Calcitonin Gene-related Peptide Innervation Research Articles

Involvement of the calcitonin gene-related peptide system in the modulation of inflamed uterus contractile function in pigs

This study analyzed severe acute endometritis action on myometrial density and distribution of protein gene product (PGP)9.5- and calcitonin gene-related peptide (CGRP)-like immunoreactive nerve fibers and calcitonin receptor-like receptor (CLR) expression, and on CGRP receptor (CGRPR) participation in uterine contractility in pigs. E. coli suspension (E. coli group) or saline (SAL group) were injected into the uteri, or only laparotomy was performed (CON group). In the E. coli group myometrium, a lack of significant changes in PGP9.5 and CGRP innervation patterns and increased CLR protein level were revealed. In all groups, compared to the pretreatment period, human αCGRP increased amplitude in the myometrium, while reducing it in endometrium/myometrium. In the E. coli group endometrium/myometrium, human αCGRP lowered amplitude vs other groups. Human αCGRP reduced frequency in CON and SAL groups and enhanced it in the E. coli group endometrium/myometrium. The frequency in E. coli group increased vs other groups. CGRPR antagonist, human αCGRP8–37, reversed (CON, SAL groups) and eliminated (E. coli group) the rise in human αCGRP-induced myometrial amplitude. In endometrium/myometrium, human αCGRP8–37 abolished (CON group) and reversed (SAL group) a decrease in frequency, and reduced the rise in frequency (E. coli group) caused by human αCGRP. Collectively, in the myometrium, endometritis did not change PGP9.5 and CGRP innervation patterns and enhanced CLR protein level. CGRPR also mediated in CGRP action on inflamed uterus contractility.

TRPV1 Channel Activated by the PGE2/EP4 Pathway Mediates Spinal Hypersensitivity in a Mouse Model of Vertebral Endplate Degeneration.

Low back pain (LBP) is the primary cause of disability globally. There is a close relationship between Modic changes or endplate defects and LBP. Endplates undergo ossification and become highly porous during intervertebral disc (IVD) degeneration. In our study, we used a mouse model of vertebral endplate degeneration by lumbar spine instability (LSI) surgery. Safranin O and fast green staining and μCT scan showed that LSI surgery led to endplate ossification and porosity, but the endplates in the sham group were cartilaginous and homogenous. Immunofluorescent staining demonstrated the innervation of calcitonin gene-related peptide- (CGRP-) positive nerve fibers in the porous endplate of LSI mice. Behavior test experiments showed an increased spinal hypersensitivity in LSI mice. Moreover, we found an increased cyclooxygenase 2 (COX2) expression and an elevated prostaglandin E2 (PGE2) concentration in the porous endplate of LSI mice. Immunofluorescent staining showed the colocalization of E-prostanoid 4 (EP4)/transient receptor potential vanilloid 1 (TRPV1) and CGRP in the nerve endings in the endplate and in the dorsal root ganglion (DRG) neurons, and western blotting analysis demonstrated that EP4 and TRPV1 expression significantly increased in the LSI group. Our patch clamp study further showed that LSI surgery significantly enhanced the current density of the TRPV1 channel in small-size DRG neurons. A selective EP4 receptor antagonist, L161982, reduced the spinal hypersensitivity of LSI mice by blocking the PGE2/EP4 pathway. In addition, TRPV1 current and neuronal excitability in DRG neurons were also significantly decreased by L161982 treatment. In summary, the PGE2/EP4 pathway in the porous endplate could activate the TRPV1 channel in DRG neurons to cause spinal hypersensitivity in LSI mice. L161982, a selective EP4 receptor antagonist, could turn down the TRPV1 current and decrease the neuronal excitability of DRG neurons to reduce spinal pain.

Low-Intensity Pulsed Ultrasound-Induced Spinal Fusion is Coupled with Enhanced Calcitonin Gene-Related Peptide Expression in Rat Model

Low-intensity pulsed ultrasound (LIPUS) has been found to accelerate fracture healing. In this study, we analyzed the role of calcitonin gene-related peptide (CGRP) in a rat spinal fusion model treated with LIPUS. The results revealed that LIPUS significantly increases bone formation, and the process was coupled with elevated CGRP innervation. CGRP was located in fibrous tissue, closely surrounding the allograft and newly formed cartilage. The density of CGRP peaked at week 3 after surgery in both the control (non-LIPUS-treated) and LIPUS-treated groups. These results suggest that LIPUS might accelerate spinal fusion by promoting sensory nerve fiber innervation.

