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Calcitonin gene-related peptide causes long-term inhibition of contraction in rat thoracic aorta through a nitric oxide-dependent pathway
Neuropeptides (1999) 33 (2), 145–154 © 1999 Harcourt Brace & Co. Ltd
Calcitonin gene-related peptide causes long-term inhibition of contraction in rat thoracic aorta through a nitric oxide-dependent pathway L. F. Lu, R. R. Fiscus Department of Physiology, Faculty of Medicine The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong, China
Summary Calcitonin gene-related peptide (CGRP) is released into the circulation during pathogenesis of endotoxin and septic shock and appears to partly mediate vascular problems of shock. To explore the function of CGRP during shock, we investigated long-term action of CGRP, alone or in combination with interleukin-1β (IL-1β), another shock mediator, in isolated rings of rat thoracic aorta. CGRP or IL-1β, by themselves, caused significant long-term (3 h) depression of contraction, while the combination of CGRP and IL-1β had no synergistic effects. Dose-response curves to phenylephrine were significantly decreased and shifted to the right when aortic rings were incubated with 1 µM CGRP for 1 h followed by 2 h incubation without CGRP. Inducible nitric oxide synthase (iNOS) inhibitors, S-methylisothiourea sulfate (SMT) and NG-nitro-L-arginine (L-NNA), completely eliminated long-term depressant effect of CGRP. Our results suggest pathology of septic shock may involve long-term inhibition of vascular contraction mediated by CGRP via expression of iNOS.
INTRODUCTION Endotoxin and hemorrhagic shock in animal models and septic shock in human patients are characterized by hypotension, hyporeactivity to vasoconstrictor agents, inadequate tissue perfusion, vascular damage, and disseminated intravascular coagulation, leading to multiple organ dysfunction and death.1 Gram negative bacterial infection is recognized as a common cause of septic shock.2 Intravenous administration of endotoxin (lipopolysaccharide, a constituent of the cell wall in gram-negative bacteria) in animals and man produces a shock-like syndrome.3,4 The pathogenesis of circulatory shock has been attributed to increased circulating levels
Received 24 August 1998 Accepted 10 February 1999 Correspondence to: Ronald R. Fiscus, Department of Physiology, Faculty of Medicine, Chinese University of Hong King, Shatin, N, T., Hong Kong, China. Tel: 852 2609 6780; Fax: 852 2603 5022; E-mail: [email protected]
of many vasoactive substances, including angiotensin II,5 catecholamines,5 prostaglandins,6 platelet activating factor,7,8 thromboxane A29,10 and endothelin.11 Interleukin-1 (IL-1) and tumor necrosis factor (TNF)12–14 are also described as mediators of septic shock. Research from our laboratory has found that plasma levels of two neuropeptides, calcitonin-gene related peptide (CGRP) and neuropeptide Y (NPY), are elevated during endotoxin shock in animal models.15,16 We have also shown CGRP levels are elevated in the plasma of human patients with septic shock caused by either gram-negative or gram-positive bacteremia.17 Based on these data and the fact that CGRP is a potent hypotensive agent, we have proposed that CGRP serves as one of the important mediators of the vascular problems in septic shock. Plasma levels of CGRP are increased as much as 22-fold after endotoxin is applied in a rat model of shock.15 This elevation of CGRP released during endotoxin shock can be sustained for at least 3 h.15 Several endogenous factors, like prostaglandins,18 lactic acid19 and nitric oxide (NO),20 which are all elevated in 145
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endotoxin shock, are believed to mediate (or enhance) the release of CGRP during shock. A likely source of the elevated CGRP in shock is the many CGRP-containing nerves located in the adventitial and medial layers of most blood vessels.21 The importance of this CGRP release in shock has been further emphasized by experiments showing that approximately 50 percent of the hypotension in endotoxin shock can be attributed to CGRP, by using a specific CGRP receptor antagonist.22 CGRP is a potent vasodilator, using either endothelium/NO-dependent or endothelium/NO-independent mechanisms of vasorelaxation.23 In the case of endothelium/NO-dependent vasorelaxation, CGRP activates a dual signal transduction mechanism involving both adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) elevations in vascular smooth muscle cells.23,24 However, whether CGRP has other effects, such as long-term actions on blood vessels, is not clear. Interleukin-1β (IL-1β) is one of the cytokines released from macrophages during circulatory shock.12 Libby and colleagues in 1986 showed that vascular smooth muscle cells exposed to very low levels of endotoxin can also express the IL-1 gene and release IL-1 into the media.25 IL-1 can produce a shock-like syndrome, characterized by hypotension and decreased vascular resistance, when administrated to animals, suggesting that it may be one of the key mediators of septic shock.26 An IL-1 receptor antagonist has been reported to reduce the mortality associated with endotoxin shock.12 IL-1-treated rat aortic rings exhibit a diminished vascular contractile response to phenylephrine and potassium chloride.12 The IL-1induced inhibition of contraction is believed to be mediated by induced synthesis of NO, one agent that is pathologically over-produced and causes hypotension during septic shock.27 Furthermore, in cultured vascular smooth muscle cells, IL-1β has been found to induce NO production and cause elevations of cGMP levels.28 A large amount of NO is produced by inducible nitric oxide synthase (iNOS), an enzyme different from the constitutive NO synthase which plays a role in the physiological regulation of blood flow and blood pressure. The over-production of NO in vascular smooth muscle can partly explain the hypotension and the vascular hyporeactivity during circulatory shock. Endotoxin, IL-1, tumor necrosis factor α (TNF α) and interferon γ (IFN γ) can induce the expression of iNOS in vascular smooth muscle cells.29 Many other factors, including cAMP-or cGMPelevating agents can augment the induction of iNOS.29,30 Schini-Kerth and colleagues have reported that CGRP (1 µM) enhances the induction of iNOS caused by IL-1β in rat aortic smooth muscle cells grown in culture, and have suggested that this interaction of CGRP and IL-1β may be an important part of the pathology of septic Neuropeptides (1999) 33(2), 145–154
shock.31 However, they found that CGRP, by itself, was not able to significantly induce iNOS or increase the production of NO in these cultured vascular smooth muscle cells.31 Because the smooth muscle cells of intact blood vessels may react differently from cultured vascular smooth muscle cells, it is still not clear what effects CGRP, either alone or in combination with IL-1β, will have on the induction of iNOS and the long-term regulation of contractility of blood vessels. In the present study, we have used freshly-isolated aortic rings (with and without endothelium) as an experimental model to investigate the actions of CGRP, alone and in combination with IL-1β, on the contractility and induction of iNOS in arteries over a 3 h period. NG-nitroL-arginine (L-NNA), a general NOS inhibitor, and S- methylisothiourea sulfate (SMT), a more selective iNOS inhibitor, were used to determine the involvement of iNOS in these responses. The data show that both CGRP and IL-1β, by themselves, can cause significant long-term depression of contraction in aortic rings. The long-term (3 h) depression of contraction of either CGRP or IL-1β was completely reversed by the addition of L-NNA, suggesting involvement of NO production. Furthermore, the CGRP-induced long-term depression of contraction was completely reversed by SMT, suggesting involvement of iNOS induction. No synergistic actions of CGRP and IL-1β were observed on inhibition of contractions. Thus, these actions of CGRP in intact rat aortic rings are clearly different from what has been found in cultured aortic smooth muscle cells.
