control was normal saline given IV at 0 and after 15 min. CT was injected IV at 4 U/kg at 0 min and 8 U/kg after 15 rain observation. Time 0 is 4 h after surgery or 3 h awake after pentobarbital anesthesia. The average body weight was 561 + 39 g (control) and 550 -+ 32 g (calcitonin).
574
PEGUERO-RIVERA AND CORDER
(23), decreased (22), or increased (33), and ours was decreased. The PVR increased in previous studies (22,23,35), and in our study also, but not significantly. The HR was unchanged (22,23) or increased (35), in contrast to our observed decreases. The CI and SVI were decreased in all the studies, including our study. Therefore, the effects that are more consistently seen are the diminution of CI, SVI, and incrementation of the PVR. In all these studies the measurements were done 30-40 min after or during anesthesia. Comparisons were between groups (23,35), same group (22), fasting (33), or not (33,35), and endotracheal intubation (38); our rats were not fasted and the comparisons were in the same group (3-h awake state). Pentobarbital anesthesia was judged acceptable by the authors for the following reasons: its convenience in administration, the consistent reproducibility of the effects of CI and SVI, and the predictable recovery trends. In order to avoid other pharmacological hemodynamic effects, we did not add any other drugs. The hemodynamic effects of CT on HR and MBP may be compared to previously reported results. In the chronic CT group, either awake or in the postsurgical anesthesia state, the SVI, CI, and HR did not change from the controls. There are no previous reports of the effect of CT on SVI and CI. The HR and MBP were reported unchanged (26,36,41). However, MBP and PVR underwent a small change in the chronic CT group, depending on the awake (PVR) or postsurgical anesthesia state (MBP). These results are suggestive of some mild blood pressure regulatory properties of salmon CT on long-term administration. Elevated CT plasma levels have been noted in the spontaneous
hypertensive rat (7). A significant lack of recovery of the pentobarbital suppressive effect on CI and SVI in the chronic CTtreated rats was evident. It has been suggested that an appreciable part of the diminution in CI on pentobarbital anesthesia is induced by inhibition of the tonic adrenomedullary secretion (22), and by interference in the myocardial calcium metabolism (33). The relationship of this pentobarbital effect with CT effect seen in the present study is unknown. One possible cause of the lack of recovery in pentobarbital anesthesia in CT rats could be that the postoperative nociceptive stimuli were altered by the CT analgesic effect (9,16,17). There are no previously reported studies on the effect of CT in rats on CI, SVI, and PVR, as measured by thermodilution catheters. The present results suggest minimal effects of CT on the nonfailing heart, but did not address any effects on a failing heart. The literature reports evidence of positive, negative, and no inotropic effects o f C T ( 10,13,20,27). In dog left atrium there is a negative CT inotropic effect, but no effect in the papillary muscle (10). Studies do report that exposure of vascular tissues to CT results in stimulation of adenyl cyclase, an enzyme involved in generating cyclic AMP (5,8,10,20,27,37), which was accompanied by alteration of myocardial cell contraction (4-6,31,37). Norepinephrinestimulated calcium influx in aorta was stimulated by CT (36). Because of the central role of calcium and cyclic A M P in cardiac function (29), it is conceivable that CT may have significant hemodynamic effects on the failing heart that were not evident in the normal heart.