Methotrexate chemotherapy triggers touch-evoked pain and increased CGRP-positive sensory fibres in the tibial periosteum of young rats

Although bone pain caused by cancer chemotherapy is a well-recognized and significant problem, with approximately 1 in 10 childhood cancer patients being reported to experience isolated bone pain along with other skeletal complications, the underlying mechanisms are poorly understood and there is no specific treatment. In this study, effects of methotrexate (MTX) treatment on pain in the hind legs and the extent of sensory innervation of the tibial bone were examined through a 20-day time course in young rats after 5 daily 0.75mg/kg MTX injections. MTX treatment increased von-Frey filament stimulation-induced mechanical allodynia and palpation nocifensive score in the tibia. MTX-treated rats showed trends in reduced loading (numbers of stands) on hind limbs after palpation, commencing early during treatment and 2weeks after the end of treatment despite no signs of ongoing pain during normal locomotion. Immunohistochemical analyses showed an increase in innervation of calcitonin gene-related peptide (CGRP)-positive sensory nerve fibres in tibial periosteum on days preceding and overlapping with those rats with touch-evoked pain responses and the bone repair phase. These data suggest that methotrexate chemotherapy triggers touch-evoked pain involving enhanced sensory nerve innervation of the bone.

Sensory re‐innervation of the rat uterus postpartum (1131.4)

Sympathetic, parasympathetic and sensory nerves almost completely disappear from the pregnant rat uterus at term but return of nerves post‐partum is not well characterized. Using immunoperoxidase staining for calcitonin gene related peptide (CGRP), we assessed post‐partum (P) sensory re‐innervation of rat uterus in whole mount preparations from days P1, P3, P5, P7, P10 and P14 (n=4/group). At P1, there were few CGRP‐immunoreactive axons, mostly at the mesometrium, where a few blood vessels were lightly innervated. CGRP‐positive axons increased at P3 and P5. There were many growth cones at both times. Muscle innervation was sparse. At P7, innervation was denser, with slightly more axons in the muscle and rare CGRP‐IR axons deep in the endometrium. There were CGRP‐immunoreactive axons in the linea uteri at the ovarian end in half the rats. At P10, the pattern of CGRP innervation was similar to P7. More CGRP‐immunoreactive axons occurred in all regions but re‐innervation of the linea uteri continued to lag behind. CGRP innervation was heaviest at P14, with blood vessels moderately to heavily innervated. Nevertheless, CGRP innervation density in all uterine layers was still much less at P14 than at oestrus. These results show that sensory innervation of the uterus has begun to return at P1. Uterine CGRP innervation increases to P14 but innervation density is still lower than in the non‐pregnant state.Grant Funding Source: Supported by NHMRC Australia

CGRP Inhibits Neurons of the Bed Nucleus of the Stria Terminalis: Implications for the Regulation of Fear and Anxiety

The bed nucleus of the stria terminalis (BNST) is thought to generate anxiety-like states via its projections to autonomic and neuroendocrine regulatory structures of the brain. However, because most BNST cells are GABAergic, they are expected to inhibit target neurons. In contrast with this, infusion of calcitonin gene-related peptide (CGRP) into BNST was reported to potentiate anxiety while activating BNST targets. The present study aimed to shed light on this paradox. The CGRP innervation of BNST originates in the pontine parabrachial nucleus and targets its anterolateral sector (BNST-AL). Thus, we investigated the effects of CGRP on BNST-AL neurons using patch recordings in vitro in male rats. CGRP did not alter the passive properties of BNST-AL cells but increased the amplitude of IPSPs evoked by stimulation of the stria terminalis (ST). However, IPSP paired-pulse ratios were unchanged by CGRP, and there was no correlation between IPSP potentiation and variance, suggesting that CGRP acts postsynaptically. Consistent with this, CGRP hyperpolarized the GABA-A reversal of BNST-AL cells. These results indicate that CGRP increases ST-evoked GABA-A IPSPs and hyperpolarizes their reversal potential through a postsynaptic change in Cl(-) homeostasis. Overall, our findings suggest that CGRP potentiates anxiety-like behaviors and increases neural activity in BNST targets, by inhibiting BNST-AL cells, supporting the conclusion that BNST-AL exerts anxiolytic effects.

Role of peptidergic nerve terminals in the skin: reversal of thermal sensation by calcitonin gene-related peptide in TRPV1-depleted neuropathy.