MATERIALS AND METHODS Preparation of isolated aortic rings The treatment of the laboratory animals and the experimental protocols of the present study adhered to the guidelines of The Chinese University of Hong Kong and were approved by an Institutional Authority for Laboratory Animal Care. Healthy, male Sprague-Dawley rats (body weight = 240–280 g) were used. Heparin (1000 U/rat) was injected intraperitoneally 15 min before rats were decapitated and exsanguinated. Their thoracic aortae were removed and placed in modified KrebsRinger-Bicarbonate (KRB) solution containing 118.5 mM NaCl, 4.74 mM KCl, 1.18 mM MgSO4, 1.18 mM KH2PO4, 2.5 mM CaCl2, 24.9 mM NaHCO3, 10 mM glucose, and 0.03 mM EDTA, and aerated with 95% O2 plus 5% CO2, as in previous experiments.24 After removing the adhering fat and connective tissue, the aortae were cut transversely into rings 4 mm long. The two rings on the outside ends of the thoracic aortae were discarded because of potential damage during handling. Usually, four to five usable rings were obtained from one thoracic aorta. The © 1999 Harcourt Brace & Co. Ltd
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endothelium was removed in some of the aortic rings by gently rubbing the intimal surface with stainless steel microforceps. The integrity or removal of the endothelium was checked by observing the vasorelaxant response to acetylcholine, described below. Measurement of Contractile and Relaxant Response in Aortic Rings The rings were suspended in organ baths containing 5 ml of KRB bubbled with 95% O2 plus 5% CO2 at 37°C using two stainless steel rods (0.275 mm diameter) and were set under a resting tension of 1 g. The KRB was replaced with fresh solution every 10 min during a 30 min equilibration period. Contractile and relaxant responses were measured isometrically using force transducers (Grass Model FT-03) and recorded on a physiological recorder (Grass Polygraph, Model 7). To test whether the endothelium was intact, each ring was stably contracted with phenylephrine (100 nM) and then exposed to acetylcholine (ACh, 100 nM). Only those rings with larger than 50% relaxant responses were considered with intact endothelium. To check whether the endothelium was successfully removed by the rubbing procedure described above, each ring was contracted with 20 nM phenylephrine until a stable contraction was obtained and then 100 nM ACh was added. Only those rings that had no response to ACh were considered without functional endothelium. To test the effects of CGRP or IL-1β, we compared the difference in the dose-response relationship of phenylephrine in the aortic rings before and after incubation with these two agents. Before measuring the dose-response relationship of phenylephrine, the aortic rings were first exposed to 1 µM phenylephrine and washed out two times in order to obtain a stable response to phenylephrine in the following steps. After measuring the dose-response relationship of phenylephrine at the beginning followed by wash out with KRB solution until the contraction of the rings returned to the baseline, the aortic rings were incubated with CGRP, IL-1β or CGRP plus IL-1β for 1 h. Then these agents were washed out and the aortic rings were incubated with pure KRB solution for an additional two hours during which time the KRB solution was replaced every 20 min. The final concentration-response determination was performed 3 h after the agents were first administered. Contractions caused by phenylephrine were tested by increasing the concentration in organ chambers in cumulative half-log increments after a steady-state response was reached to each increment. To test the involvement of NOS, NG-nitro-L-arginine (L-NNA) or S-methylisothiourea sulfate (SMT) was added before the beginning of the final dose response determination. © 1999 Harcourt Brace & Co. Ltd
Chemicals and drugs Rats were supplied by a colony of Sprague-Dawley rats from the Laboratory Animal Service Center, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. Synthetic rat CGRP (α-CGRP) was purchased from Bachem Inc. (Torrance, CA, USA) and Sigma Chemical Company (St. Louis, MO, USA). IL-1β (Human, Recombinant) and SMT were purchased from Calbiochem-Novabiochem Corporation (San Diego, CA, USA). L-NNA, L-phenylephrine (hydrochloride), acetylcholine (Chloride), and heparin (sodium salt) were purchased from Sigma Chemical Company. Statistical analysis The data were analyzed using one-way ANOVA and further analyzed using the Student-Newman-Keuls (S-NK) test for multiple comparisons between treatment groups. A P value of <0.05 was used to indicate significant difference between treatment group means. The data are presented as mean values ± the standard error of the mean (SEM). The ‘n’ values given in the figure legends represent the number of individual rats used for providing the aortic rings in each treatment group. RESULTS Effects of interleukin-1β on NOS induction in rings of rat thoracic aorta The effects of IL-1β in rat aortic rings were observed as a positive control to show the inhibition of contractility through induction of iNOS expression. IL-1β was able to cause significant depression of phenylephrine-induced contractions in rat aortic rings both with and without endothelium at a time point as early as 3 h (Figs 1A & B). To compare the effect of IL-1β in these two groups (i.e. with and without endothelium), the magnitude of inhibition was compared at the concentrations of phenylephrine for each group that elicited a similar level of contraction in the controls, which were 50–70% of the maximum contraction to phenylephrine. These concentrations were 10–7 M in rings with endothelium and 3×10–8M in rings without endothelium. In rat aortic rings with endothelium, 10 ng/ml IL-1β inhibited the contraction to 10–7M phenylephrine from a control of 51.0±4.9% to 2.5±0.5% of the maximum contraction (n=5). The same concentration of IL-1β caused inhibition of contraction to 3×10–8 M phenylephrine from a control of 54.9±5.3% to 3.7±1.7% in aortic rings without endothelium (n=5). The changes of maximum contractions to phenylephrine were 91.3±3.9% to 22.6±9.7% and 87.7±3.4% to 44.2±3.6% in aortic rings with and without endothelium, respectively. Neuropeptides (1999) 33(2), 145–154
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Fig. 1 Effect of IL-1β on phenylephrine-induced contractions in rat thoracic aorta with (A, n=5) or without endothelium (B, n=5). The rat aortic rings were incubated with control KRB or 10 ng/ml IL-1β for 1 h, followed by 2 h in KRB solution. 100 µM L-NNA were added in some of the organ baths to investigate the involvement of nitric oxide synthase. The data here show the changes of contractions in the rings treated with control (▲), IL-1β (●), L-NNA (■ ■ ) or IL-1β plus L-NNA (■).
The NOS inhibitor L-NNA reversed most of the depression of contraction caused by IL-1β. When endothelium was intact, L-NNA reversed the contraction to 10–7M phenylephrine of IL-1β treated rings up to 80.2±12.4%, which was even bigger than the time control. This is because L-NNA is an inhibitor of both constitutive NOS and inducible NOS. In aortic rings without endothelium, L-NNA reversed the IL-1β depressed contraction in response to 3×10–8M phenylephrine up to 70.1±3.4%.
n=7). In rat aortic rings without endothelium, the dose response curves of phenylephrine given by the groups treated by IL-1β itself (4 ng/ml) and IL-1β (4 ng/ml) in combination with CGRP (1 µM) were almost completely the same (Fig.3). As with aortic rings with endothelium, there was no statistically significant difference between responses to IL-1β alone or in combination with CGRP (P>0.05, n=6). Long-term effect of CGRP on rat thoracic aorta
Effects of interleukin-1 β and CGRP in combination in rat thoracic aorta Treating aortic rings with IL-1β combined with CGRP did not cause any synergistic effects on inhibition of contraction. Thus, CGRP did not appear to augment the induction of iNOS caused by IL-1β in rat aortic rings either with or without endothelium (Figs 2 & 3). In rat aortic rings with endothelium, the decrease of contraction to 10–7 M phenylephrine caused by 0.5 ng/ml IL-1β was from 53.1±4.5% to 19.2±6.2%, whereas the change of contraction to the same dose of phenylephrine caused by 0.5 ng/ml IL-1β together with 1 µM CGRP was 53.1±4.5% to 13.4±4.5% (Fig. 2B). There was no statistically significant difference between the group with IL-1β and the group with the combination of IL-1β and CGRP (P>0.05, Neuropeptides (1999) 33(2), 145–154
Exposure of aortic rings with endothelium to CGRP (1 µM) for 1 h, followed by a 2-h incubation with control KRB solution, significantly depressed the contractions to phenylephrine at all concentrations (Fig. 4) (P<0.05, n=6). These data represent a different group of rats from that in Figure 2. For example, CGRP depressed contractions to 10–7M phenylephrine from a control of 47.5±5.8% to 22.09±5.4%. The change of maximum contractions to phenylephrine was from a control of 86.5±4.0% to 70.6±7.3% in the presence of CGRP pretreatment. The EC50 value of phenylephrine was shifted about 3-fold from a control of 7.25×10–8M to 2.05×10–7M. The long-term depression of contraction caused by CGRP can also be seen in Fig.2 representing another group of rats. According to the dose response © 1999 Harcourt Brace & Co. Ltd
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Fig. 2 Effect of IL-1β in combination with CGRP on phenylephrine-induced contractions in rat aorta with endothelium. A: the changes of dose response relationship to phenylephrine after treatment with control KRB solution (▲ ▲), IL-1β 0.5 ng/ml (■ ■), CGRP 1 µM (●) or IL-1β plus CGRP (■) (n=7). B: the change of contractions to 10–7 M phenylephrine after the incubation. (*P<0.05, comparing to the control, n=7).