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cells during blockade of sodium and potassium permeability. Fiziol. Zh. SSSR 61(2):251-6; 1975. Parker, G. W.; Martin, D. G. Technique for cardiovascular monitoring in awake tethered rats. Lab. Anim. Sci. 39(5):463--467; 1989. Parker, J. L.; Adams, H. R. The influence of chemical restraining agents on cardiovascular function: A review. Lab. Anita. Sci. 28(5): 575-583; 1978. Resnick, L. M. Calciotropic hormones in human and experimental hypertension. Am. J. Hypertens. 3:171S-178S; 1990. Schaefer, C. F.; Brackett, D. J.; Biber, B.; Lerner, M. R.; Holaday, J. W.; Wilson, M. F.; Fagraeus, L. Respiratory and cardiovascular effects of thyrotropin-releasing hormone as modified by isoflurane, enflurane, pentobarbital, and ketamine. Regul. Pept. 24(3):269-282; 1989. Schleiffer, R.; Helwig, J. J.; Pernot, F.; Gairard, A. Vascular effects of calcitonin and parathyroid hormone. In: Doepfner, W., ed. Calcitonin 1984. The Netherlands: Amsterdam Excerpta Medica; 1986: 15-24. Smirnov, P. A.; Chaikovskaia, E. V.; Shestak, V. S. Influence of thyrocalcitonin on the adenosine triphosphate activity and the electrolyte balance of the rat myocardium. Farmakol. Toksikol. 39(1): 44-45; 1976. Sokolov, I. K.; Belen'ki, I. E. E.; Tunitskaia, T. A.; Kalugina, G. E.; Briskin, A. I. Effect of thyrocalcitonin on the recovery process in adrenaline lesion of the myocardium. Farmakol. Toksikol. 39(2): 167-173; 1976. Takamata, A.; Nose, H.; Mack, G. W.; Morimoto, T. Control of total peripheral resistance during hyperthermia in rats. J. Appl. Physiol. 69(3): 1087-1092; 1990. Takayanagi, N.; Tozuka, T.; Toda, N. Antihypertensive action of elcalcitonin. Nippon Yakurigaku Zasshi 82(5):383-93; 1983. Wegener, L. L.; McCarron, D. A. Acute intravenous caleitonin: Failure to modify systemic blood pressure. Peptides 9:1191-93; 1988.
0196-9781/92 $5.00 + .00 Copyright© 1992 PergamonPressLtd.
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Hemodynamic Effects of Calcitonin in the Normal Rat ALFREDO
M. P E G U E R O - R I V E R A 1 A N D C L I N T O N
N. C O R D E R 2
Department of Clinical Pharmacology, Oklahoma Medical Research Foundation, Oklahoma City, OK R e c e i v e d 13 N o v e m b e r 1991 PEGUERO-RIVERA, A. M. AND C. N. CORDER. Hemodynamic effects ofcalcitonin in the normal rat. PEPTIDES 13(3) 571-575, 1992.--calcitonin (CT) was administered acutely (IV 4-8 U/kg) and chronically (SC 2 U/kg/day × 150 day) to normal male rats. Measurements included heart rate (HR), mean blood pressure (MBP), cardiac index (CI), peripheral vascular resistance (PVR), and stroke volume index (SVI). The MBP was higher in CT rats examined under pentobarbital anesthesia. Upon awakening from anesthesia, rats chronically on CT exhibited impaired recovery of CI and SVI. Hemodynamic effects were not seen in rats acutely treated with CT. Heart weight was unchanged in chronic treatment with CT. Therefore, CT had minimal hemodynamic effects in the normal male rat. Rat
Calcitonin
Cardiac output
Blood pressure
It appears that there are no controlled studies in which the hemodynamic effects of CT on cardiac output (CO), peripheral vascular resistance (PVR), and stroke volume, as measured by thermodilution catheter, have been determined. There is an indirect clinical evidence in a condition known as Paget's disease in which the CO is elevated due to incremental changes in blood flow in the bone following accelerated bone turnover. Calcitonin is utilized to treat this bone condition. The CO tended to decrease from 7.6 to 6.2 l/rain with chronic porcine CT (19), and from 5.5 to 4.9 1/min/m 2 with chronic salmon CT (l 1). Therefore, CT apparently has a hemodynamic effect in patients with Paget's disease. In order to further elucidate the effects of CT on normal heart function, we carried out the following study in which salmon CT was given acutely and chronically in normal male rats in which a thermodilution catheter was utilized for hempdynamic measurements.
CALCITONIN (CT, thyrocalcitonin) is commercially available as human CT and salmon CT. Calcitonin participates with parathyroid hormone in maintenance of blood calcium levels (3,8,34). In the kidney, CT either increased or decreased the excretion of phosphate and calcium (2). It may also decrease the nociceptive response (9,16,17). Calcitonin had variable effects on the cardiovascular system in rats when administered acutely [salmon CT (26,36,41), human CT (26,41)] or chronically [eel CT (40)], and in man, when given acutely [human CT (15,18), salmon (15), and porcine CT (13)], and in dogs [porcine (1)]. The CT effects were accompanied by the stimulation of adenyl cyclase, an enzyme involved in the generation of cyclic AMP (5,8,10,20,27,37). Other effects of CT that are suspected include blood pressure regulatory properties, as evidenced in the young spontaneously hypertensive rat in which hypertension and cardiac hypertrophy were associated with high plasma levels of CT (7,34). The literature reports evidence of CT-induced positive and negative inotropic effects, or no effect at all (10,13,20,27). Calcitonin affected the electrical activity of the myocardium, which was accompanied by diminution of Na+-K ÷ ATPase activity, alteration of the cyclic AMP system, and myocardial cell contraction (4-6,31,37). Also, there was evidence in the isolated atrium of the dog, but not in the papillary muscle, of a CTinduced negative inotropic effect (10). Additionally, intravenous infusion of epinephrine or isoproterenol that resulted in myocarditis and death in rats could be reversed by CT (14,38).