To investigate the contribution of peptidergic intraepidermal nerve fibers (IENFs) to nociceptive responses after depletion of the thermal-sensitive receptor, transient receptor potential vanilloid subtype 1 (TRPV1), we took advantage of a resiniferatoxin (RTX)-induced neuropathy which specifically affected small-diameter dorsal root ganglion (DRG) neurons and their corresponding nerve terminals in the skin. Thermal hypoalgesia (p<0.001) developed from RTX-treatment day 7 (RTXd7) and became normalized from RTXd56 to RTXd84. Substance P (SP)(+) and TRPV1(+) neurons were completely depleted (p = 0.0001 and p<0.0001, respectively), but RTX had a relatively minor effect on calcitonin gene-related peptide (CGRP)(+) neurons (p = 0.029). Accordingly, SP(+) (p<0.0001) and TRPV1(+) (p = 0.0008) IENFs were permanently depleted, but CGRP(+) IENFs (p = 0.012) were only transiently reduced and had recovered by RTXd84 (p = 0.83). The different effects of RTX on peptidergic neurons were attributed to the higher co-localization ratio of TRPV1/SP than of TRPV1/CGRP (p = 0.029). Thermal hypoalgesia (p = 0.0018) reappeared with an intraplantar injection of botulinum toxin type A (botox), and the temporal course of withdrawal latencies in the hot-plate test paralleled the innervation of CGRP(+) IENFs (p = 0.0003) and CGRP contents in skin (p = 0.01). In summary, this study demonstrated the preferential effects of RTX on depletion of SP(+) IENFs which caused thermal hypoalgesia. In contrast, the skin was reinnervated by CGRP(+) IENFs, which resulted in a normalization of nociceptive functions.

Lack of migraine in headaches of familial dysautonomia patients

Familial Dysautonomia (FD) is an autosomal recessive genetic disease where autonomic and sensory functions are defective affecting many body systems including the vascular. Plasma level of the neurotransmitter Calcitonin Gene Related Peptide (CGRP) is decreased in FD patients. This compound has been implicated to take part in the pathogenesis of migraine. We aimed to evaluate the symptoms of headaches in FD patients and to test the hypothesis that these patients will have a low incidence of migrainous headache. Sixty-five FD patients were evaluated by a medical headache questionnaire. Mean age was 23.73+10.82years (mean 21years) and there were 37 males (57%).Thirty-eight patients (58.5%) described having episodic headache conforming to criteria of tension headache, and in 17 of those 38 (44.7%) headache were dependent on changes in blood pressure, except from one patient who had complaints that matched diagnosis of acephalic migraine. None of the patients had symptoms compatible with migraine or cluster headache. Results show that the headache is a very common complaint in FD, there is lack of migraine symptoms in this group. This might be attributed to defective sensory innervation and deficiency of CGRP. FD could be regarded as a human model for CGRP deficiency when studying the pathogenesis of migraine.

The Mechanism of Calcitonin Gene-Related Peptide–Containing Nerve Innervation

Calcitonin gene-related peptide (CGRP) is a major neurotransmitter and CGRP-containing primary sensory neurons play an important role in nociception and potent vasodilation. CGRP-containing nerves in mesenteric arteries are decreased in pathological animal models (hypertension, diabetes, and atherosclerosis). In apolipoprotein E–knockout mice, which have atherosclerosis and peripheral sensory nerve defects, nerve growth factor (NGF)-mediated CGRP nerve facilitation was down-regulated, which may have been caused by the impairment of the Akt–NO–cGMP pathway. In addition, NGF-mediated CGRP neurite outgrowth was decreased in fructose-induced insulin-resistant rats. We recently discovered that renin–angiotensin inhibitors improved CGRP innervation in spontaneously hypertensive rats, indicating that rescuing CGRP nerve innervation might improve pathophysiological conditions. To find a novel reagent that facilitates CGRP nerves, a new model, phenol-injured perivascular nerve model rats, was established. Adrenomedullin, hepatocyte growth factor, and angiotensin II type 2 receptor activation induced CGRP nerve distribution in phenol-injured rats. Furthermore, in insulin-resistant model rats, the down-regulation of CGRP nerves was likely due to the depression of phosphoinositide 3-kinase (PI3K)-dependent Akt activation. Administration of candesartan improves CGRPergic function via the PI3K–Akt pathway in insulin-resistant rats. Thus, clarification of the mechanisms of CGRP nerve defects may constitute future therapeutic targets.

Involvement of Calcitonin Gene-Related Peptide Innervation in the Promoting Effect of Low-Intensity Pulsed Ultrasound on Spinal Fusion Without Decortication

