Mechanism of bradykinin-induced plasma extravasation in the rat knee joint.

1. We have investigated the mechanism of bradykinin (BK)-induced plasma extravasation into the knee joint of the anaesthetized rat. Accumulation of [125I]-human serum albumin within the synovial cavity was used as a marker of increased vascular permeability. 2. Perfusion with BK (1 microM) produced significant plasma extravasation into the knee which was inhibited by co-perfusion of the selective bradykinin B2 receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin (Hoe 140, 200 nM). 3. The bradykinin B1 receptor agonist, [des-Arg9]-BK (up to 100 mM), did not induce plasma extravasation into the knee joint, over this time period. 4. Chemical sympathectomy by chronically administered 6-hydroxydopamine (6-OHDA) did not inhibit bradykinin-induced plasma extravasation. Acute intra-articular perfusion with 6-OHDA (to stimulate transmitter release from sympathetic nerve terminals) at concentrations up to 50 mM did not induce significant plasma extravasation. Intra-articular perfusion of 100 mM 6-OHDA induced significant plasma extravasation but produced severe systemic toxicity. 5. The selective neurokinin1 (NK1) receptor antagonist, RP67580 (230 nmol kg-1), or receptor antagonists for the mast cell products histamine and 5-hydroxytryptamine did not significantly inhibit BK-induced plasma extravasation. 6. Co-perfusion of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) (1 mM) did not significantly inhibit the response to BK. 133Xe clearance from L-NAME (1 mM)-injected joints was significantly (P < 0.05) reduced compared to D-NAME injected joints, suggesting a reduction in blood flow as a result of decreased basal NO production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of tachykinin-induced plasma protein extravasation by calcitonin gene-related peptide

The effect of neuropeptides on plasma protein extravasation was investigated in the abdominal skin of rats. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) induced extravasation with a threshold dose of about 1 pmol. Calcitonin gene-related peptide (CGRP) was ineffective up to 6 pmol. However, when CGRP was injected together with either of the tachykinins extravasation was potentiated. A dose of 6 pmol CGRP shifted the dose-response curve of SP to the left by a factor of about 100. The vasoconstrictor neuropeptide Y (NPY, 12 pmol) reduced the extravasation caused by SP or SP plus 6 pmol CGRP. These results indicate that all 3 tachykinins currently known to be present in sensory neurons induce plasma protein extravasation, i.e. mimic one sign of neurogenic inflammation. This activity is potentiated in the presence of CGRP which coexists with SP and NKA indicating that neurogenic inflammation may be augmented by these interactions.     

Specific neurokinin receptors mediate plasma extravasation in the rat knee joint.

1 Plasma extravasation in the rat knee joint was induced by intra-articular injection of neurokinins and specific neurokinin receptor agonists. 2 Pronounced plasma extravasation was produced by substance P (SP, 4-185 microM) and to a lesser extent by neurokinin-B (NKB, 83-413 microM), whereas neurokinin-A (NKA, 88-440 microM) and calcitonin gene-related peptide (CGRP, 26-130 microM) had no significant effect. 3 The specific neurokinin1 receptor agonist [Sar9, Met(O2)11]-substance P (NK1 agonist) in doses of 0.4-70 microM appeared to be more potent than SP in eliciting plasma extravasation. The neurokinin2 receptor agonist [Nle10]-neurokinin A4-10 (NK2 agonist) was not effective at 70 microM but produced a small and significant effect at 330 microM, whereas the neurokinin3 receptor agonist [MePhe7]-neurokinin B (NK3 agonist) was without effect at 40 microM or 400 microM. 4 Injections of SP or NKA into the synovial cavity of the rat knee were equally effective in producing marked plasma extravasation in remote sites such as the forelimb and hindlimb paws. 5 Co-administration experiments showed that the effects of SP were synergistic with NKA or the NK1 receptor agonist, but not with CGRP or the NK2 receptor agonist. 6 The rank order of potency was NK1 agonist greater than or equal to SP greater than NKB greater than NK2 agonist suggesting that NK1 receptors mediate plasma extravasation in the rat knee joint.     