Fig. 3 Effect of IL-1β in combination with CGRP on phenylephrine-induced contractions in rat aorta without endothelium. A: the changes of dose response relationship to phenylephrine after treatment with control KRB solution (■), CGRP 1 µM (■ ■), IL-1β 4 ng/ml (●) or IL-1β 4 ng/ml plus CGRP 1 µM (● ●) (n=6). B: the change of contractions to 3×10–8 M phenylephrine after the incubation. (*P<0.05, comparing to the control, n=6).
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relationship of phenylephrine, the inhibition of contraction caused by 1 µM CGRP was almost the same as that induced by 0.5 ng/ml IL-1β in aortic rings with endothelium (Fig. 2). Effects of L-NNA and SMT on the long-term inhibition of contraction caused by CGRP
Fig. 4 The long-term effects of CGRP on phenylephrine-induced contractions in rat aorta with endothelium. Rat aortic rings were incubated with control KRB solution (■) or 1 µM CGRP (◆) for 1 h, followed by 2 h incubation without CGRP.
If L-NNA or SMT was added before measuring the dose response relationship of phenylephrine after 1 h exposure to CGRP and a subsequent 2 h incubation in KRB solution, the depression of contractions caused by CGRP was completely reversed (Figs 5 & 6). There was no difference between the contractions of time control and CGRP treated rings when L-NNA or SMT was added. Thus pretreatment with L-NNA or SMT completely blocked the long-term depressant effects of CGRP. DISSCUSSION In the present study, we have made two important observations. The first is that CGRP cannot synergistically
Fig. 5 Effect of L-NNA on the long-term inhibition of contraction caused by CGRP. The rat aortic rings were incubated with control KRB or 1 µM CGRP for 1 h, followed by 2 h in KRB solution. 100 µM L-NNA were added in some of the organ baths to investigate the involvement of nitric oxide synthase. A: the changes of dose response to phenylephrine in the rings treated with control (● ●), CGRP (●), L-NNA (■ ■ ) or LNNA plus CGRP (■) (n=9). B: the difference of the contractions to 10–7 M phenylephrine after the treatments (**P<0.05, comparing with the control; *P<0.05 comparing with the groups added with L-NNA).
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Fig. 6 Effect of SMT on the long-term inhibition of contraction caused by CGRP. The rat aortic rings were incubated with control KRB or 1 µM CGRP for 1 h, followed by 2 h in KRB solution. 100 µM SMT were added in some of the organ baths to investigate the involvement of nitric oxide synthase. A: the changes of dose response to phenylephrine in the rings treated with control (■), CGRP (▲), SMT (▼) or SMT plus CGRP (●) (n=9). B: the difference of the contractions to 10–7 M phenylephrine after the treatments (**P<0.05, comparing with the control; *P<0.05 comparing with the groups added with SMT).
augment the iNOS induction caused by IL-1β in intact rings of rat thoracic aorta. This result is different from what has been found in cultured vascular smooth muscle cells.31 The second observation is that CGRP by itself can cause a long-term inhibition of contraction in rat thoracic aorta. Like the immediate vasorelaxant effects of CGRP in rat aorta, this long-term effect of CGRP was found to be completely dependent on endothelium. The mechanism of this response appears to involve induction of iNOS, because the response was blocked by inhibitors of iNOS. In cultured rat aortic vascular smooth muscle cells, Schini-Kerth and colleagues have found that CGRP, in a concentration-dependent manner from 0.3 to 3 µM, enhanced the release of nitrite (a stable oxidation product of NO) caused by IL-1β. Furthermore, the formation of L-citrulline from L-arginine, an indication of NOS activity, caused by IL-1β was enhanced by CGRP (1 µM). Also the cultured vascular smooth muscle cells treated by IL-1β in combination with CGRP (1 µM) had a more marked effect on inhibition of platelet aggregation. Based on the further observation that cAMP-dependent © 1999 Harcourt Brace & Co. Ltd
vasodilators, forskolin and isoproterenol, and the activator of the cAMP-dependent protein kinase, Sp-cAMPS, also enhanced the release of nitrite and the formation of L-citrulline evoked by IL-1β, Schini-Kerth and colleagues concluded that CGRP potentiates the IL-1β induced expression of iNOS via a cAMP-dependent mechanism. However, in our experiments measuring the contraction of aortic rings using an organ bath system, we did not find any synergistic effect of IL-1β and CGRP on inhibiting the contractility of smooth muscle. A possible explanation for the difference in response between our data and the data of Schini-Kerth and colleagues is that there may be some differences between the smooth muscle cells in culture and those from fresh tissue. The characteristics of the cells may have undergone changes during culturing. For example, in the work of Sirsjo and colleagues,32 it was found that in rat smooth muscle cells in culture and in aortic strips, there were clear differences in the mechanism of induction of mRNA for iNOS. iNOS mRNA expression was weak in cultured vascular smooth muscle cells when exposed to either interferon-γ (IFNγ) or LPS, but the combination LPS + Neuropeptides (1999) 33(2), 145–154
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IFNγ enhanced the expression. In aortic strips, in contrast, LPS alone induced a pronounced expression, with no further increase by IFNγ. Furthermore, cycloheximide potentiated the expression of iNOS mRNA in vascular smooth muscles cells in culture stimulated with LPS + IFNγ, but attenuated the responses in aortic strips. It seems likely that there are differences in the intracellular signaling pathways for the induction of iNOS mRNA between mature vascular smooth muscle cells in the vessel wall and the phenotypically modulated cells grown in cultures. Thus, the difference of CGRP’s effect in cultured smooth muscle cells and in aortic rings may also be caused by these differences of the intracellular signaling pathways for the induction of iNOS mRNA in these two cases. Instead of synergistically enhancing the IL-1β-induced iNOS expression in rat thoracic aorta, we found that CGRP by itself can cause a long-term inhibition of contraction in aortic rings with endothelium. In a previous study from our laboratory,24 we had found that CGRP caused an endothelium- and EDRF(NO)- dependent vasorelaxation in rat aortic rings immediately after it was applied to the organ bath. In our present work, the rat aortic rings were incubated with CGRP for 1 h followed by 2 h incubation without CGRP. Thus, CGRP was washed out 2 h before measuring the second dose-response relationship of phenylephrine. One can argue that the long-term vasorelaxant effect of CGRP may simply represent a residual effect of the immediate vasorelaxant effect observed with exposure to CGRP. However, in our experience, the residual relaxant effect of CGRP declines gradually and disappears in about 20–30 min after CGRP is washed out. In the present study, we have shown that the depression of contraction appears at a 2 h time point after CGRP is washed out. This indicates that CGRP is causing an unexpected long-term depressant effect in aortic rings and appears to involve a mechanism that require 2 h to develop. This time frame is similar to that of the depression of contraction caused by IL-1β via induction of iNOS. Thus, we hypothesized that the CGRP-induced long-term depression of contractions may also involve iNOS induction in rat aortic rings. The results of our experiments with L-NNA, a general NOS inhibitor, and SMT, a more selective iNOS inhibitor, have supported this proposed mechanism of the long-term depressant effect of CGRP. Both inhibitors completely blocked the long-term depression of contraction caused by CGRP, suggesting involvement of iNOS induction. The concentration of CGRP around the smooth muscle cells in aorta and other arteries may reach a very high level during certain pathologies, such as endotoxin shock. We have already detected a dramatic elevation of plasma level of CGRP during endotoxin shock.15–17 On Neuropeptides (1999) 33(2), 145–154
