Doses Of Calcitonin Gene-related Peptide Research Articles

CGRP antibody rescues tactile sensitization induced by mild traumatic brain injury in mice

Post-traumatic headache (PTH) after mild traumatic brain injury (mTBI) is debilitating and hard to diagnose. Unfortunately, PTH is highly prevalent in active duty personnel, Veterans, as well as sport athletes. The high incidence and chronic nature of PTH underscores the need to study these problems in animal models in order to develop new and effective treatments. Patients suffering from PTH often present the same symptoms as migraine patients, which can be studied in mice. We explored hypersensitivity to a tactile stimulation of the periorbital facial and plantar paw areas in a model of mTBI in mice using von Frey filaments. This mild closed head impact (mCHI) is induced by a weight (30 g) dropped onto the intact skull once a day over 3 consecutive days, after which mice develop a transient cephalic hypersensitivity between day 1 and day 5 after the injury, and then recover to normal thresholds. Additionally, after mice recover from this transient cephalic hypersensitivity, an injection of normally non-noxious triggers such as a low dose of calcitonin gene-related peptide (CGRP) or a low dose of sodium nitroprusside (SNP, a nitric oxide donor) can induce tactile hypersensitivity. This can last for at least 13 weeks after injury. Treatment with a CGRP-blocking monoclonal antibody after injury, but prior to CGRP or SNP administration can alleviate this sensitization. Preliminary data indicate that after injuries, administration of CGRP is also able to induce light aversion at usually non-noxious dim light intensities. In summary, we report that mice are primed by a mild TBI that sensitizes them to subsequent migraine triggers in a CGRP-dependent manner. Dept. Defense W81XWH-16-1-0071, W81XWH-16-1-0211 to AFR; VA-ORD (RRD IHA Research Award and Pilot Grant from Iowa City VA Center for the Prevention and Treatment of Visual Loss to ASW.

Effects of Calcitonin Gene-related Peptide on Dental Pulp Stem Cell Viability, Proliferation, and Differentiation

IntroductionPulpitis is an inflammation of dental pulp caused by bacterial proliferation near or within pulpal tissues. In advanced stages, when the inflammation is associated with pulp necrosis, pulp preservation is dependent on dental pulp stem cells (DPSCs) that can differentiate into odontoblastlike cells and produce reparative dentin. In this study, we evaluated the influence of sensory neurons through calcitonin gene-related peptide (CGRP) on DPSC viability and proliferation and the ability of DPSCs to differentiate into mineralizing cells. MethodsCommercially available DPSCs were treated with varying doses of CGRP, and metabolic activity, viability, proliferation, and cell death were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays, trypan blue staining, 5-bromo-2'-deoxyuridine cell proliferation assay, and caspase-3 staining, respectively. DPSC differentiation was assessed with alizarin red staining and by quantifying messenger RNA expression of odontoblast makers. ResultsCGRP induced a dose-dependent decrease of DPSC metabolic activity that was prevented by the CGRP receptor antagonist CGRP 8-37. The decrease in the proportion of live cells induced by CGRP is associated with a decrease of cell proliferation but not with caspase-3–dependent apoptosis. Interestingly, dexamethasone-induced DPSC differentiation into mineralizing cells was neither inhibited nor enhanced by CGRP treatment. ConclusionsThe neuropeptide CGRP has an inhibitory effect on DPSC proliferation but does not enhance or inhibit the differentiation of DPSCs into mineralizing cells. This suggests that CGRP might negatively influence the ability of DPSCs to contribute to regenerative or tissue repair processes.

Involvement of CGRP in peripheral nerve injury-induced behavioral hypersensitivity and chemokine production (CCR5P.260)