Calcitonin gene-related peptide increases survival of a musculocutaneous critical flap in the rat

Calcitonin gene-related peptide (CGRP) was shown to increase the survival area of ischaemic tissue from 45% in control animals to about 90% in treated animals. This effect was demonstrated in a musculocutaneous flap model in the rat. The concentrations used were 2 X 10(5) times lower than those known to cause an increase in skin blood flow under normal conditions. Treatment with one single dose up to 36 h postoperatively was found to increase the flap survival area. It is suggested that the mechanism/s by which CGRP increases survival of ischaemic tissue may be different from vasodilation or that the sensitivity is altered during ischaemic conditions and that CGRP may be a powerful tool to reduce ischemia in various clinical conditions.     

Calcitonin gene-related peptide (CGRP) causes redistribution of blood flow in humans

Summary In normal human subjects (n=6), blood flow in the common carotid artery, assessed with an ultrasonic duplex-scanning unit, was increased up to 152% of basal levels by 60-min infusions of human calcintonin gene-related peptide I (CGRP) 80 pmol·kg^–1·h^–1, but it was not affected by 20 pmol·kg^–1·h^–1 CGRP or 88 pmol·kg¹·h^–1 human calcitonin.In the superior mesenteric artery, on the other hand, blood flow was reduced by 80 pmol·kg^–1·h^–1 CGRP to 58% of the basal level, but not by 20 pmol·kg^–1·h^–1 CGRP or with 88 pmol·kg^–1·h^–1 calcitonin.Blood flow in the abdominal aorta remained largely unchanged under the same conditions.Skin blood flow, assessed by a laser Doppler unit, was increased up to 682% of the basal level by 80 pmol·kg^–1·h^–1 CGRP, but not by 20 pmol·kg^–1·h^–1 CGRP or calcitonin.Thus CGRP increased regional blood flow to the brain and the skin at the expense of the gastrointestinal tract.     

Calcitonin gene-related peptide in the joint: contributions to pain and inflammation

Arthritis is the commonest cause of disabling chronic pain, and both osteoarthritis (OA) and rheumatoid arthritis (RA) remain major burdens on both individuals and society. Peripheral release of calcitonin gene-related peptide (CGRP) contributes to the vasodilation of acute neurogenic inflammation. Contributions of CGRP to the pain and inflammation of chronic arthritis, however, are only recently being elucidated. Animal models of arthritis are revealing the molecular and pathophysiological events that accompany and lead to progression of both arthritis and pain. Peripheral actions of CGRP in the joint might contribute to both inflammation and joint afferent sensitization. CGRP and its specific receptors are expressed in joint afferents and up-regulated following arthritis induction. Peripheral CGRP release results in activation of synovial vascular cells, through which acute vasodilatation is followed by endothelial cell proliferation and angiogenesis, key features of chronic inflammation. Local administration of CGRP to the knee also increases mechanosensitivity of joint afferents, mimicking peripheral sensitization seen in arthritic joints. Increased mechanosensitivity in OA knees and pain behaviour can be reduced by peripherally acting CGRP receptor antagonists. Effects of CGRP pathway blockade on arthritic joint afferents, but not in normal joints, suggest contributions to sensitization rather than normal joint nociception. CGRP therefore might make key contributions to the transition from normal to persistent synovitis, and the progression from nociception to sensitization. Targeting CGRP or its receptors within joint tissues to prevent these undesirable transitions during early arthritis, or suppress them in established disease, might prevent persistent inflammation and relieve arthritis pain.     

Calcitonin gene-related peptide receptors in human gastrointestinal epithelia.

1. The secretory responses to calcitonin gene-related peptide (CGRP) receptor agonists have been characterized in two human adenocarcinoma cell lines, namely HCA-7 and Colony-29 (Col-29) epithelia. These cells form polarized epithelial layers when grown on permeable supports and allow changes in electrogenic ion transport in response to agonists to be monitored continuously. 2. alpha-CGRP (rat and human sequences), rat beta-CGRP and human [Tyr0]CGRP applied to the basolateral surface were found to be full agonists, causing prolonged increases in short-circuit current. Concentration-response curves exhibited EC50 values of 0.6-1.5 nM in HCA-7 cells. The same agonists were less effective in Col-29 epithelia, the EC50 values ranging from 1 to 10 nM in these cells. [Cys(ACM)2,7]CGRP was effective in both cell lines and was more potent in HCA-7 cells. 3. CGRP receptors were preferentially located on the basolateral surface in both cell types. Addition of r alpha-CGRP to the apical domain produced significantly smaller secretory responses (8.1% in HCA-7 and 29.2% in Col-29) compared with those produced following basolateral application (100%). 4. In both cell lines r alpha-CGRP-elevated short-circuit current was inhibited by the loop diuretic piretanide (200 microM) and by somatostatin (100 nM). Pretreating epithelia with the cyclo-oxygenase inhibitor, piroxicam (5 microM) had no significant effect upon CGRP responses in either cell line. 5. Rat alpha-CGRP (0.2 nM) responses in HCA-7 epithelia were inhibited by the C-terminal fragment CGRP(8-37) (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin gene-related peptide and its role in migraine pathophysiology