the basis of immunocytochemistry works,21 the perivascular CGRP immunoactive nerve fibers are distributed in high density along the arteries. Some of these nerve fibers project into the medial-adventitial border, thus allowing for CGRP to be released in close proximity to the vascular smooth muscle cells of the medial layer of arteries. During the shock state, there is a large elevation of CGRP release from perivascular sensory nerves.15,16 CGRP likely accumulates to high concentrations in the area of arterial smooth muscle cells during septic shock, and this response can be sustained for at least several hours. This sustained high concentration of CGRP during shock may play an additional role besides the immediate vasodilatory effect. For example, CGRP may also contribute to long-term inhibition of contraction via the induction of iNOS that may be an important part of the pathogenesis of septic shock. The induction of iNOS by CGRP could be mediated by the elevations of cAMP and cGMP levels caused by CGRP that is known to occur in smooth muscle cells of rat aortic rings.23,24 Some evidence has already been reported that cAMP and cGMP can cause or at least participate in the induction of iNOS in vascular smooth muscle. For example, Imai and colleagues33 have found that 8-bromo-cAMP, a membrane-permeable cAMP derivative, stimulated NO2– and NO3– production and increased steady-state levels of iNOS mRNA in rat vascular smooth muscle cells in a time- and dose-dependent manner. They also found that L-NMMA, a NOS inhibitor, completely blocked the 8-bromo-cAMPinduced NO2– and NO3– production. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine) all stimulated NO2– and NO3– production. In the study of Koide and colleagues,29 it was shown that an elevation of intracellular cAMP, particularly in combination with inflammatory cytokines, increased the levels of iNOS mRNA in vascular smooth muscle cells. Similar results have been reported by Durante and colleagues.34 Cyclic GMP can also upregulate NOS expression in vascular smooth muscle cells. Inoue and colleagues35 reported that 8-bromo-cGMP, an cell-permeable analogue of cGMP, induced a time- and dose-dependent enhancement of IL-1-induced NO production and iNOS mRNA expression in vascular smooth muscle cells. Human atrial natriuretic peptide (ANP), which stimulates cGMP in vascular smooth muscle cells, also enhanced NO release. Marumo and colleagues30 also found that the natriuretic peptides, ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), all augmented the induction of iNOS through cGMP elevations in vascular smooth muscle cells. CGRP can cause a dual elevation of both cAMP and cGMP levels inside the vascular smooth muscle cells of © 1999 Harcourt Brace & Co. Ltd
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intact rat aorta possessing healthy endothelium.23,24 Exposure to 100 nM CGRP for 1 min can cause two-fold and 9.3-fold elevations of cAMP and cGMP levels, respectively, in rat aortic rings when the endothelium is intact and healthy.36 Thus, the sustained accumulation of CGRP in the smooth muscle layer of blood vessels during the development of shock could cause elevations of intracellular cAMP and cGMP levels in smooth muscle cells, potentially contributing to the induction of iNOS in the smooth muscle cells. In conclusion, the data of our present study have shown that CGRP cannot synergistically augment the iNOS induction by IL-1 in intact rat thoracic aorta, which is different form what has been found in cultured vascular smooth muscle cells. The reason may be the mechanisms of iNOS induction are different in cultured cells and normal vascular tissues. CGRP by itself can cause a long-term inhibition of contraction, which can be reversed by two NOS inhibitors, L-NNA and SMT. The data suggest that CGRP by itself can cause iNOS induction in vascular smooth muscle. The iNOS induction by CGRP may be mediated by the cAMP and cGMP elevations inside the vascular smooth muscle cells caused by CGRP, known to be released during certain pathologies, such as septic shock. The resulting long-term depression of contraction could be responsible, in part, for the prolonged hypotension and vascular hyporesponsiveness to vasoconstrictors that normally occurs in the later stages of septic shock. ACKNOWLEDGEMENT We would like to thank the expert technical assistant provide by Mr Alex W. K. Tu. This research project was supported by an RGC Earmarked Grant from the Research Grants Council of Hong Kong (No. CUHK 266/96M) awarded to R. R. F. REFERENCES 1. Wright CE, Rees DD, Moncada S. Protective and pathological roles of nitric oxide in endotoxin shock. Cardiovasc Res 1992; 26: 48–57. 2. Root RK, Jacob R. Septicaemia and septic shock. In: Wilson JD, Braunwald E, Isselbacher KJ, Harrison’s Principles of internal medicine. 12th ed. New York: Mcgraw-Hill, 1991: 502–507. 3. Parratt JR. Myocardial and circulatory effects of E. coli endotoxin; modification of responses to catecholamines. Br J Pharmacol 1973; 47: 12–25. 4. Suffredini AF, Fromm RE, Parker MM et al. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med 1989; 321: 280–287. 5. Hall RC, Hodge RL. Vasoactive hormones in endotoxin shock: a comparative study in cats and dogs. J Physiol (Lond). 1971; 213: 69–84. 6. Parratt JR, Sturgess RM. E. coli endotoxin shock in the cat; treatment with indomethacin. Br J Pharmacol 1975; 53: 485–488.
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7. Boughton-Smith NK, Hutcheson I, Whittle BJ. Relationship between PAF-acether and thromboxane A2 biosynthesis in endotoxin-induced intestinal damage in the rat. Prostaglandins 1989; 38: 319–333. 8. Doebber TW, Wu MS, Robbins JC, Choy BM, Chang MN, Shen TY. Platelet activating factor (PAF) involvement in endotoxininduced hypotension in rats. Studies with PAF-receptor antagonist kadsurenone. Biochem Biophys Res Commun 1985; 127, No. 3: 799–808. 9. Fletcher JR, Ramwell PW, Harris RH. Thromboxane, prostacyclin, and hemodynamic events in primate endotoxin shock. Advances in shock research 1981; 5: 143–148: 143–148. 10. Cook JA, Wise WC, Halushka PV. Elevated thrombaxane levels in the rat during endotoxic shock. Protective effects of imidazole, 13-azaprostanoic acid, or essential fatty acid deficiency. J Clin Invest 1980; 65: 227–230. 11. Sugiura M, Inagami T, Kon V. Endotoxin stimulates endothelinrelease in vivo and in vitro as determined by radioimmunoassay. Biochem Biophys Res Commun. 1989; 161: 1220–1227. 12. Beasley D, Cohen RA, Levinsky NG. Interleukin-1 inhibits contraction of vascular smooth muscle. J Clin Invest 1989; 83: 331–335. 13. McKenna TM. Prolonged exposure of rat aorta to low levels of endotoxin in vitro results in impaired contractility. Association with vascular cytokine release. J Clin Invest 1990; 86: 160–168. 14. Tracey KJ, Fong Y, Hesse DG et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 1987; 330: 662–664. 15. Wang X, Jones SB, Zhou Z, Han C, Fiscus RR. Calcitonin generelated peptide (CGRP) and neuropeptide Y (NPY) levels are elevated in plasma and decreased in vena cava during endotoxic shock in the rat. Circ Shock 1992; 36: 21–30. 16. Arden WA, Fiscus RR, Wang X et al. Elevations in circulating calcitonin gene-related peptide correlate with hemodynamic deterioration during endotoxic shock in pigs. Circ Shock 1994; 42: 147–153. 17. Joyce CD, Fiscus RR, Wang X, Dries DJ, Morris RC, Prinz RA. Calcitonin gene-related peptide levels are elevated in patients with sepsis. Surgery 1990; 108: 1097–1101. 18. Wang X, Han C, Jones SB, Yang L, Fiscus RR. Calcitonin generelated peptide release in endotoxicosis may be mediated by prostaglandins. Shock 1995; 3: 34–39. 19. Wang X, Fiscus RR. Lactic acid potentiates bradykinin- and low pH-induced release of CGRP from rat spinal cord slices. Am J Physiol 1997; 273 (Endocrinol. 36): E92–E98. 20. Wang X, Wu Z, Tang Y, Fiscus RR, Han C. Rapid nitric oxideand prostaglandin-dependent release of calcitonin gene-related peptide (CGRP) triggered by endotoxin in rat mesenteric arterial bed. Br J Pharmacol 1996; 118: 2164–2170. 21. Uddman R, Edvinsson L, Ekblad E, Hakanson R, Sundler F. Calcitonin gene-related peptide (CGRP): perivascular distribution and vasodilatory effects. Regul Pept. 1986; 15: 1–23. 22. Huttemeier PC, Ritter EF, Benveniste H. Calcitonin gene-related peptide mediates hypotension and tachycardia in endotoxic rats. Am J Physiol 1993; 265: H767–H769. 23. Fiscus RR. Molecular mechanisms of endothelium-mediated vasodilation. Sem Thromb Hemost 1988; Supplement 14: 12–22. 24. Fiscus RR, Zhou HL, Wang X et al. Calcitonin gene-related peptide (CGRP)-induced cyclic AMP, cyclic GMP and vasorelaxant responses in rat thoracic aorta are antagonized by blockers of endothelium-derived relaxant factor (EDRF). Neuropeptides 1991; 20 (2): 133–143.