Abstract Previously, we have observed increased expression of Calcitonin Gene Related Peptide (CGRP), a pro-nociceptive neuropeptide, along with selected cytokines and chemokines, in the lumbar spinal cord following spinal nerve L5 transection (L5Tx, a murine model of neuropathic pain) in BALB/c mice. To further study the effects of CGRP in neuropathic pain, we intrathecally (i.t.) injected a CGRP antagonist (CGRP8-37) into BABL/c mice following L5Tx. Mice injected with CGRP8-37 displayed a reduced L5Tx-induced mechanical hypersensitivity, but not heat hypersensitivity. CGRP8-37 also caused significant reduction of the level of lumbar spinal cord CCL5 (RANTES) post-L5Tx when compared to vehicle injected-mice and non-injected mice. i.t. injection of a CCL5 neutralizing antibody significantly inhibited L5Tx-induced mechanical hypersensitivity. To test whether CGRP played its pro-nociceptive role through stimulating glial response, mixed glial cells were treated with various doses of CGRP. CGRP induced a slight increase of CCL5 24 hours post-treatment, which was correlated with the increased expression of MAP Kinase (MAPK) signaling pathway molecules: p38 and JNK. Thus, CGRP may promote MAPK-mediated glial activation and the subsequent production of CCL5, which potentiate behavioral hypersensitivity following L5Tx.

Genetic Enhancement of Calcitonin Gene-Related Peptide-Induced Central Sensitization to Mechanical Stimuli in Mice

Calcitonin gene-related peptide (CGRP) is a key player in migraine. To address the role of CGRP in mechanical allodynia, which is a common feature of migraine, we used CGRP-sensitized transgenic mice. These mice have elevated nervous-system expression of the human receptor activity-modifying protein-1 (hRAMP1) subunit of the CGRP receptor. Under baseline conditions, the nestin/hRAMP1 mice and control littermates had similar hindpaw withdrawal thresholds to von Frey filaments. The effect of CGRP was tested using a filament that elicited a withdrawal response on 20% of its presentations. Following intrathecal injection of 1 nmol CGRP in the nestin/hRAMP1 mice, the response frequency was 80% within 30 minutes. The antagonist CGRP(8-37) blocked the increased response. In control littermates, a 5-fold higher dose of CGRP was required to elicit a similar response. In contrast to intrathecal injection, peripheral CGRP did not increase the mechanical responses. Intraplantar injection of capsaicin was used to test the efficacy of endogenous CGRP. Capsaicin increased mechanical responses in the nestin/hRAMP1 and control mice, although a higher dose was required in controls. In contrast to control mice, there was also a contralateral paw response in nestin/hRAMP1 mice, which is consistent with central sensitization. In this study we show central CGRP-induced mechanical allodynia that is enhanced by overexpression of RAMP1 in nervous system. These data suggest that hypersensitivity to CGRP could be a potential mechanism underlying central sensitization in migraine and point to CGRP-receptor antagonists as a possible therapy for other pain disorders.

Improvement of the Closed Cranial Window Model in Rats by Intracarotid Infusion of Signalling Molecules Implicated in Migraine

Intravital microscopy on a closed cranial window allows one to measure change in the diameter of cranial blood vessels after intravenous (i.v.) administration of pharmacodynamic substances. Putative targets being pursued in migraine are large vasodilating peptide molecules such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase polypeptide (PACAP)-38. High i.v. doses are required to study their craniovascular pharmacology. Unfortunately, this leads to a drop in blood pressure (BP) that subsequently causes blood vessels to dilate by autoregulation. Hence it is difficult to decipher what effect is caused by direct receptor agonist interaction or contributed by autoregulation. In the present study we infused substances with an ingenious indwelling catheter in the common carotid artery in rats. Intracarotidly seven-, 12- and 17-fold lower doses of CGRP, PACAP-38 and capsaicin were required, respectively, compared with i.v. infusion to induce the same dilation in dural artery. Dilating intracarotid (i.c.) doses caused no or a minimal fall in BP, whereas equi-responsive i.v. doses caused a marked BP reduction. The CGRP blocking potential of olcegepant was amplified by > 20 times on i.c. infusion. Pial artery responses to CGRP did not change with i.c. infusion, demonstrating that dilations after i.v. CGRP are mediated by autoregulation rather than through specific receptors. We applied CGRP topically, which induced concentration-dependent dural vasodilation, but no effect on pial artery or on BP. In conclusion, this new approach offers an improvement of the existing model by allowing more accurate assessment of effects of pharmaca on the cranial vasculature without inducing significant systemic effects.