METHOD Male Wistar Kyoto rats (Charles River Company), weighing approximately 180 g, were separated into two different treatment groups. They were housed approximately four per cage at 2225°C, fed rat chow (Purina Mills Inc.) and tap water ad lib. The day/night cycle was 12/12 h, respectively. The animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Chronic subcutaneous injections were carded out with salmon calcitonin (Calcimar, Rorer Phar-
Present address: Department of Anesthesiology, University of Puerto Rico Medical School, Medical Science Campus, GPO Box 5067, San Juan, Puerto Rico, 00936. 2 Requests for reprints should be addressed to Dr. Clinton N. Corder, Department of Clinical Pharmacology, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104-5047.
571
572 maceuticals, Lot# CR-4002C-201039/-89), 2 U per kg body weight. Dosing was selected according to known chronic metabolic effects on rats (12,28). The solution was diluted with 0.9% saline daily and freshly injected. For the subsequent intravenous study outline in Table 2, CT was diluted to 8 U/ml and 16 U/ ml with normal saline and the appropriate volume was injected intravenously; controls received equal volumes of normal saline. Rats were anesthetized with sodium pentobarbital given IP at 40 mg/kg and restrained supine on an operating board; 10 mg/kg of sodium pentobarbital was given supplementally before the end of surgery based upon the corneal reflex and body movement. Under an aseptic technique, three incisions were made: ventral neck (longitudinal, right side, 2 cm), dorsal neck (sagittal, middle side, l cm) and in the right groin area (oblique, parallel to the femoral vessels, 2 cm). Subsequent ligatures and sutures were done with 3-0 silk. A 15 cm polyethylene tubing (i.d. 0.86 mm, o.d. 1.27 mm, 70 tzl, PE90, Clay Adams) and a 15 cm thermistor (1.5 F, bead 0.5 ram, specially manufactured by Clen Leiber, Edwards Lab, San Diego, CA) were passed subcutaneously from the dorsal to the ventral incision and a 17.5 cm PE90 tubing was passed from the groin incision to the dorsal neck incision. The thermistor placement was through a 0.35 × 7 cm plastic sleeve to protect the thermistor bead, after which the sleeve was removed. By blunt dissection of the ventral neck area, the right external jugular vein and right carotid artery were isolated with ligatures in the proximal and distal sides. The PE90 tubing was passed 3.5 cm intravenously into the right superior vena cava, and the thermistor was introduced intraarterially with the tip 3-3.5 cm into the aortic arch. Ligatures were placed around the catheter and thermistor, and the incision was closed. The iliac artery was cannulized with a 4 cm PE50 (i.d. 0.58 mm, o.d. 0.66 mm) tubing to an intraarterial distance of 1.5 cm placing it in the abdominal aorta. It was secured with a ligature and connected to the PE90 tube coming out subcutaneously from the dorsal neck, and the incision was sutured. The two PE90 catheters and thermistor exiting the dorsal neck were sutured with closing of the dorsal incision. The catheters were filled with 0.9% saline containing heparin at 12.5 U/ml and the ends plugged with metal wire. The thermistor was connected to a thermodilution cardiac output computer (Edwards 9520) and the calculated curve by the computer was depicted in a polygraph (Grass Model 7). The iliac artery catheter was connected in a PE90-PE50-PE90 interphase to a strain gauge transducer (P23XL Viggo-Spectramed) for mean blood pressure and heart rate measurements. The venous catheter was used for injections of 200 #l (500 #1 Hamilton syringe) of 0.9% saline at 22-25°C for cardiac output measurements. Measurements were recorded in triplicate and the mean values were taken for each time point: the coefficient of variation of sequential determination of cardiac index was 6.9%. The catheter was flushed with 0.9% saline containing 12.5 U heparin/ml between triplicate measurements. The catheters and the thermistor were protected by wrapping the rat's neck, chest, and back with surgical tape, in such a way that there was no interference to respiration. The rats were unrestrained and the four extremities free for movement. Blood temperature was monitored by the thermistor probe and maintained between 36-38°C, with a heat lamp of 60 watts. The surgical preparation time was about 1 h. The initial hemodynamic measurements were made while the rats were still under pentobarbital anesthesia. They were then moved to a regular unrestricted cage of 61 X 46 × 41 cm to regain consciousness, which took approximately 3 h. Although there would still be residual effects from the incomplete elimination (half-life 15-48 h) of barbiturates from the animal, and stress of the surgical procedures,
