Although induced hypotension can reduce blood loss and improve surgical conditions, it occasionally causes depression of splanchnic organ blood flow with inadequate perfusion of the vital organs as a severe complication [1,2]. Calcitonin gene-related peptide (CGRP), an endogenous peptide involved in several biological processes, induces hypotension by vasodilatation . Despite these properties, calcitonin gene-related peptide has not yet been accepted as a vasodilating agent for clinical use because it augments sympathetic nervous activity and increases plasma catecholamine concentrations [3,4]. Hypotension and/or increased sympathetic activity cause a redistribution of blood flow to the vital organs and alter organ perfusion. Numerous studies have reported an increase in the splanchnic blood flow during administration of calcitonin gene-related peptide [5-7]. However, some studies have reported either no change  or a decrease  in splanchnic organ circulation during hypotension induced by this peptide. As the influence of hypotension, induced by the calcitonin gene-related peptide, on splanchnic organ blood flow is variable, its safety needs to be clarified. The present study was designed to determine the differences in the effects of various degrees of hypotension induced by calcitonin gene-related peptide. We examined the systemic haemodynamics and splanchnic organ blood-flow responses in dogs anaesthetized with isoflurane during hypotension induced with either calcitonin gene-related peptide or sodium nitroprusside (SNP), a commonly used vasodilating agent.
Our animal Experimental Committee approved the study. Thirty healthy adult mongrel dogs of either gender (18.7 ± 0.5 kg, mean ± SEM) were anaesthetized with sodium pentobarbital (25 mg kg−1 intravenously (i.v.)). After tracheal intubation, the lungs were mechanically ventilated with a volume-cycled animal ventilator (Harvard Respirator®; South Natrick, MA, USA) to maintain normocapnia. Anaesthesia was maintained with 1.3% isoflurane in oxygen via an Ohmeda Vaporizer® (BOC Health Care, Windlesham, UK). End-tidal isoflurane and CO2 concentrations were measured continuously, but ventilation was not adjusted to keep end-tidal CO2 constant (Capnomac Ultima®; Datex, Helsinki, Finland).
Catheters were placed in the left femoral artery for continuous systemic arterial pressure monitoring and blood sampling, and in the right femoral vein for drug administration. Normal saline (NaCl 0.9%) was infused at a rate of 7 mL kg−1 h−1. A 7-F flow-directed pulmonary artery catheter (Swan-Ganz thermodilution catheter®; Baxter Healthcare, Irvine, CA, USA) was advanced into the pulmonary artery via the right external jugular vein for the measurement of right atrial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure and cardiac output. Cardiac output was measured in triplicate by thermodilution using 5 mL cold temperature-monitored normal saline. The results were recorded with a computerized system (MTC6210®; Nihon Kohden, Tokyo, Japan). Heart rate was continuously monitored by calculation, using a cardiotachometer (AT601G®; Nihon Kohden), from lead II of the electrocardiogram. A 7-F pig-tailed catheter (Schneider, Minneapolis, MN, USA) was passed into the left ventricle via the right femoral artery and measured the left ventricular pressure. The left ventricular maximum rate of pressure change (LV dP/dtmax) was derived electrically from the left ventricular pressure wave signal with an electronic differentiator (EQ601G®; Nihon Kohden). Each pressure-monitoring catheter was connected to a pressure transducer (Uniflow, Baxter Healthcare). The animals were placed in the supine position during the measurements and the zero reference was levelled at the mid-point of the chest. Mean arterial pressure and mean pulmonary artery pressure were determined electronically. Arterial blood samples were drawn anaerobically from the femoral catheter and analysed for pHa, PaCO2, PaO2 and base excess using a blood-gas analyser (Radiometer ABL505®; Copenhagen, Denmark).
Laparotomy was performed through a midline incision and the left kidney, liver and pancreas were carefully isolated. Platinum electrodes (Standard needle type 100 μm diameter, UHE-100®; Unique Medical, Tokyo, Japan) were placed in the cortex of the left kidney, the left lobe of the liver and the body of the pancreas, respectively. These platinum electrodes were introduced to a depth of 3-6 mm from the surface of the organs. Three silver-silver chloride reference electrodes (Plate type UHE-001®; Unique Medical) were placed subcutaneously in the animal's flank close to the kidney, liver and pancreas, respectively. The abdomen was closed when these procedures had been completed. The platinum electrodes were connected to hydrogen detection systems (Digital UH-meter MHG-D1®) and recorders (Desk recorder U-288®; both Unique Medical). Splanchnic organ blood flow was measured by the hydrogen clearance method as previously described [8,9].