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25. Libby P, Ordovas JM, Birinyl LK, Auger KR, Dinarello CA. Inducible interleukin-1 gene expression in human vascular smooth muscle cells. J Clin Invest 1986; 78: 1432–1438. 26. Okusawa S, Gelfand JA, Ikejima T, Connolly RJ, Dinarello CA. Interleukin 1 induces a shock-like state in rabbits. Synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition. J Clin Invest 1988; 81: 1162–1172. 27. French JF, Lambert LE, Dage DC. Nitric oxide synthase inhibitors inhibit interleukin-1 beta-induced depression of vascular smooth muscle. J Pharmacol Exp Ther 1991; 259: 260–264. 28. Beasley D, Schwartz JH, Brenner BM. Interleukin 1 induces prolonged L-arginine-dependent cyclic guanosine monophosphate and nitrite production in rat vascular smooth muscle cells. J Clin Invest 1991; 87: 602–608. 29. Koide M, Kawahara Y, Nakayama I, Tsuda T, Yokoyama M. Cyclic AMP-elevating agents induce an inducible type of nitric oxide synthase in cultured vascular smooth muscle cells. Synergism with the induction elicited by inflammatory cytokines. J Biol Chem 1993; 268: 24959–24966. 30. Marumo T, Nakaki T, Hishikawa K et al. Natriuretic peptideaugmented induction of nitric-oxide synthase through cyclic guanosine 3′, 5′-monophosphate elevation in vascular smooth muscle cells. Endocrinology 1995; 136: 2135–2142.
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31. Schini-Kerth VB, Fisslthaler B, Busse R. CGRP enhances induction of NO synthase in vascular smooth muscle cells via a cAMP-dependent mechanism. Am J Physiol 1994; 267: H2483H2490. 32. Sirsjo A, Soderkvist P, Sundqvist T, Carlsson M, Ost M, Gidlof A. Different induction mechanisms of mRNA for inducible nitric oxide synthase in rat smooth muscle cells in culture and in aortic strips. FEBS Lett 1994; 338: 191–196. 33. Imai T, Hirata Y, Kanno K, Marumo F. Induction of nitric oxide synthase by cyclic AMP in rat vascular smooth muscle cells. J Clin Invest 1994; 93: 543–549. 34. Durante W, Cheng K, Schafer AI. Cyclic nucleotide regulation of interleukin-1 beta induced nitric oxide synthase expression in vascular smooth muscle cells. Thromb Res 1994; 75: 63–71. 35. Inoue T, Fukuo K, Nakahashi T, Hata S, Morimoto S, Ogihara T. cGMP upregulates nitric oxide synthase expression in vascular smooth muscle cells. Hypertension 1995; 25: 711–714. 36. Hao H, Fiscus RR, Wang X, Diana JN. Nω-Nitro-L-arginine inhibits vasodilations and elevations of both cyclic AMP and cyclic GMP levels in rat aorta induced by calcitonin generelated peptide (CGRP). Neuropeptides 1994; 26: 123–131.
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Calcitonin gene-related peptide causes long-term inhibition of contraction in rat thoracic aorta through a nitric oxide-dependent pathway L. F. Lu, R. R. Fiscus Department of Physiology, Faculty of Medicine The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong, China
Summary Calcitonin gene-related peptide (CGRP) is released into the circulation during pathogenesis of endotoxin and septic shock and appears to partly mediate vascular problems of shock. To explore the function of CGRP during shock, we investigated long-term action of CGRP, alone or in combination with interleukin-1β (IL-1β), another shock mediator, in isolated rings of rat thoracic aorta. CGRP or IL-1β, by themselves, caused significant long-term (3 h) depression of contraction, while the combination of CGRP and IL-1β had no synergistic effects. Dose-response curves to phenylephrine were significantly decreased and shifted to the right when aortic rings were incubated with 1 µM CGRP for 1 h followed by 2 h incubation without CGRP. Inducible nitric oxide synthase (iNOS) inhibitors, S-methylisothiourea sulfate (SMT) and NG-nitro-L-arginine (L-NNA), completely eliminated long-term depressant effect of CGRP. Our results suggest pathology of septic shock may involve long-term inhibition of vascular contraction mediated by CGRP via expression of iNOS.
INTRODUCTION Endotoxin and hemorrhagic shock in animal models and septic shock in human patients are characterized by hypotension, hyporeactivity to vasoconstrictor agents, inadequate tissue perfusion, vascular damage, and disseminated intravascular coagulation, leading to multiple organ dysfunction and death.1 Gram negative bacterial infection is recognized as a common cause of septic shock.2 Intravenous administration of endotoxin (lipopolysaccharide, a constituent of the cell wall in gram-negative bacteria) in animals and man produces a shock-like syndrome.3,4 The pathogenesis of circulatory shock has been attributed to increased circulating levels
Received 24 August 1998 Accepted 10 February 1999 Correspondence to: Ronald R. Fiscus, Department of Physiology, Faculty of Medicine, Chinese University of Hong King, Shatin, N, T., Hong Kong, China. Tel: 852 2609 6780; Fax: 852 2603 5022; E-mail: [email protected]
of many vasoactive substances, including angiotensin II,5 catecholamines,5 prostaglandins,6 platelet activating factor,7,8 thromboxane A29,10 and endothelin.11 Interleukin-1 (IL-1) and tumor necrosis factor (TNF)12–14 are also described as mediators of septic shock. Research from our laboratory has found that plasma levels of two neuropeptides, calcitonin-gene related peptide (CGRP) and neuropeptide Y (NPY), are elevated during endotoxin shock in animal models.15,16 We have also shown CGRP levels are elevated in the plasma of human patients with septic shock caused by either gram-negative or gram-positive bacteremia.17 Based on these data and the fact that CGRP is a potent hypotensive agent, we have proposed that CGRP serves as one of the important mediators of the vascular problems in septic shock. Plasma levels of CGRP are increased as much as 22-fold after endotoxin is applied in a rat model of shock.15 This elevation of CGRP released during endotoxin shock can be sustained for at least 3 h.15 Several endogenous factors, like prostaglandins,18 lactic acid19 and nitric oxide (NO),20 which are all elevated in 145
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endotoxin shock, are believed to mediate (or enhance) the release of CGRP during shock. A likely source of the elevated CGRP in shock is the many CGRP-containing nerves located in the adventitial and medial layers of most blood vessels.21 The importance of this CGRP release in shock has been further emphasized by experiments showing that approximately 50 percent of the hypotension in endotoxin shock can be attributed to CGRP, by using a specific CGRP receptor antagonist.22 CGRP is a potent vasodilator, using either endothelium/NO-dependent or endothelium/NO-independent mechanisms of vasorelaxation.23 In the case of endothelium/NO-dependent vasorelaxation, CGRP activates a dual signal transduction mechanism involving both adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) elevations in vascular smooth muscle cells.23,24 However, whether CGRP has other effects, such as long-term actions on blood vessels, is not clear. Interleukin-1β (IL-1β) is one of the cytokines released from macrophages during circulatory shock.12 Libby and colleagues in 1986 showed that vascular smooth muscle cells exposed to very low levels of endotoxin can also express the IL-1 gene and release IL-1 into the media.25 IL-1 can produce a shock-like syndrome, characterized by hypotension and decreased vascular resistance, when administrated to animals, suggesting that it may be one of the key mediators of septic shock.26 An IL-1 receptor antagonist has been reported to reduce the mortality associated with endotoxin shock.12 IL-1-treated rat aortic rings exhibit a diminished vascular contractile response to phenylephrine and potassium chloride.12 The IL-1induced inhibition of contraction is believed to be mediated by induced synthesis of NO, one agent that is pathologically over-produced and causes hypotension during septic shock.27 Furthermore, in cultured vascular smooth muscle cells, IL-1β has been found to induce NO production and cause elevations of cGMP levels.28 A large amount of NO is produced by inducible nitric oxide synthase (iNOS), an enzyme different from the constitutive NO synthase which plays a role in the physiological regulation of blood flow and blood pressure. The over-production of NO in vascular smooth muscle can partly explain the hypotension and the vascular hyporeactivity during circulatory shock. Endotoxin, IL-1, tumor necrosis factor α (TNF α) and interferon γ (IFN γ) can induce the expression of iNOS in vascular smooth muscle cells.29 Many other factors, including cAMP-or cGMPelevating agents can augment the induction of iNOS.29,30 Schini-Kerth and colleagues have reported that CGRP (1 µM) enhances the induction of iNOS caused by IL-1β in rat aortic smooth muscle cells grown in culture, and have suggested that this interaction of CGRP and IL-1β may be an important part of the pathology of septic Neuropeptides (1999) 33(2), 145–154