Calcitonin Gene-Related Peptide Receptor Antagonism Does Not Affect the Severity of Myocardial Ischemia during Atrial Pacing in Dogs with Coronary Artery Stenosis

Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that also has potent vasodilator activity. There are conflicting preclinical reports regarding the effect of CGRP receptor antagonism in the setting of myocardial ischemia. The present study was conducted in a canine model in which regional myocardial ischemia was reproducibly evoked by serial periods of atrial pacing (80 beats per min above baseline rate) in the presence of a 40% stenosis of the left anterior descending (LAD) coronary artery. Ischemia severity was quantitated by changes in unipolar epicardial electrograms (EG) recorded in the area of ischemia. In validation studies, the calcium entry blocker diltiazem reduced ischemia severity (before versus after treatment: DeltaEG, 1.92 +/- 0.23 versus 0.54 +/- 0.24 mV; p < 0.05) and tended to increase LAD flow (7.7 +/- 0.7 versus 9.4 +/- 1.4 ml/min; p = 0.10), whereas the coronary constrictor serotonin increased ischemia severity (before versus after treatment: DeltaEG, 2.11 +/- 0.44 versus 4.90 +/- 1.46 mV; p < 0.05) concomitant with a reduction in LAD flow (9.1 +/- 1.1 versus 5.4 +/- 1.5 ml/min; p < 0.05). A 30 microg/kg/min i.v. infusion test dose of the CGRP receptor antagonist CGRP((8-37)) was validated by demonstrating complete block of the depressor effects of exogenous i.v. 0.03 to 0.3 microg/kg CGRP. This dose of CGRP((8-37)), administered either intravenously or intra-atrially, had no effect on ischemia severity or paced LAD flow, indicating no intrinsic effect of CGRP receptor antagonism on the severity of acute myocardial ischemia. Likewise, the administration of a hemodynamically active dosing regimen of CGRP (0.03 microg/kg/min i.v.) had no effect on paced coronary flow or ischemia severity, suggesting no major role of CGRP in regulating ischemic blood flow.

MIGRAINE PATHOPHYSIOLOGY

MIGRAINE PATHOPHYSIOLOGY

Involvement of calcitonin gene-related peptide in control of human fetoplacental vascular tone.

Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental functions during pregnancy. The present study was designed to examine the existence of CGRP-A receptor components, the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1), in the human placenta and the vasoactivity of CGRP in the fetoplacental circulation. Immunofluorescent staining of the human placenta in term labor using polyclonal anti-CRLR and RAMP1 antibodies revealed that labeling specifically concentrated in the vascular endothelium and the underlying smooth muscle cells in the umbilical artery/vein, chorionic artery/vein, and stem villous vessels as well as in the trophoblast layer of the placental villi. In vitro isometric force measurement showed that CGRP dose dependently relaxes the umbilical artery/vein, chorionic artery/vein, and stem villous vessels. Furthermore, CGRP-induced relaxation of placental vessels are inhibited by a CGRP receptor antagonist (CGRP8-37), ATP-sensitive potassium (KATP) channel blocker (glybenclamide), and cAMP-dependent protein kinase A inhibitor (Rp-cAMPS) and partially inhibited by a nitric oxide inhibitor (Nomega-nitro-l-arginine methyl ester). We propose that CGRP may play a role in the control of human fetoplacental vascular tone, and the vascular dilations in response to CGRP may involve activation of KATP channels, cAMP, and a nitric oxide pathway.

Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism

Although accumulated data suggest that calcitonin gene-related peptide (CGRP) produces anabolic effects in skeletal tissue by directly acting on osteogenic cells, neither the distribution of CGRP receptor subtypes nor the associated cellular signaling pathways are well understood. In this study, we have pharmacologically and biochemically characterized CGRP-binding sites in immature human osteoblastic MG63 cells. In a [ 125I]CGRP whole-cell-binding assay, nonlinear regression curve-fitting analysis demonstrated a single binding site ( K D=405±29 pM; 13,100±223 sites per cell). Immunocytochemical and Western blot analyses demonstrated that 48-, 52-, and 120-kDa forms of the calcitonin receptor-like receptor (CRLR) and a 15-kDa form of the receptor-activity-modifying protein-1 (RAMP-1) was expressed on the plasma membrane. CGRP strongly stimulated cellular cAMP production and this effect was antagonized not only by an antagonist of the subtype-1 CGRP (CGRP 1) receptor, CGRP-(8-37), but by an agonist of the putative subtype-2 CGRP (CGRP 2) receptor, [Cys(Acm) 2,7]-CGRP, that also itself acted as a weak agonist. In contrast to published data, CGRP dose- and time-dependently dephosphorylated and inactivated extracellular signal response kinase (ERK). This action was blocked by CGRP-(8-37), by an inhibitor of cAMP-dependent protein kinase (H-89), or by an inhibitor of protein phosphatases (vanadate). Prolonged CGRP treatments significantly suppressed DNA synthesis at 27 h, but up-regulated type I collagen. Both these actions were blocked by CGRP-(8-37) and mimicked by a specific inhibitor of ERK (PD98059). In summary, our data suggest that the CGRP receptors in MG63 cells meet many, but not all, of the classical criteria used to define CGRP 1 receptors. These receptors that functioned in a pharmacologically distinct manner could inhibit cell proliferation, and were substantially more sensitive to a CGRP 2 receptor agonist than are typical CGRP 1 receptors. These receptor proteins were not exactly matched with the known components of a CGRP 1 receptor that have been reported. Therefore, it is possible that the CGRP receptors expressed in immature osteoblastic human MG63 cells represent a variation of the known CGRP 1 receptor.