Hydrogen gas, at approximately 10%, was added to the inspired gas for 1 min until near saturation in the tissues when this inhalation was terminated and the washout curve of hydrogen was recorded through an appropriate amplifier unit (MHG-D1U®; Unique Medical). In the current study, the digital UH-meter measured both mono- and biexponentials with a proprietary computer program incorporated in the UH-meter and excluded recirculation effects.
The 30 dogs were divided into three groups (n = 10 each). Human calcitonin gene-related peptide (∝) was used for the study (donated by Asahi Chemical Industry, Japan). Dogs in the CGRP60 and CGRP50 groups received an infusion of 0.001% calcitonin gene-related peptide (calcitonin gene-related peptide in 0.1% bovine serum albumin in normal saline). Dogs in the sodium nitroprusside group received an infusion of 0.02% sodium nitroprusside (sodium nitroprusside in normal saline, which was protected from photodegradation). After at least 60 min of haemodynamic stabilization, the baselines were measured. The infusions of calcitonin gene-related peptide and sodium nitroprusside were increased in a step-wise fashion until the desired mean arterial pressure was attained. Mean arterial pressure was reduced to 60 mmHg in the CGRP60 and sodium nitroprusside groups, and to 50 mmHg in the CGRP50 group. All variables were measured every 30 min during and after the periods of induced hypotension. Additional haemodynamic variables were measured 10 min after termination of the study drugs.
Data were expressed as mean ± SEM. Inter- and intragroup comparisons of haemodynamic variables and splanchnic organ blood flows were analysed by ANOVA with Dunnett's test. P < 0.05 was considered as significant.
A mean arterial pressure of 60 mmHg was achieved within 4.7 ± 0.9 min in the CGRP60 group and after 8.8 ± 1.4 min in the sodium nitroprusside group. A mean arterial pressure of 50 mmHg was achieved within 8.2 ± 0.8 min in the CGRP50 group. The dose of calcitonin gene-related peptide and sodium nitroprusside required to maintain hypotension was 155 ± 41 ng kg−1 min−1 in the CGRP60 group, 412 ± 89 ng kg−1 min−1 in the CGRP50 group, and 8.1 ± 1.1 μg kg−1 min−1 in the sodium nitroprusside group, respectively.
The time-course of haemodynamic variables is shown in Tables 1-3. Mean arterial pressure in all three groups decreased throughout the periods of observation. After termination of the study drugs, mean arterial pressure in the CGRP60 group was higher than in the CGRP50 group, but lower than in the sodium nitroprusside group. The heart rate remained unchanged during the hypotensive period in all three groups. The cardiac index in the CGRP60 group increased and in the sodium nitroprusside group it remained unchanged, whereas in the CGRP50 group it decreased during hypotension. Mean pulmonary artery and pulmonary capillary wedge pressures remained unchanged in the CGRP60 group, whereas they decreased during the hypotensive period in the CGRP50 and sodium nitroprusside groups. Systemic vascular resistance was reduced in all three groups throughout the observation. After the study drugs were terminated, SVR was higher in the sodium nitroprusside group than in the CGRP60 group. Left ventricular dP/dtmax remained unchanged in the CGRP60 group but it was reduced in the CGRP50 and sodium nitroprusside groups during the hypotensive period.
The time-course of splanchnic organ blood flow is shown in Table 4. Renal blood flow was maintained fairly well in the CGRP60 group during the hypotensive period, but it was reduced in the CGRP50 and sodium nitroprusside groups throughout the periods of observation. Hepatic blood flow decreased in all three groups during the hypotensive period, but in the CGRP50 group it had not returned to baseline values after 30 min. The reduction in hepatic blood flow in both calcitonin gene-related peptide groups was associated with the dose-dependent decrease in mean arterial pressure. Pancreatic blood flow did not change significantly in the CGRP60 group, whereas it was reduced throughout the observation in the CGRP50 and sodium nitroprusside groups.
The time-course of arterial blood-gas status is shown in Table 5. PaCO2 increased in the CGRP60 group, whereas it remained unchanged during the hypotensive period in the CGRP50 and sodium nitroprusside groups. Base excess decreased in all three groups throughout the period of observation; however, the acidosis was less severe in the sodium nitroprusside group than in the CGRP60 group.
The findings showed that hypotension induced by calcitonin gene-related peptide or sodium nitroprusside is associated with a reduction of systemic vascular resistance and with varying changes in cardiac index and stroke volume index. The differences between the CGRP60, CGRP50 and sodium nitroprusside groups in the effects on stroke volume index may reflect the dilatation of peripheral and pulmonary vessels caused by the larger dose of calcitonin gene-related peptide or sodium nitroprusside. Further, the observed change in splanchnic organ blood flow during induced hypotension in both calcitonin gene-related peptide groups showed a dose-dependent reduction corresponding to the decrease in mean arterial pressure.