Migraine is a common neurological disorder that is associated with an increase in plasma calcitonin gene-related peptide (CGRP) levels. CGRP, a neuropeptide released from activated trigeminal sensory nerves, dilates intracranial blood vessels and transmits vascular nociception. Therefore, it is propounded that: (i) CGRP may have an important role in migraine pathophysiology, and (ii) inhibition of trigeminal CGRP release or CGRP-induced cranial vasodilatation may abort migraine. In this regard, triptans ameliorate migraine headache primarily by constricting the dilated cranial blood vessels and by inhibiting the trigeminal CGRP release. In order to explore the potential role of CGRP in migraine pathophysiology, the advent of a selective CGRP receptor antagonist was obligatory. The introduction of di-peptide CGRP receptor antagonists, namely BIBN4096BS (1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl] pentyl] amino]-1-[(3,5-dibromo-4-hydroxyphenyl) methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-, [R-(R*,S*)]-), is a breakthrough in CGRP receptor pharmacology and can be used as a tool to investigate the role of CGRP in migraine headaches. Preclinical investigations in established migraine models that are predictive of antimigraine activity have shown that BIBN4096BS is a potent CGRP receptor antagonist and that it has antimigraine potential. Indeed, a recently published clinical study has reported that BIBN409BS is effective in treating acute migraine attacks without significant side effects. The present review will discuss mainly the potential role of CGRP in the pathophysiology of migraine and the various treatment modalities that are currently available to target this neuropeptide.     

Effects of interactions of naturally-occurring neuropeptides on blood flow in the rat knee joint.

1. Changes of blood flow in the rat knee joint, measured by laser Doppler flowmetry, were produced by topical application of naturally-occurring neuropeptides to the joint capsule. 2. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) all produced dose-dependent transient vasodilatation of the rat knee joint microvasculature. NKB showed significantly smaller vasodilator responses compared to SP and NKA which were similar in their potencies. 3. Calcitonin gene-related peptide (CGRP) produced dose-dependent vasodilatation which was more pronounced than that produced by the neurokinins. The rank order of potency was: CGRP > SP = NKA > NKB. The vasodilator effect of CGRP was also more prolonged and this extended phase was abolished by co-administration of SP. 4. Cross-tachyphylaxis was not observed with the different neurokinins, but SP and NKA showed novel antagonistic effects on NKB-induced vasodilatation. 5. Co-administration of 1 nmol of the specific NK1 receptor antagonist, CP-96345, with 1 nmol of each of the neurokinins produced significant inhibition of the vasodilator response to SP but did not affect vasodilator responses to NKA and NKB. Co-administration of CP-96345 with the neurokinins plus superfusion of the rat knee joint with a solution containing 0.1 mM CP-96345 further reduced the vasodilator responses to SP but again the vasodilator responses to NKA and NKB were not significantly altered. 6. The results suggest that multiple neurokinin receptor types may be present in the rat knee joint which could mediate the vasodilator responses of the different neurokinins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitonin gene-related peptide in rat arterial and venous vessels: sensitivity to capsaicin, bradykinin and FMLP.

Sensitivity of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) to capsaicin was investigated in different arterial and venous tissues (mesenteric, renal and femoral artery and vein and carotid artery and jugular vein) of the rat. In vivo capsaicin pre-treatment depleted or reduced tissue CGRP-LI in all the vessels examined with the exception of the carotid artery. Likewise, in vitro exposure to capsaicin evoked release of CGRP-LI from all vessels, excluding the carotid artery. Remarkable amount of CGRP-LI sensitive to capsaicin was present in both mesenteric artery and vein as compared to the other vascular tissues. Endogenous (bradykinin) or exogenous (N-formyl-methionyl-leucyl-phenylalanine) proinflammatory agents, were found to release CGRP-LI from mesenteric veins.     

Actions of locally administered adrenoceptor agonists on increased plasma protein extravasation and blood flow in guinea-pig skin.