PEGUERO-RIVERA AND CORDER the rats were awake, walked, and had withdrawal reflexes to confrontation. Awake (acute recovery) measurements were then made with the rats unrestrained. These procedures, with modifications, were previously reported (21,24,25,30,32). The following hemodynamic measurements were made and/ or calculated: heart rate (bpm) (HR): number of arterial pulsations in six seconds at 25 mm/s paper speed times ten; mean blood pressure (mmHg) (MBP): an electronic mean of t/2 amplitude of the high frequency response at 0. l (Operational Manual, Grass Model 7 Polygraph); cardiac output (ml/min) (CO): automatic calculation dial (Edwards 9520 Computer) of the time integral of the dilution curve; also depicted graphically by the Grass polygraph for qualitative inspection; cardiac index (ml/ min/100 g) (CI): [cardiac output (ml/min) × 1000] + [body weight (kg/20) × 25] (25 is the factor of conversion of the injectate volume to the ideal volume 5 cc/0.02 cc at cardiac constant of 0.306 and temperature 22-25°C); stroke volume index (#lfloeat/100 g) (SVI): (CI × 1000) + HR; and peripheral vascular resistance (mmHg/ml/min/100 g) (PVR): MBP/CI. At the end of the study, animals were heparinized with heparin IV 150 U/kg, then sacrificed with sodium pentobarbital 100 mg/kg, IV. The catheters and thermistor were removed after their placement was confirmed. The heart was excised, rinsed with saline, and blotted. The wet heart was immediately weighed. All the data was analyzed using the Student t-test (Table 1) or analysis of variance for multiple comparisons (Table 2). The null hypothesis was rejected at p < 0.05, unless otherwise stated. RESULTS Results of the chronic subcutaneous administration of salmon CT are shown in Table t. The body weights increased to 547 g in control, and heart to body weight ratio was 2.52 × 10-31 The CT-treated rats had a significantly decreased heart to body weight ratio, suggesting a relatively decreased heart size, even though the heart weights were not different. The control HR was about 404 bpm and MBP was 113 mmHg. This was accompanied by a CI of 30.6 ml/min/100 g and SVI of 75.2 #lfoeat/100 g. The PVR was calculated at 3.81 mmHg/ml/min/100 g. These were the values on the control rats approximately 30 min after the end of surgery and still under sodium pentobarbital anesthesia at 40 mg/kg of body weight with a supplemental dose of l0 rag/ kg for maintenance. Shown in the anesthetized column are the corresponding values for the CT-treated rats, The results in this parallel treatment group indicated that CT rats, just after the surgical procedure, had the same HR as in controls, but the MBP may have been slightly elevated at 123 mmHg (17 = 0.09). Other parameters (CI, PVR, and SVI) were no different from the parallel control. In the awake columns (Table 1), the measurements are shown approximately 3 h after the animals awoke. These animals were unrestrained in their cage with no previous conditioning. There was no difference between control and CT treatment on HR, MBP, CI, and SVI. However, there was an insignificant elevation of PVR in the CT-treated group, 3.96 vs. 3.58 mmHg/ml/min/ 100 g (p = 0.06). Therefore, in the group chronically treated with salmon CT, there is some evidence of increase in the PVR and possible increase in MBP, but in neither case was the change significant. Pentobarbital anesthesia decreased CI and SVI by 12% and 7%, respectively, in the control animals (anesthetized versus awake columns, Table l). The HR was diminished about 5% by pentobarbital in both control and CT rats, but was only significant in control rats. This was contrary to the expected va-