We found that the changes in mean pulmonary artery and pulmonary capillary wedge pressures during hypotension with calcitonin gene-related peptide were related to dosage. Thus, it appears that the smaller dose of calcitonin gene-related peptide causes dilatation of the resistance vessels, with a minimal effect on the capacitance vessels, with consequent increase of venous return. In addition, calcitonin gene-related peptide receptors are more numerous around arteries than around veins . The pulmonary vascular beds appear to be sensitive to the larger dose of calcitonin gene-related peptide, which dilated the pulmonary vessels and affected cardiac ventricular-filling pressures. A lower dose of calcitonin gene-related peptide did not seem to induce the same responses of the pulmonary vasculature, although it was a potent vasodilator of pulmonary arteries and veins that were correlated to the distribution of the calcitonin gene-related peptide receptors in the pulmonary vasculature . Higher plasma calcitonin gene-related peptide concentrations - at the larger dose of calcitonin gene-related peptide in the CGRP50 group - are probably required to bind to the calcitonin gene-related peptide receptors in the pulmonary venous beds where calcitonin gene-related peptide-containing nerve fibres have been demonstrated by immunochemical techniques . The CGRP50 group had a lower cardiac index than the CGRP60 group. This was attributed to the reduced stroke volume index because there were no changes in heart rate. The differences in the response of the stroke volume index to hypotension between the groups may be explained mainly by the dissimilar changes in cardiac ventricular-filling pressures. Mechanisms responsible for the reduction in stroke volume index at the larger dose (in the CGRP50 group) may result from pulmonary vasodilatation or venodilatation. Sodium nitroprusside is known to dilate the arterial and venous beds and cause a balanced pre- and afterload reduction, resulting in hypotension with an unchanged cardiac index .
The reductions of left ventricular dP/dtmax in the CGRP60 group, associated with the decreases in mean arterial pressure and left ventricular end-diastolic pressure, were much smaller than in the other groups. However, it is unlikely that the reductions of LV dP/dtmax could be secondary to the reduction in cardiac contractility, because a positive inotropic effect of calcitonin gene-related peptide has been uniformly reported in earlier studies [4,14]. Therefore, other mechanisms may be responsible for the observed reductions in LV dP/dtmax in the CGRP50 and sodium nitroprusside groups.
The renal vascular bed appears to be sensitive to calcitonin gene-related peptide since renal blood flow is maintained during low-dose calcitonin gene-related peptide-induced hypotension. Calcitonin gene-related peptide has been shown to decrease renal vascular resistance and to increase renal blood flow . The findings in the present and previous studies [8,9] indicate that calcitonin gene-related peptide has a direct vasodilating activity on the renal vasculature during hypotension when the peptide is infused at a rate that decreases mean arterial pressure to 60 mmHg. In contrast, high-dose calcitonin gene-related peptide-induced hypotension caused a reduction in renal blood flow, most likely due to the decreases in mean arterial pressure and cardiac index. Likewise, hypotension induced by sodium nitroprusside reduced renal blood flow in this study because the drug has only a weak vasodilating effect on the renal vasculature [16,17]. Because hypotension within the autoregulatory pressure range of renal vasculature has been shown to result in redistribution of blood flow from the outer cortical layers to the juxtamedullary layers , mechanisms responsible for the reduction of renal blood flow may cause a maldistribution within the autoregulation process.
It is thought that calcitonin gene-related peptide is a potent vasodilator in the splanchnic circulation [6,7]. However, the increase in blood flow due to vasodilatation of the hepatic vascular beds during hypotension induced by the calcitonin gene-related peptide is less appreciated because previous studies have reported either no change  or a reduction  of hepatic blood flow. Hepatic blood flow was unaffected by any differences between the high- and the low-dose calcitonin gene-related peptide groups. The reasons for the observed reduction in hepatic blood flow reported in the calcitonin gene-related peptide groups remain speculative, but most likely it is attributable to redistribution to other dilated vascular beds in the vital organs when the perfusion pressure decreases. These findings suggest that the various degrees of hypotension induced by the calcitonin gene-related peptide may impair the maintenance of splanchnic organ blood flow during isoflurane anaesthesia. Studies investigating the hepatic circulation during sodium nitroprusside-induced hypotension found it to be increased [17,19], decreased [20,21] or unchanged . The findings in the present study suggest that the liver may be at risk of hypoperfusion, but not of ischaemic damage.
In conclusion, the results show that the lower dose of calcitonin gene-related peptide acted as an arteriolar vasodilator while a higher dose dilated both the arteriolar and venous systems. In addition, the hypotension induced by the calcitonin gene-related peptide, with regard to the splanchnic vascular beds, provided a smaller margin of safety against profound hypotension.
The authors thank Yoshie Hirakawa for secretarial assistance. This research was supported in part by a grant from Showa University.
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