shock.31 However, they found that CGRP, by itself, was not able to significantly induce iNOS or increase the production of NO in these cultured vascular smooth muscle cells.31 Because the smooth muscle cells of intact blood vessels may react differently from cultured vascular smooth muscle cells, it is still not clear what effects CGRP, either alone or in combination with IL-1β, will have on the induction of iNOS and the long-term regulation of contractility of blood vessels. In the present study, we have used freshly-isolated aortic rings (with and without endothelium) as an experimental model to investigate the actions of CGRP, alone and in combination with IL-1β, on the contractility and induction of iNOS in arteries over a 3 h period. NG-nitroL-arginine (L-NNA), a general NOS inhibitor, and S- methylisothiourea sulfate (SMT), a more selective iNOS inhibitor, were used to determine the involvement of iNOS in these responses. The data show that both CGRP and IL-1β, by themselves, can cause significant long-term depression of contraction in aortic rings. The long-term (3 h) depression of contraction of either CGRP or IL-1β was completely reversed by the addition of L-NNA, suggesting involvement of NO production. Furthermore, the CGRP-induced long-term depression of contraction was completely reversed by SMT, suggesting involvement of iNOS induction. No synergistic actions of CGRP and IL-1β were observed on inhibition of contractions. Thus, these actions of CGRP in intact rat aortic rings are clearly different from what has been found in cultured aortic smooth muscle cells.
MATERIALS AND METHODS Preparation of isolated aortic rings The treatment of the laboratory animals and the experimental protocols of the present study adhered to the guidelines of The Chinese University of Hong Kong and were approved by an Institutional Authority for Laboratory Animal Care. Healthy, male Sprague-Dawley rats (body weight = 240–280 g) were used. Heparin (1000 U/rat) was injected intraperitoneally 15 min before rats were decapitated and exsanguinated. Their thoracic aortae were removed and placed in modified KrebsRinger-Bicarbonate (KRB) solution containing 118.5 mM NaCl, 4.74 mM KCl, 1.18 mM MgSO4, 1.18 mM KH2PO4, 2.5 mM CaCl2, 24.9 mM NaHCO3, 10 mM glucose, and 0.03 mM EDTA, and aerated with 95% O2 plus 5% CO2, as in previous experiments.24 After removing the adhering fat and connective tissue, the aortae were cut transversely into rings 4 mm long. The two rings on the outside ends of the thoracic aortae were discarded because of potential damage during handling. Usually, four to five usable rings were obtained from one thoracic aorta. The © 1999 Harcourt Brace & Co. Ltd
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endothelium was removed in some of the aortic rings by gently rubbing the intimal surface with stainless steel microforceps. The integrity or removal of the endothelium was checked by observing the vasorelaxant response to acetylcholine, described below. Measurement of Contractile and Relaxant Response in Aortic Rings The rings were suspended in organ baths containing 5 ml of KRB bubbled with 95% O2 plus 5% CO2 at 37°C using two stainless steel rods (0.275 mm diameter) and were set under a resting tension of 1 g. The KRB was replaced with fresh solution every 10 min during a 30 min equilibration period. Contractile and relaxant responses were measured isometrically using force transducers (Grass Model FT-03) and recorded on a physiological recorder (Grass Polygraph, Model 7). To test whether the endothelium was intact, each ring was stably contracted with phenylephrine (100 nM) and then exposed to acetylcholine (ACh, 100 nM). Only those rings with larger than 50% relaxant responses were considered with intact endothelium. To check whether the endothelium was successfully removed by the rubbing procedure described above, each ring was contracted with 20 nM phenylephrine until a stable contraction was obtained and then 100 nM ACh was added. Only those rings that had no response to ACh were considered without functional endothelium. To test the effects of CGRP or IL-1β, we compared the difference in the dose-response relationship of phenylephrine in the aortic rings before and after incubation with these two agents. Before measuring the dose-response relationship of phenylephrine, the aortic rings were first exposed to 1 µM phenylephrine and washed out two times in order to obtain a stable response to phenylephrine in the following steps. After measuring the dose-response relationship of phenylephrine at the beginning followed by wash out with KRB solution until the contraction of the rings returned to the baseline, the aortic rings were incubated with CGRP, IL-1β or CGRP plus IL-1β for 1 h. Then these agents were washed out and the aortic rings were incubated with pure KRB solution for an additional two hours during which time the KRB solution was replaced every 20 min. The final concentration-response determination was performed 3 h after the agents were first administered. Contractions caused by phenylephrine were tested by increasing the concentration in organ chambers in cumulative half-log increments after a steady-state response was reached to each increment. To test the involvement of NOS, NG-nitro-L-arginine (L-NNA) or S-methylisothiourea sulfate (SMT) was added before the beginning of the final dose response determination. © 1999 Harcourt Brace & Co. Ltd
Chemicals and drugs Rats were supplied by a colony of Sprague-Dawley rats from the Laboratory Animal Service Center, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. Synthetic rat CGRP (α-CGRP) was purchased from Bachem Inc. (Torrance, CA, USA) and Sigma Chemical Company (St. Louis, MO, USA). IL-1β (Human, Recombinant) and SMT were purchased from Calbiochem-Novabiochem Corporation (San Diego, CA, USA). L-NNA, L-phenylephrine (hydrochloride), acetylcholine (Chloride), and heparin (sodium salt) were purchased from Sigma Chemical Company. Statistical analysis The data were analyzed using one-way ANOVA and further analyzed using the Student-Newman-Keuls (S-NK) test for multiple comparisons between treatment groups. A P value of <0.05 was used to indicate significant difference between treatment group means. The data are presented as mean values ± the standard error of the mean (SEM). The ‘n’ values given in the figure legends represent the number of individual rats used for providing the aortic rings in each treatment group. RESULTS Effects of interleukin-1β on NOS induction in rings of rat thoracic aorta The effects of IL-1β in rat aortic rings were observed as a positive control to show the inhibition of contractility through induction of iNOS expression. IL-1β was able to cause significant depression of phenylephrine-induced contractions in rat aortic rings both with and without endothelium at a time point as early as 3 h (Figs 1A & B). To compare the effect of IL-1β in these two groups (i.e. with and without endothelium), the magnitude of inhibition was compared at the concentrations of phenylephrine for each group that elicited a similar level of contraction in the controls, which were 50–70% of the maximum contraction to phenylephrine. These concentrations were 10–7 M in rings with endothelium and 3×10–8M in rings without endothelium. In rat aortic rings with endothelium, 10 ng/ml IL-1β inhibited the contraction to 10–7M phenylephrine from a control of 51.0±4.9% to 2.5±0.5% of the maximum contraction (n=5). The same concentration of IL-1β caused inhibition of contraction to 3×10–8 M phenylephrine from a control of 54.9±5.3% to 3.7±1.7% in aortic rings without endothelium (n=5). The changes of maximum contractions to phenylephrine were 91.3±3.9% to 22.6±9.7% and 87.7±3.4% to 44.2±3.6% in aortic rings with and without endothelium, respectively. Neuropeptides (1999) 33(2), 145–154
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Fig. 1 Effect of IL-1β on phenylephrine-induced contractions in rat thoracic aorta with (A, n=5) or without endothelium (B, n=5). The rat aortic rings were incubated with control KRB or 10 ng/ml IL-1β for 1 h, followed by 2 h in KRB solution. 100 µM L-NNA were added in some of the organ baths to investigate the involvement of nitric oxide synthase. The data here show the changes of contractions in the rings treated with control (▲), IL-1β (●), L-NNA (■ ■ ) or IL-1β plus L-NNA (■).