CALCITONIN AND CARDIAC OUTPUT
573
TABLE 1 EFFECT OF CHRONICALLY INJECTED SALMON CALCITONIN ON HEMODYNAMICS IN THE RAT Anesthetized Measurement
Control [9l
Heart weight (g) Body weight (g) Heart/Body Weight × 10-3 HR (bpm) MBP (mmHg) CI (ml/min/100 g) PVR (mmHg/ml/min/100 g) SVI (#l/beat/100 g)
1.37 547 2.52 404 113 30.6 3.81 75.2
___0.05 + 24 + 0.09 + 7 +__3 ___2.1 ___0.26 +__4.7
Awake
Calcitonin [7]
Control [9]
Calcitonin [7]
1.40 + 0.06 577 ___23 2.44 +__0.12" 392 + 17 123 --- 6:~ 30.0 + 2.4 4.21 + 0.28 77.4 + 6.2
---423 + 10t 122 _+ 6 34.8 + 2.3f 3.58 + 0.22 81.2 + 4.9I"
--409 + 15 126 +__5 31.9 + 1.1 3.96 + 0.09§ 77.4 + 1.9
Number of subjects in brackets. Values are mean +- standard error. CT treatment was for 150 days at 2 U salmon calcitonin/kg weight SC. Anesthesia was with pentobarbital 40 mg/kg + 10 mg/kg. Anesthesia observations were 30 min after the surgery was completed. Awake observations about 3 h later with animals conscious. Heart rate (HR), mean blood pressure (MBP), cardiac index (CI), peripheral vascular resistance (PVR), stroke volume index (SVI). *t*§ Control vs. calcitonin within anesthetic state (i.e., anesthetized or awake): * p < 0.01, § p < 0.066, ~tp < 0.09. Anesthetized vs. awake within group (i.e., control or calcitonin): 'f p < 0.05.
golytic effect o f pentobarbital. Therefore, the effect o f pentobarbital anesthesia is m o r e suppressive o n h e m o d y n a m i c s in Cq" rats t h a n in control rats; i.e., the control rats recover CI a n d SVI at a b o u t 3 h postanesthesia, whereas CT-treated rats r e m a i n suppressed. T h e effect of acutely a d m i n i s t e r e d s a l m o n C T is s h o w n in Table 2. In this case, there were control rats t h a t h a d never received C T a n d were awake approximately 4 h after surgery. Calcitonin at 4 U / k g was given at 0 t i m e a n d t h e n 5 a n d 15 m i n u t e s values were recorded; C T 8 U / k g was repeated d u r i n g this total o f 3 5 - m i n observation period. A parallel control group o f rats received saline injections. T h e r e were n o statistical differences between CT- a n d saline-treated groups with respect to HR, MBP, CI, PVR, a n d SVI. In s u m m a r y , the effects o f C T a d m i n i s t e r e d chronically are evident, b u t it depends u p o n w h e t h e r o n e is looking at the effect u n d e r pentobarbital anesthesia or in the postsurgical awake animal.
DISCUSSION The aortic thermodilution m e t h o d used in this study is similar to that which has b e e n utilized a n d p r o v e n reliable for meas u r e m e n t o f CI in conscious a n d anesthetized rats (21,24,25,30). T h e CI values range from 21 to 58 m l / m i n / 1 0 0 g b.wt. depending o n strain, grade, a n d type o f stress, a n d anesthetic t e c h n i q u e (32). For the Wistar strain 40 (30), 28 (24), a n d 36 (39) m l / m i n / 100 g values have been reported a n d are similar to o u r values o f 35. T h e accuracy a n d reproducibility o f this m e t h o d to detect h e m o d y n a m i c changes o n pharmacological or physiological challenges in the rat model have m a d e it reliable (35,39). T h e h e m o d y n a m i c effects o f s o d i u m p e n t o b a r b i t a l n o t e d in this study are similar to previous results. Pentobarbital was administered IP at 50 m g / k g (23) or IV at 30 m g / k g a n d meas u r e m e n t s were d o n e 2 days after t h o r a c o t o m y (22); IP 40 m g / kg with m a i n t e n a n c e o f IP 20 m g / k g followed by c o n t i n u o u s IV infusion of 6 m g / k g / h (35); a n d in this study 40 m g / k g IV with m a i n t e n a n c e o f 10 mg/kg. T h e M B P was reported as u n c h a n g e d
TABLE 2 EFFECT OF ACUTELY INJECTED SALMON CALCITONIN ON HEMODYNAMICS IN THE AWAKE RAT Group
Time (min)
HR (bpm)
MBP (mmHg)
CI (ml/min/lO0 g)
PVR (mmHg/ml/min/lO0 g)
SVI (#lfoeat/lO0 g)
Control (n = 4)
0 5 15 20 35
418 436 431 440 435
+ 14 + 20 + 17 + 12 +__11
114 116 120 119 123
+ 3 + 2 + 3 + 1 ___3
34.8 35.5 36.0 36.0 39.0
+__1.5 + 1.2 + 2.4 + 1.9 ___1.7
3.32 3.28 3.39 3.33 3.15
___0.19 ___0.13 ___0.30 ___0.19 +__0.10
83.4 84.5 84.0 81.8 86.0