1 Bradykinin-induced increased plasma protein extravasation (IPPE) and blood flow have been assessed in guinea-pig skin by isotopic methods. 2 alpha-Adrenoceptor agonists inhibited IPPE and reduced cutaneous blood flow. The potency of alpha-agonists as inhibitors of IPPE correlated with their vasoconstrictor effects. The actions of noradrenaline on both IPPE and blood flow were blocked by phentolamine but not by propranolol. 4 beta-Adrenoceptor agonists inhibited IPPE at doses which either increased or caused little change in cutaneous blood flow. Isoprenaline inhibition of IPPE was reduced by propranolol but was unaffected by phentolamine. 5 The inhibitory action of alpha-agonists on IPPE can be explained by a reduction in blood flow to the affected site. Beta agonist inhibition is not due to effects on blood flow but is probably caused by a reduction in permeability of the microvessels.     

Interactions between the tachykinins and calcitonin gene-related peptide lead to the modulation of oedema formation and blood flow in rat skin.

1. The mechanisms involved in tachykinin-induced oedema were investigated in rat skin and interactions between the tachykinins and calcitonin gene-related peptide (CGRP) were studied. 2. Intradermal injections of the tachykinins, substance P, neurokinin A and neurokinin B, stimulated local oedema formation which was in each case potentiated by co-injection of the vasodilator CGRP. Oedema induced by substance P, in the presence and absence of CGRP, was significantly inhibited by pretreatment of rats with a combination of the histamine H1 antagonist, mepyramine, and the 5-hydroxytryptamine antagonist, methysergide. Oedema induced by neurokinin A or B was not inhibited by this pretreatment. 3. Intradermally-injected CGRP induced a long lasting increase in local blood flow, which was measured with a laser Doppler blood flow meter. The simultaneous injection of substance P, but not of the structurally-related neurokinins, caused a loss of the prolonged vasodilator activity of CGRP. 4. These results show that oedema induced by substance P is partially dependent on mast cell amines and that only substance P causes a loss of the prolonged vasodilator activity of CGRP. 5. We suggest that the ability of substance P to prevent the persistent vasodilator activity of CGRP may be a direct consequence of substance P-induced activation of mast cells.     

Calcitonin gene-related peptide (CGRP)-evoked inotropism during hyper- and hypo-sensory-motor innervation in rat atria

1 Positive inotropic responses to calcitonin gene-related peptide (CGRP) were evaluated in atria isolated from in vivo rat models of hyper-sensory-motor innervation (following neonatal guanethidine treatment) and hypo-sensory-motor innervation (following neonatal capsaicin treatment), to explore the hypothesis that functional responsiveness of atrial myocardium to CGRP may correlate with tissue levels of the sensory-motor neurotransmitters. Comparative evaluation of inotropic responses to CGRP following in vitro treatment of atria with guanethidine was also performed. 2 Following long-term guanethidine treatment, positive inotropic responses to CGRP were significantly attenuated, while supersensitivity to the sympathetic transmitter noradrenaline was shown. Maximal inotropic responses to CGRP (30 nM ) were 214.0 ± 28.1 (n = 8) and 146.8 ±21.7 mg (n = 8; P < 0.01) increase of the basal contractile tension in control and treated preparations, respectively. The pD₂ values for noradrenaline were 6.71 ± 0.12 (n = 8) and 7.26 ± 0.13 (n = 6; P < 0.01) in control and treated atria, respectively. Acute application of guanethidine in vitro did not modify the positive inotropism by CGRP or the β-adrenoceptor agonist isoprenaline. 3 Sensory-motor hypoinnervation following chronic treatment with capsaicin did not affect the inotropic responses to CGRP. Neither guanethidine nor capsaicin treatment affected the contractile apparatus of myocytes, as demonstrated by similar basal contractile tension as well as calcium-evoked inotropic responses in control and treated preparations. 4 In summary, increased sensory-motor innervation, following long-term sympathectomy with guanethidine, resulted in attenuation of the inotropic responses of the rat atrium to CGRP, while no changes in the inotropic responses were seen following sensory-motor denervation with capsaicin. Down-regulation of CGRP receptors or altered post-receptor signalling may be involved in the reduced responsiveness to CGRP.     

Calcitonin gene-related peptide desensitizes skeletal muscle arterioles to substance P in vivo.