The NOS inhibitor L-NNA reversed most of the depression of contraction caused by IL-1β. When endothelium was intact, L-NNA reversed the contraction to 10–7M phenylephrine of IL-1β treated rings up to 80.2±12.4%, which was even bigger than the time control. This is because L-NNA is an inhibitor of both constitutive NOS and inducible NOS. In aortic rings without endothelium, L-NNA reversed the IL-1β depressed contraction in response to 3×10–8M phenylephrine up to 70.1±3.4%.
n=7). In rat aortic rings without endothelium, the dose response curves of phenylephrine given by the groups treated by IL-1β itself (4 ng/ml) and IL-1β (4 ng/ml) in combination with CGRP (1 µM) were almost completely the same (Fig.3). As with aortic rings with endothelium, there was no statistically significant difference between responses to IL-1β alone or in combination with CGRP (P>0.05, n=6). Long-term effect of CGRP on rat thoracic aorta
Effects of interleukin-1 β and CGRP in combination in rat thoracic aorta Treating aortic rings with IL-1β combined with CGRP did not cause any synergistic effects on inhibition of contraction. Thus, CGRP did not appear to augment the induction of iNOS caused by IL-1β in rat aortic rings either with or without endothelium (Figs 2 & 3). In rat aortic rings with endothelium, the decrease of contraction to 10–7 M phenylephrine caused by 0.5 ng/ml IL-1β was from 53.1±4.5% to 19.2±6.2%, whereas the change of contraction to the same dose of phenylephrine caused by 0.5 ng/ml IL-1β together with 1 µM CGRP was 53.1±4.5% to 13.4±4.5% (Fig. 2B). There was no statistically significant difference between the group with IL-1β and the group with the combination of IL-1β and CGRP (P>0.05, Neuropeptides (1999) 33(2), 145–154
Exposure of aortic rings with endothelium to CGRP (1 µM) for 1 h, followed by a 2-h incubation with control KRB solution, significantly depressed the contractions to phenylephrine at all concentrations (Fig. 4) (P<0.05, n=6). These data represent a different group of rats from that in Figure 2. For example, CGRP depressed contractions to 10–7M phenylephrine from a control of 47.5±5.8% to 22.09±5.4%. The change of maximum contractions to phenylephrine was from a control of 86.5±4.0% to 70.6±7.3% in the presence of CGRP pretreatment. The EC50 value of phenylephrine was shifted about 3-fold from a control of 7.25×10–8M to 2.05×10–7M. The long-term depression of contraction caused by CGRP can also be seen in Fig.2 representing another group of rats. According to the dose response © 1999 Harcourt Brace & Co. Ltd
CGRP causes inhibition of contraction in rat thoracic aorta 149
Fig. 2 Effect of IL-1β in combination with CGRP on phenylephrine-induced contractions in rat aorta with endothelium. A: the changes of dose response relationship to phenylephrine after treatment with control KRB solution (▲ ▲), IL-1β 0.5 ng/ml (■ ■), CGRP 1 µM (●) or IL-1β plus CGRP (■) (n=7). B: the change of contractions to 10–7 M phenylephrine after the incubation. (*P<0.05, comparing to the control, n=7).
Fig. 3 Effect of IL-1β in combination with CGRP on phenylephrine-induced contractions in rat aorta without endothelium. A: the changes of dose response relationship to phenylephrine after treatment with control KRB solution (■), CGRP 1 µM (■ ■), IL-1β 4 ng/ml (●) or IL-1β 4 ng/ml plus CGRP 1 µM (● ●) (n=6). B: the change of contractions to 3×10–8 M phenylephrine after the incubation. (*P<0.05, comparing to the control, n=6).
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relationship of phenylephrine, the inhibition of contraction caused by 1 µM CGRP was almost the same as that induced by 0.5 ng/ml IL-1β in aortic rings with endothelium (Fig. 2). Effects of L-NNA and SMT on the long-term inhibition of contraction caused by CGRP
Fig. 4 The long-term effects of CGRP on phenylephrine-induced contractions in rat aorta with endothelium. Rat aortic rings were incubated with control KRB solution (■) or 1 µM CGRP (◆) for 1 h, followed by 2 h incubation without CGRP.
If L-NNA or SMT was added before measuring the dose response relationship of phenylephrine after 1 h exposure to CGRP and a subsequent 2 h incubation in KRB solution, the depression of contractions caused by CGRP was completely reversed (Figs 5 & 6). There was no difference between the contractions of time control and CGRP treated rings when L-NNA or SMT was added. Thus pretreatment with L-NNA or SMT completely blocked the long-term depressant effects of CGRP. DISSCUSSION In the present study, we have made two important observations. The first is that CGRP cannot synergistically
Fig. 5 Effect of L-NNA on the long-term inhibition of contraction caused by CGRP. The rat aortic rings were incubated with control KRB or 1 µM CGRP for 1 h, followed by 2 h in KRB solution. 100 µM L-NNA were added in some of the organ baths to investigate the involvement of nitric oxide synthase. A: the changes of dose response to phenylephrine in the rings treated with control (● ●), CGRP (●), L-NNA (■ ■ ) or LNNA plus CGRP (■) (n=9). B: the difference of the contractions to 10–7 M phenylephrine after the treatments (**P<0.05, comparing with the control; *P<0.05 comparing with the groups added with L-NNA).
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Fig. 6 Effect of SMT on the long-term inhibition of contraction caused by CGRP. The rat aortic rings were incubated with control KRB or 1 µM CGRP for 1 h, followed by 2 h in KRB solution. 100 µM SMT were added in some of the organ baths to investigate the involvement of nitric oxide synthase. A: the changes of dose response to phenylephrine in the rings treated with control (■), CGRP (▲), SMT (▼) or SMT plus CGRP (●) (n=9). B: the difference of the contractions to 10–7 M phenylephrine after the treatments (**P<0.05, comparing with the control; *P<0.05 comparing with the groups added with SMT).