_+ 4.9 + 5.2 + 8.1 _ 6.2 ___4.9
Calcitonin (n = 4)
0 5 15 20 35
428 435 435 430 430
+ 21 ___ 18 +__21 ___ 16 + 18
122 124 125 126 130
+ 11 +__12 ___12 + 12 + 13
35.5 34.3 36.8 37.8 40.0
+ 4.8 _+ 5.3 _+ 5.2 + 2.8 ___3.4
3.52 3.69 3.45 3.06 3.24
___0.25 ___0.20 + 0.15 ___0.28 + 0.10
82.5 78.0 83.3 87.3 92.0
+_ 8.9 + 9.8 + 8.8 +__4.2 ___4.7
Values are mean _+ standard error. Control was normal saline given IV at 0 and after 15 min. CT was injected IV at 4 U/kg at 0 min and 8 U/kg after 15 rain observation. Time 0 is 4 h after surgery or 3 h awake after pentobarbital anesthesia. The average body weight was 561 + 39 g (control) and 550 -+ 32 g (calcitonin).
574
PEGUERO-RIVERA AND CORDER
(23), decreased (22), or increased (33), and ours was decreased. The PVR increased in previous studies (22,23,35), and in our study also, but not significantly. The HR was unchanged (22,23) or increased (35), in contrast to our observed decreases. The CI and SVI were decreased in all the studies, including our study. Therefore, the effects that are more consistently seen are the diminution of CI, SVI, and incrementation of the PVR. In all these studies the measurements were done 30-40 min after or during anesthesia. Comparisons were between groups (23,35), same group (22), fasting (33), or not (33,35), and endotracheal intubation (38); our rats were not fasted and the comparisons were in the same group (3-h awake state). Pentobarbital anesthesia was judged acceptable by the authors for the following reasons: its convenience in administration, the consistent reproducibility of the effects of CI and SVI, and the predictable recovery trends. In order to avoid other pharmacological hemodynamic effects, we did not add any other drugs. The hemodynamic effects of CT on HR and MBP may be compared to previously reported results. In the chronic CT group, either awake or in the postsurgical anesthesia state, the SVI, CI, and HR did not change from the controls. There are no previous reports of the effect of CT on SVI and CI. The HR and MBP were reported unchanged (26,36,41). However, MBP and PVR underwent a small change in the chronic CT group, depending on the awake (PVR) or postsurgical anesthesia state (MBP). These results are suggestive of some mild blood pressure regulatory properties of salmon CT on long-term administration. Elevated CT plasma levels have been noted in the spontaneous
hypertensive rat (7). A significant lack of recovery of the pentobarbital suppressive effect on CI and SVI in the chronic CTtreated rats was evident. It has been suggested that an appreciable part of the diminution in CI on pentobarbital anesthesia is induced by inhibition of the tonic adrenomedullary secretion (22), and by interference in the myocardial calcium metabolism (33). The relationship of this pentobarbital effect with CT effect seen in the present study is unknown. One possible cause of the lack of recovery in pentobarbital anesthesia in CT rats could be that the postoperative nociceptive stimuli were altered by the CT analgesic effect (9,16,17). There are no previously reported studies on the effect of CT in rats on CI, SVI, and PVR, as measured by thermodilution catheters. The present results suggest minimal effects of CT on the nonfailing heart, but did not address any effects on a failing heart. The literature reports evidence of positive, negative, and no inotropic effects o f C T ( 10,13,20,27). In dog left atrium there is a negative CT inotropic effect, but no effect in the papillary muscle (10). Studies do report that exposure of vascular tissues to CT results in stimulation of adenyl cyclase, an enzyme involved in generating cyclic AMP (5,8,10,20,27,37), which was accompanied by alteration of myocardial cell contraction (4-6,31,37). Norepinephrinestimulated calcium influx in aorta was stimulated by CT (36). Because of the central role of calcium and cyclic A M P in cardiac function (29), it is conceivable that CT may have significant hemodynamic effects on the failing heart that were not evident in the normal heart.
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36.
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