Infusion of pregnant rats with calcitonin gene-related peptide (CGRP)(8-37), a CGRP receptor antagonist, increases blood pressure and fetal mortality and decreases fetal growth.

Calcitonin gene-related peptide (CGRP) is the most potent endogenous vasodilatory peptide, and is involved in the regulation of blood flow to vital organs. We have previously shown that CGRP may be involved in vascular adaptations that occur during pregnancy, and that steroid hormones may be involved in these mechanisms. We hypothesized that endogenous CGRP is required for maintaining blood pressure and fetoplacental growth in pregnant rats, and that progesterone will enhance CGRP effects. The vasodilatory effects of CGRP are known to be inhibited by a competitive CGRP receptor antagonist, the C-terminal fragment CGRP(8-37). In the present study, we investigated whether continuous s.c. infusion of CGRP(8-37) to pregnant rats will reduce fetoplacental growth and increase systolic blood pressure. We also assessed whether progesterone will alter the effects of CGRP(8-37) on blood pressure during postpartum. Groups of five pregnant rats were s.c. infused with varying doses of CGRP(8-37) from Day 17 of pregnancy. Daily systolic blood pressures, pup weight, mortality at term delivery, and fetoplacental weights on Day 20 of gestation were measured. CGRP(8-37) at a dose of 0.083 mg day(-1) kg(-1) body weight (BW) showed no effects; however, doses of 0.33 and 1.33 mg day(-1) kg(-1) BW increased (P < 0.05) blood pressure during pregnancy, and these elevated blood pressures persisted during postpartum with the highest dose used. Progesterone (2 mg per injection, twice a day; s.c.) treatment significantly elevated blood pressure in rats infused with CGRP(8-37) during postpartum, suggesting that progesterone regulates CGRP-induced vascular effects. CGRP(8-37) infusion caused significant reductions in pup weight with an increase in mortality rate, and these effects were dose-dependent. Placental and fetal weights were also decreased prior to term on Day 20 of gestation, 72 h after CGRP(8-37) infusion, indicating effects on uteroplacental tissues. Therefore, we suggest that endogenous CGRP plays an important role in maintaining normal fetoplacental development, fetal survival, and vascular adaptations during pregnancy.

Blood Pressure and Cardiovascular Tone: Role of CGRP Family of Peptides

Calcitonin gene-related peptide (CGRP) belongs to a family of peptides (adrenomedullin, amylin and calcitonin), most of which have significant effects on the cardiovascular system[1]. CGRP is the most potent endogenous vasodilatory peptide and is the most studied of the peptides in this family[2]. CGRP and its receptors are widely distributed throughout the vascular system. Localization of i-CGRP and its receptors in the heart, correlates well with the postulated functions of this peptide (i.e., increase in heart rate, atrial contractility, and increase in coronary blood flow). CGRP and ADM have direct effects on capacitance blood vessels, and administration leads to peripheral vasodilation and reduced vascular resistance. Injection or an infusion of physiological doses of CGRP and ADM has both significant effects on the blood pressure; cardiac performance, and blood supply to critical organs. Effects of CGRP (and its antagonists) have been examined as potential therapeutic agents in controlling hypertension, cardiac failure, cardiac arrhythmia, migraine headaches, Renaud’s syndrome, preeclampsia, etc. Animal studies have revealed that in the presence of progesterone, administration of CGRP can alleviate both premature delivery and preeclampsia-like syndrome. The role of CGRP as a potential therapeutic agent and its mechanism of actions in alleviating pregnancy-induced hypertension will be discussed in detail[3].