augment the iNOS induction caused by IL-1β in intact rings of rat thoracic aorta. This result is different from what has been found in cultured vascular smooth muscle cells.31 The second observation is that CGRP by itself can cause a long-term inhibition of contraction in rat thoracic aorta. Like the immediate vasorelaxant effects of CGRP in rat aorta, this long-term effect of CGRP was found to be completely dependent on endothelium. The mechanism of this response appears to involve induction of iNOS, because the response was blocked by inhibitors of iNOS. In cultured rat aortic vascular smooth muscle cells, Schini-Kerth and colleagues have found that CGRP, in a concentration-dependent manner from 0.3 to 3 µM, enhanced the release of nitrite (a stable oxidation product of NO) caused by IL-1β. Furthermore, the formation of L-citrulline from L-arginine, an indication of NOS activity, caused by IL-1β was enhanced by CGRP (1 µM). Also the cultured vascular smooth muscle cells treated by IL-1β in combination with CGRP (1 µM) had a more marked effect on inhibition of platelet aggregation. Based on the further observation that cAMP-dependent © 1999 Harcourt Brace & Co. Ltd
vasodilators, forskolin and isoproterenol, and the activator of the cAMP-dependent protein kinase, Sp-cAMPS, also enhanced the release of nitrite and the formation of L-citrulline evoked by IL-1β, Schini-Kerth and colleagues concluded that CGRP potentiates the IL-1β induced expression of iNOS via a cAMP-dependent mechanism. However, in our experiments measuring the contraction of aortic rings using an organ bath system, we did not find any synergistic effect of IL-1β and CGRP on inhibiting the contractility of smooth muscle. A possible explanation for the difference in response between our data and the data of Schini-Kerth and colleagues is that there may be some differences between the smooth muscle cells in culture and those from fresh tissue. The characteristics of the cells may have undergone changes during culturing. For example, in the work of Sirsjo and colleagues,32 it was found that in rat smooth muscle cells in culture and in aortic strips, there were clear differences in the mechanism of induction of mRNA for iNOS. iNOS mRNA expression was weak in cultured vascular smooth muscle cells when exposed to either interferon-γ (IFNγ) or LPS, but the combination LPS + Neuropeptides (1999) 33(2), 145–154
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IFNγ enhanced the expression. In aortic strips, in contrast, LPS alone induced a pronounced expression, with no further increase by IFNγ. Furthermore, cycloheximide potentiated the expression of iNOS mRNA in vascular smooth muscles cells in culture stimulated with LPS + IFNγ, but attenuated the responses in aortic strips. It seems likely that there are differences in the intracellular signaling pathways for the induction of iNOS mRNA between mature vascular smooth muscle cells in the vessel wall and the phenotypically modulated cells grown in cultures. Thus, the difference of CGRP’s effect in cultured smooth muscle cells and in aortic rings may also be caused by these differences of the intracellular signaling pathways for the induction of iNOS mRNA in these two cases. Instead of synergistically enhancing the IL-1β-induced iNOS expression in rat thoracic aorta, we found that CGRP by itself can cause a long-term inhibition of contraction in aortic rings with endothelium. In a previous study from our laboratory,24 we had found that CGRP caused an endothelium- and EDRF(NO)- dependent vasorelaxation in rat aortic rings immediately after it was applied to the organ bath. In our present work, the rat aortic rings were incubated with CGRP for 1 h followed by 2 h incubation without CGRP. Thus, CGRP was washed out 2 h before measuring the second dose-response relationship of phenylephrine. One can argue that the long-term vasorelaxant effect of CGRP may simply represent a residual effect of the immediate vasorelaxant effect observed with exposure to CGRP. However, in our experience, the residual relaxant effect of CGRP declines gradually and disappears in about 20–30 min after CGRP is washed out. In the present study, we have shown that the depression of contraction appears at a 2 h time point after CGRP is washed out. This indicates that CGRP is causing an unexpected long-term depressant effect in aortic rings and appears to involve a mechanism that require 2 h to develop. This time frame is similar to that of the depression of contraction caused by IL-1β via induction of iNOS. Thus, we hypothesized that the CGRP-induced long-term depression of contractions may also involve iNOS induction in rat aortic rings. The results of our experiments with L-NNA, a general NOS inhibitor, and SMT, a more selective iNOS inhibitor, have supported this proposed mechanism of the long-term depressant effect of CGRP. Both inhibitors completely blocked the long-term depression of contraction caused by CGRP, suggesting involvement of iNOS induction. The concentration of CGRP around the smooth muscle cells in aorta and other arteries may reach a very high level during certain pathologies, such as endotoxin shock. We have already detected a dramatic elevation of plasma level of CGRP during endotoxin shock.15–17 On Neuropeptides (1999) 33(2), 145–154
the basis of immunocytochemistry works,21 the perivascular CGRP immunoactive nerve fibers are distributed in high density along the arteries. Some of these nerve fibers project into the medial-adventitial border, thus allowing for CGRP to be released in close proximity to the vascular smooth muscle cells of the medial layer of arteries. During the shock state, there is a large elevation of CGRP release from perivascular sensory nerves.15,16 CGRP likely accumulates to high concentrations in the area of arterial smooth muscle cells during septic shock, and this response can be sustained for at least several hours. This sustained high concentration of CGRP during shock may play an additional role besides the immediate vasodilatory effect. For example, CGRP may also contribute to long-term inhibition of contraction via the induction of iNOS that may be an important part of the pathogenesis of septic shock. The induction of iNOS by CGRP could be mediated by the elevations of cAMP and cGMP levels caused by CGRP that is known to occur in smooth muscle cells of rat aortic rings.23,24 Some evidence has already been reported that cAMP and cGMP can cause or at least participate in the induction of iNOS in vascular smooth muscle. For example, Imai and colleagues33 have found that 8-bromo-cAMP, a membrane-permeable cAMP derivative, stimulated NO2– and NO3– production and increased steady-state levels of iNOS mRNA in rat vascular smooth muscle cells in a time- and dose-dependent manner. They also found that L-NMMA, a NOS inhibitor, completely blocked the 8-bromo-cAMPinduced NO2– and NO3– production. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine) all stimulated NO2– and NO3– production. In the study of Koide and colleagues,29 it was shown that an elevation of intracellular cAMP, particularly in combination with inflammatory cytokines, increased the levels of iNOS mRNA in vascular smooth muscle cells. Similar results have been reported by Durante and colleagues.34 Cyclic GMP can also upregulate NOS expression in vascular smooth muscle cells. Inoue and colleagues35 reported that 8-bromo-cGMP, an cell-permeable analogue of cGMP, induced a time- and dose-dependent enhancement of IL-1-induced NO production and iNOS mRNA expression in vascular smooth muscle cells. Human atrial natriuretic peptide (ANP), which stimulates cGMP in vascular smooth muscle cells, also enhanced NO release. Marumo and colleagues30 also found that the natriuretic peptides, ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), all augmented the induction of iNOS through cGMP elevations in vascular smooth muscle cells. CGRP can cause a dual elevation of both cAMP and cGMP levels inside the vascular smooth muscle cells of © 1999 Harcourt Brace & Co. Ltd
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intact rat aorta possessing healthy endothelium.23,24 Exposure to 100 nM CGRP for 1 min can cause two-fold and 9.3-fold elevations of cAMP and cGMP levels, respectively, in rat aortic rings when the endothelium is intact and healthy.36 Thus, the sustained accumulation of CGRP in the smooth muscle layer of blood vessels during the development of shock could cause elevations of intracellular cAMP and cGMP levels in smooth muscle cells, potentially contributing to the induction of iNOS in the smooth muscle cells. In conclusion, the data of our present study have shown that CGRP cannot synergistically augment the iNOS induction by IL-1 in intact rat thoracic aorta, which is different form what has been found in cultured vascular smooth muscle cells. The reason may be the mechanisms of iNOS induction are different in cultured cells and normal vascular tissues. CGRP by itself can cause a long-term inhibition of contraction, which can be reversed by two NOS inhibitors, L-NNA and SMT. The data suggest that CGRP by itself can cause iNOS induction in vascular smooth muscle. The iNOS induction by CGRP may be mediated by the cAMP and cGMP elevations inside the vascular smooth muscle cells caused by CGRP, known to be released during certain pathologies, such as septic shock. The resulting long-term depression of contraction could be responsible, in part, for the prolonged hypotension and vascular hyporesponsiveness to vasoconstrictors that normally occurs in the later stages of septic shock. ACKNOWLEDGEMENT We would like to thank the expert technical assistant provide by Mr Alex W. K. Tu. This research project was supported by an RGC Earmarked Grant from the Research Grants Council of Hong Kong (No. CUHK 266/96M) awarded to R. R. F. REFERENCES 1. Wright CE, Rees DD, Moncada S. Protective and pathological roles of nitric oxide in endotoxin shock. Cardiovasc Res 1992; 26: 48–57. 2. Root RK, Jacob R. Septicaemia and septic shock. In: Wilson JD, Braunwald E, Isselbacher KJ, Harrison’s Principles of internal medicine. 12th ed. New York: Mcgraw-Hill, 1991: 502–507. 3. Parratt JR. Myocardial and circulatory effects of E. coli endotoxin; modification of responses to catecholamines. Br J Pharmacol 1973; 47: 12–25. 4. Suffredini AF, Fromm RE, Parker MM et al. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med 1989; 321: 280–287. 5. Hall RC, Hodge RL. Vasoactive hormones in endotoxin shock: a comparative study in cats and dogs. J Physiol (Lond). 1971; 213: 69–84. 6. Parratt JR, Sturgess RM. E. coli endotoxin shock in the cat; treatment with indomethacin. Br J Pharmacol 1975; 53: 485–488.
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