Calcitonin gene-related peptide (CGRP) was tested for possible interactions with the effects of substance P on rabbit skeletal muscle arterioles in vivo. Both CGRP and substance P dose-dependently increased arteriolar diameter. However, pretreatment with CGRP made the arterioles insensitive to substance P, and also prevented the formation of aggregates of leukocytes and platelets normally seen after substance P. These results suggest that CGRP, in addition to its direct effects, may act as a modulator of the effects of substance P in neurogenic inflammation.     

Calcitonin gene-related peptide relaxed rat tail artery helical strips in vitro in an intracellular calcium-dependent manner

The vasoactive properties of calcitonin gene-related peptide (CGRP) were examined using the helical strip of the rat tail artery as a model. CGRP was found to inhibit norepinephrine-induced contraction but not KCl- or vasopressin-induced contraction. No significant inhibition occurred in either KCl- or norepinephrine-stimulated strips when Ca²⁺ was added incrementally to otherwise Ca²⁺-free bathing solution. However, a dose-related inhibition to norepinephrine-stimulated contraction in Ca²⁺-free bathing solution was observed. This strongly suggests that CGRP exerts its effect by inhibiting the mobilization of intracellular Ca²⁺.     

The calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS reduces neurogenic increases in dural blood flow

In an in vivo preparation of the exposed rat cranial dura mater electrical field stimulation causes increases in blood flow that are mainly due to the vasodilatory effect of calcitonin gene-related peptide (CGRP) released from meningeal afferents. In this preparation the effect of BIBN4096BS, a non-peptide competitive antagonist of CGRP receptors, was examined. Additionally, in an in vitro preparation of the hemisected rat skull the effect of BIBN4096BS on CGRP release stimulated by activation of meningeal afferents was analysed. Injection of BIBN4096BS at cumulative doses of 300 microg/kg and 900 microg/kg caused dose-dependent inhibition of the electrically evoked blood flow increases. The basal blood flow and vital parameters were not significantly changed by any dose. In the hemisected skull BIBN4096BS at 10(-6) M did not alter the CGRP release evoked by depolarizing K(+) concentrations or antidromic electrical stimulation of the trigeminal ganglion. We conclude that neurogenic increases in dural blood flow are reduced by BIBN4096BS without changing basal vascular parameters. This peripheral effect may be important with regard to CGRP receptor inhibition as an antimigraine strategy.     

降钙素基因相关肽(CGRP)抗体治疗偏头痛

降钙素基因相关肽(Calcitonin gene-related peptide,CGRP)是参与偏头痛发病机制的一种重要的神经肽,具有扩血管的作用。针对CGRP的治疗在临床试验中获得了显著的效果,而且不引起血管收缩,对心血管疾病患者相对安全。最早合成的药物是小分子量CGRP受体拮抗剂,主要用于偏头痛急性期的治疗,但因肝脏毒性使其研发过程屡屡受挫,目前尚无该类药物获得上市批准。随后出现的CGRP单克隆抗体在预防治疗发作性偏头痛和慢性偏头痛的临床试验中显示出充分的疗效和安全性,目前已经有3个单克隆抗体获得上市批准。本文主要对CGRP与偏头痛的关系以及CGRP单克隆抗体治疗偏头痛进行综述。     

Indomethacin potentiates endotoxin-induced blood flow reduction and histological injury in rat gastric mucosa.

1. The effect of the intravenous administration of lipopolysaccharide from Salmonella typhosa endotoxin on arterial blood pressure (BP), gastric mucosal blood flow (GMBF) and gastric damage was studied in anaesthetized rats. The effect of the inhibition of endogenous prostaglandin generation by indomethacin on these parameters was also investigated in this model of endotoxin shock. 2. A similar and dose-dependent percentage of reduction in BP and GMBF was observed 5 min after a bolus injection of 20 or 30 mg kg-1 endotoxin. A transient recovery in GMBF at 15 min was observed followed by a second fall at 30 min, at a time when BP was slowly increasing. 3. Pretreatment with indomethacin (5 mg kg-1, s.c.) one hour before the administration of 30 mg kg-1 endotoxin, significantly augmented the reduction in GMBF without affecting the reduction in BP. 4. The gastric damage, assessed histologically, was similar and confined to the superficial mucosa 30 min after the administration of 20 or 30 mg kg-1 endotoxin. The histologically-assessed damage was significantly greater in indomethacin pretreated rats injected with 30 mg kg-1 endotoxin. 5. These findings suggest that endogenous prostaglandin generation plays a protective role in endotoxin-induced gastric mucosal microcirculatory disturbances and mucosal damage.