Alleviation of mechanical and thermal allodynia by CGRP 8-37 in a rodent model of chronic central pain

CGRP 8-37 is a truncated version of calcitonin gene-related peptide (CGRP) that binds to the CGRP receptor with similar affinity but does not activate the receptor and is a highly selective CGRP receptor antagonist. CGRP and activation of its receptor appear to play a role in peripheral inflammatory and neuropathic models of pain although there is considerable controversy. The aim of this study was to examine possible anti-nociceptive effects of CGRP 8-37 on a model of chronic central neuropathic pain known to develop weeks after spinal hemisection. Adult male Sprague–Dawley rats were given a spinal hemisection ( N=34) or a sham surgery ( N=10) at the T13 spinal segment. An externally accessible PE-10 intrathecal catheter that terminated at T13 was used for drug delivery. Animals were allowed to recover for 4 weeks at which time the hemisected animals displayed mechanical and thermal allodynia bilaterally, in both forelimbs and hindlimbs. CGRP 8-37 was delivered just prior to a testing session in 1, 5, 10, or 50 nM doses in artificial cerebral spinal fluid in 10 μl volumes. CGRP 8-37 was effective in alleviating mechanical and thermal allodynia in a dose-dependent manner ( P<0.05). The 50 nM dose was most efficacious for both forelimb and hindlimb responses ( P<0.05). The period of efficacy was 10 min to onset for a duration of 20 min. Post-drug washout responses were not statistically significant compared to pre-drug responses. The sham control groups demonstrated no statistically significant difference at any dose of CGRP 8-37 when compared to pre-surgical baseline values. In conclusion, CGRP 8-37 is effective in abolishing mechanical and thermal allodynia produced by spinal hemisection. Consequently, the CGRP receptor may play a role in chronic central neuropathic pain and offers a novel therapeutic approach to managing chronic central pain.

Effects of calcitonin gene-related peptide on tissue survival, blood flow and neutrophil recruitment in experimental skin flaps

Local administration of calcitonin gene-related peptide (CGRP) has been shown to improve tissue survival in surgical skin flaps. Moreover, topical CGRP has been demonstrated to exert anti-inflammatory effects in different animal models of skin inflammation. The aim of the present study was to establish whether systemic treatment with low doses of CGRP may improve survival and reduce neutrophil accumulation in surgical skin flaps.Using a well-established dorsal skin-flap model in the rat, we found that intraperitoneal (i.p.) pretreatment with low doses of CGRP dose-dependently increased flap survival. Thus, in untreated animals flap survival at day 7 after surgery was 42%, as compared to 44%, 60%, 69% and 73% survival after a single preoperative i.p. injection of 10–15, 10–12, 10–9and 10–6mol CGRP, respectively (P< 0.05 versus control for the three highest doses). The three effective doses had no detectable effects on either flap blood flow (laser Doppler) or mean arterial blood pressure. On the other hand, 5 × 10–9mol CGRP i.p. significantly reduced the marked surgery-induced accumulation of flap myeloperoxidase (a marker for neutrophil recruitment) without affecting the circulating neutrophil count.Taken together, our findings demonstrate that low systemic doses of CGRP can cause a major improvement in skin-flap survival in the rat, possibly via inhibition of surgically induced neutrophil recruitment.

Effects of calcitonin gene-related peptide on vascular resistance in rats: role of sex steroids.

It has been demonstrated in reflex-intact animals that the sensitivity to calcitonin gene-related peptide (CGRP) is increased during pregnancy and that this action is mediated by sex steroids but not by nitric oxide (NO). We assessed the effects of CGRP in the following groups of anesthetized ganglion-blocked rats: 1) pregnant, 2) ovariectomized, and 3) ovariectomized and treated with estradiol and progesterone. Changes in mean arterial pressure (MAP) were assessed after the administration of varying doses of CGRP. Decreases in MAP after CGRP administration were significantly greater in pregnant rats and ovariectomized rats administered sex steroids than in ovariectomized controls. The CGRP antagonist CGRP8-37 produced a pressor response of similar magnitude in both pregnant and ovariectomized rats. We also assessed the effects of CGRP and the modulating role of NO in the isolated uterine vascular bed preparation. CGRP reduced perfusion pressure to a greater degree in ovariectomized animals treated with sex steroids than in ovariectomized animals. This response was attenuated by pretreatment with an NO synthesis inhibitor. CGRP8-37 produced a similar increase in perfusion pressure in both groups. We conclude that 1) the increased vascular sensitivity observed during pregnancy or after treatment with sex steroids is in part mediated by NO, and 2) CGRP8-37 has a vasoconstrictor action of its own.

Pregnancy and steroid hormones enhance the vasodilation responses to CGRP in rats.

We recently reported that calcitonin gene-related peptide (CGRP) reversed the hypertension induced by nitric oxide inhibition in pregnant rats and that this effect appeared to be progesterone dependent. In the present study, we examined whether the vasodilator responses to CGRP are increased during pregnancy and whether these responses are steroid hormone dependent. Three groups of ovariectomized (Ovx) rats (n = 4-8 rats/group) were studied 3 days after daily treatment (subcutaneous injection) with progesterone (P; 2 mg/injection, twice daily for 3 days, in 0.2 ml of sesame oil), 17beta-estradiol (E; 2.5 microgram/injection, twice daily for 3 days, in 0.2 ml of sesame oil), or vehicle (sesame oil). A fourth group (n = 6 rats) of pregnant rats was studied on day 19 of gestation. A fifth group of adult, nonpregnant rats (n = 6 rats), regardless of stage of estrous cycle, was also used in this study. Mean arterial blood pressure (MAP) was continuously monitored in fully awake and free-moving instrumented rats. MAP was measured before and after administration of either saline or varying bolus doses of CGRP (9-360 pmol/kg body wt). CGRP produced a dose-dependent decrease in MAP in all rats with a significant (P < 0.05) reduction in MAP beginning with a CGRP dose of 90 pmol/kg and with maximal effects observed at 360 pmol/kg. Decreases in MAP in response to CGRP were significantly (P < 0.05) greater in pregnant compared with nonpregnant rats. Similarly to pregnant rats, Ovx rats given both E and P treatments produced greater decreases in MAP in response to CGRP at 90, 180, and 360 pmol/kg doses compared with both ovary-intact and Ovx nonpregnant rats, which were not different from each other. In summary, these data show that 1) the hypotensive effects of CGRP are dose dependent and 2) the hypotensive effects of CGRP are enhanced during pregnancy and in Ovx rats treated with either E or P. Therefore, we suggest that the decrease in vascular tone that is seen during pregnancy may be mediated, at least in part, by a sex steroid hormone-induced increase in the vascular sensitivity to the vasodilator effects of CGRP.

Modulation of synovial blood flow by the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8-37).

1. The effect of the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8-37) on blood flow in the knee joint of the anaesthetized rat was investigated. 2. Synovial blood flow in both exposed and intact, skin-covered knees was measured by laser Doppler perfusion imaging. 3. Topical application of CGRP(8-37) caused a dose-dependent fall in synovial blood flow in the exposed knee joint of the rat. At low (1.5 nmol) doses of CGRP(8-37) there was no significant effect on synovial blood flow. In rats treated with 7.5 nmol CGRP(8-37) there was a fall in synovial blood flow (maximum effect at 10 min: -28.8 +/- 4.6%; n=7), which returned to resting levels within 30 min. The highest dose (15 nmol) of antagonist used in this study caused a marked (maximum at 10 min: -35.6 +/- 9.3%; n=8), and prolonged (up to 30 min) fall in blood flow. 4. Ten days after surgical denervation, CGRP(9-37) (15 nmol, topical) had no significant effect on blood flow in the rat exposed knee joint (change in flux at 10 min: -5.1+/-3.6%; n=4). This suggests that CGRP(8-37) acts selectively to antagonize the actions of a neurally derived product, probably CGRP, on the rat synovial vasculature. 5. In skin-covered knee joints, intra-articular injection of CGRP(8-37) (15 nmol; bolus) elicited a significant fall in synovial blood flow (maximum effect at 10 min: -15.5 +/- 5.8%; n=6). 6. CGRP (0.01, 0.1 or 1.0 nmol; topical) caused a dose-dependent increase in exposed knee joint blood flow, which was attenuated by co-administration of 1.5 nmol CGRP(8-37). For example, 1 nmol CGRP elicited a peak increase in flux at 10 min of 94.7 +/- 31.8% (n=8) and 28.8 +/- 8.9% (n=7) in the absence and presence of CGRP(8-37), respectively. The vasodilator responses induced by acetylcholine (ACh) (10 nmol, topical; n=4-5) or sodium nitroprusside (SNP) (10 nmol, topical; n=4-5) were unaltered in the presence of CGRP(8-37) (1.5 nmol, topical). 7. Thus, the CGRP receptor antagonist CGRP(8-37) elicits vasoconstriction in the rat synovium. This suggests that the endogenous, basal release of CGRP may play a physiological role in the regulation of blood flow in the rat knee joint.

Spatial heterogeneity of the effects of calcitonin gene-related peptide (CGRP) on the microvasculature of ligaments in the rabbit knee joint.

1. Experiments were performed in anaesthetized rabbits to examine the effects of calcitonin gene-related peptide (CGRP) and the CGRP antagonist CGRP8-37 on blood flow to the medial collateral ligament of the knee joint. 2. Topical application of CGRP (10(-13) to 10(-9) mol) to the exposed external surface of eight knee joints resulted in dose-dependent dilatation of vessels in both the ligament and the joint capsule. The magnitude of this response varied significantly in different regions of the medial collateral ligament, with the 10(-9) mol dose of CGRP giving the maximum response (101.5 +/- 25.3% increase) at the femoral insertion site of the medial collateral ligament and lowest (23.1 +/- 8.8%) at the tibial insertion site. 3. Topical application of CGRP8-37 (0.1, 1 and 10 nmol) produced dose-dependent constriction of vessels in the ligament and the joint capsule in five knees, with a trend towards the greatest effect occurring at the femoral insertion site (45.8 +/- 8.1% reduction in blood flow). With the 10 nmol dose, the vasoconstrictor response at the femoral insertion site differed significantly (P<0.05) from the responses obtained at the tibial insertion and joint capsule sites. 4. Topical application of CGRP8-37 (0.1, 1 and 10 nmol) to four chronically denervated knees produced substantially smaller vasoconstrictor responses at all sites. At the femoral insertion site, where 10 nmol CGRP8-37 normally produces a 45.8 +/- 8.1% reduction in blood flow (n=8), ten days following denervation this response was reduced to 6.5 +/- 6.1%, this difference being significant (P=0.01). 5. Adrenaline was applied topically to augment blood vessel tone, in order to establish how effectively co-administration of CGRP would offset this increase in tone. Adrenaline (10(-10) mol) produced vasoconstriction at all sites (n=6). In the capsule this vasoconstriction was virtually abolished when CGRP (10(-9) mol) was co-administered with adrenaline but in the ligament vasodilatation occurred at all sites. This vasodilatation was significantly greater at the femoral insertion site compared to the tibial insertion and mid ligament sites (P<0.05 for both) and the capsule (P<0.01). 6. Topical application of substance P (10(-10) or 10(-9) mol) failed to elicit dilatation of ligament blood vessels. 7. These results suggest that endogenous CGRP may play an important role in regulating blood flow to different structures in and around the knee joint.

Calcitonin gene-related peptide in viscerosensitive response to colorectal distension in rats.

The role of calcitonin gene-related peptide (CGRP) on colorectal distension-induced visceral pain was investigated in conscious rats. Intracolonic administration of acetic acid (0.6%) resulted in a significantly increased number of abdominal contractions in response to colorectal balloon distension from 5.8 +/- 1.2 in controls to 16.6 +/- 1.0 in acetic acid-treated animals (P < 0.05), evidencing sensitization of visceral afferent pathways and subsequently visceral hyperalgesia. This sensitization phenomenon was not observed in animals previously treated with systemic capsaicin. Likewise, in animals not treated with capsaicin, use of an intravenous antagonist for CGRP [human CGRP-(8-37)], completely reversed the sensitizing effects of acetic acid. Furthermore, intravenous administration of CGRP dose dependently increased the number of abdominal contractions in response to colorectal distension from 3.0 +/- 1.1 (CGRP 250 ng) to 17.0 +/- 1.2 (CGRP 500 ng, P < 0.05), as previously observed in acetic acid-treated animals. Finally, intrathecal administration of hCGRP-(8-37) (mid-lumbar) also resulted in a total dose-dependent reversal of CGRP (500 ng) or acetic acid-induced visceral hypersensitivity. These results demonstrate that CGRP plays a major role in this model of visceral afferent nerve sensitization from gastrointestinal origin.

Calcitonin Gene-related Peptide Increases Pulmonary Blood Flow in Fetal Sheep. † 1602

Calcitonin gene-related peptide (CGRP) is a potent vasodilator. We studied the hemodynamic effects of exogenously administered CGRP on pulmonary blood flow in 7 near-term fetal sheep. Catheters were inserted into the left pulmonary artery (LPA) to administer drugs, and into the main pulmonary and carotid arteries, as well as the left atrium, to measure pressures. An ultrasonic flow transducer was placed around the LPA to measure flow continuously. In addition, LPA resistance was calculated. In each examination, we first studied the hemodynamic effects of a single injection of CGRP into the LPA, compared with those produced by acetylcholine; then we studied the effects of 4 additional sequentially repeated injections of the same dose of CGRP, each injection separated by 5 min. The first injection of CGRP (1.36± 0.13 μg/kg [mean ± SD]) increased LPA blood flow significantly, from 17 ± 10 to 120 ± 21 mL/min, and decreased main pulmonary and carotid arterial pressures, from 58 ± 5 to 48± 6 mm Hg and from 56 ± 3 to 46 ± 5 mm Hg, respectively, with a concomitant decrease in LPA resistance from 1.57 to 0.24 mm Hg · mL· min-1. These changes were restored to baseline within 5 min, and were quite similar to those with acetylcholine (0.55 ± 0.06 μg/kg). With repeated injections of CGRP, LPA blood flow increased significantly in a stepwise fashion with concomitant decreases in LPA resistance. Heart rate(from pressure signal) also increased in a stepwise fashion, while pulmonary and carotid arterial pressures showed no significant decrease. We conclude that exogenously administered CGRP has a pulmonary vasodilatory effect in fetal sheep and increases fetal pulmonary blood flow, and that this effect is cumulative with repeated injections. We also suggest that repeated injections of this peptide probably increase fetal heart rate through a direct positive chronotropic effect, and not through a secondary response following a decrease in systemic blood pressure. These findings suggest a role for endogenous CGRP in the remarkable decrease in pulmonary vascular resistance during the transition from fetal to neonatal life.

Receptor subtypes mediating renal actions of calcitonin gene-related peptide

We previously reported that the renal arterial infusions of non-hypotensive doses of calcitonin gene-related peptide (CGRP) caused renal vasodilatation and increases in glomerular filtration rate at a low dose, but renal vasoconstriction, natriuresis and kaliuresis at a high dose. In the present study, we examined the effects of the specific CGRP 1 receptor antagonist CGRP-(8–37) (1 and 10 nmol/kg) and the putative CGRP receptor antagonist, [Tyr 0]CGRP-(28–37) (3 and 30 nmol/kg), on the renal vascular and tubular effects of CGRP in inactin-anaesthetized Sprague-Dawley rats. Renal arterial infusion of single doses of CGRP (0.3–300 pmol/kg per min) did not significantly alter mean arterial pressure or heart rate. However, during the continuous renal arterial infusion of either CGRP-(8–37) or [Tyr 0]CGRP-(28–37), a high dose of CGRP (300 pmol/kg per min) paradoxically reduced arterial pressure and increased heart rate. CGRP-(8–37) completely but [Tyr 0]CGRP-(28–37) incompletely inhibited the vasodilatation and increments in glomerular filtration rate elicited by low doses of CGRP. Both blockers abolished the renal vasoconstriction but did not inhibit diuresis, natriuresis and kaliuresis elicited by a high but non-hypotensive dose of CGRP. On the basis that CGRP-(8–37) is a competitive CGRP 1 receptor antagonist, our results suggest: (1) the renal vascular effect of CGRP is completely mediated via the activation of CGRP 1 receptors, (2) the renal tubular effects of CGRP are not mediated via CGRP 1 receptors, and (3) [Tyr 0]CGRP-(28–37) is a CGRP 1 receptor antagonist with potency and efficacy less than those of CGRP-(8–37).