A prospective left-right comparison designed experiment using a rabbit posterolateral intertransverse process fusion model. To investigate the involvement of calcitonin gene-related peptide (CGRP) innervation in the promoting effect of low intensity pulsed ultrasound stimulation (LIPUS) on spinal fusion without decortication. Sensory neuropeptide CGRP is involved in bone repair and ectopic ossification. Comparison of CGRP innervations in ectopic bone between sham LIPUS and LIPUS sides can help us to understand the relationship between sensory nerve innervation and LIPUS. A total of 27 New Zealand white rabbits underwent bilateral posterolateral intertransverse process fusion with implantation of porous poly-D,L-lactic acid blocks loaded with 1.25 μg recombinant human bone morphogenetic protein-4 solution. One side was provided LIPUS daily whereas the other side served as control. Animals were killed and the operated lumbar vertebrae were harvested for histomorphologic evaluation at 3 days (n = 3), 1 week (n = 6), 3 weeks (n = 6), 7 weeks (n = 6), and 12 weeks (n = 6) following surgery, respectively. LIPUS accelerated the invasion of CGRP-positive nerve fibers during ectopic ossification spatially and temporally. Spatially, CGRP-positive nerve fibers were also observed in the new formed cartilage and bone tissues on LIPUS side, whereas they were only detected in the fibrous tissue and bone marrow on sham LIPUS side. Temporally, the density of CGRP-positive nerve fibers was significantly higher on the LIPUS side when compared with the sham LIPUS side. LIPUS promoted the invasion of CGRP sensory nerve in ectopic bone, which may in turn contribute to the promoting effect of LIPUS on ectopic ossification.

Temporal and Spatial CGRP Innervation in Recombinant Human Bone Morphogenetic Protein Induced Spinal Fusion in Rabbits

Animal experiment using a rabbit posterolateral intertransverse process fusion model. To explore the temporal and spatial distribution of sensory nerve fibers expressing calcitonin-gene related peptide (CGRP) during spinal fusion induced by recombinant human bone morphogenetic protein-4 and the role of the CGRP innervation in ectopic bone formation and remodeling. Sensory neuropeptide CGRP involved in local bone turnover has been evidenced but its underlying mechanism is poorly understood. Knowledge in the CGRP innervation in ectopic bone induced by bone morphogenetic proteins can help us to understand its role in bone turnover. Twenty-seven New Zealand white rabbits underwent single level posterolateral intertransverse process fusion of the lumbar vertebrae with implantation of porous poly-d,l-lactic acid blocks loaded with 1.25 microg recombinant human bone morphogenetic protein-4 solution. Animals were killed and the operated lumbar vertebrae were harvested for histomorphological evaluation at 3 days (n = 3), 1 week (n = 6), 3 weeks (n = 6), 7 weeks (n = 6), and 12 weeks (n = 6) following surgery, respectively. New cartilage presented at 1 week postimplantation adjacent to the implant, reached a peak volume at week 3 followed by a drop till week 12 after its ossification. Trabeculae-like woven bone structure presented at week 3. CGRP-positive nerve fibers regenerated already at 3 days postimplantation, reached its peak density at week 3. The CGRP-positive fibers presented both in fibrous tissues adjacent to proliferating cartilages and in bone marrow of newly formed trabecular bone. The observed spatial and temporal regeneration of CGRP-positive nerve fibers in ectopic bone formation suggested CGRP innervation is associated with ectopic osteogenesis.

Proton Acts as a Neurotransmitter for Nicotine-Induced Adrenergic and Calcitonin Gene-Related Peptide-Containing Nerve-Mediated Vasodilation in the Rat Mesenteric Artery

Nicotine stimulates presynaptic nicotinic acetylcholine receptors in perivascular adrenergic nerves and releases unknown transmitter(s) that activate transient receptor potential vanilloid-1 (TRPV1) located on calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves, resulting in vasodilation. The present study investigated a potential transmitter transmitting between perivascular adrenergic nerves and CGRPergic nerves. Rat mesenteric vascular beds without endothelium were contracted by perfusion with Krebs' solution containing methoxamine, and the perfusion pressure and pH levels of the perfusate were measured. Nicotine perfusion for 1 min induced concentration-dependent vasodilation and lowered pH levels, which were abolished by cold-storage denervation of preparations, guanethidine (adrenergic neuron blocker), and mecamylamine (nicotinic alpha(3)beta(4)-acetylcholine receptor antagonist). Capsazepine (TRPV1 antagonist) blunted nicotine-induced vasodilation, but had no effect on the reduction of pH. Injection of hydrochloric acid (HCl) and perfusion of Krebs' solution at low pH (6.0-7.2) induced vasodilation. HCl-induced vasodilation was inhibited by cold-storage denervation, capsazepine, capsaicin (CGRP depletor), and CGRP(8-37) (CGRP receptor antagonist). Perfusion of adrenergic transmitter metabolites (normetanephrine and 3-methoxydopamine), but not of other metabolites, induced vasodilation, which was not inhibited by capsaicin treatment. Immunohistochemical staining of mesenteric arteries showed dense innervation of CGRP- and TRPV1-immunopositive nerves, with both immunostainings appearing in the same neuron. Mesenteric arteries were densely innervated by neuropeptide Y-immunopositive nerves, which coalesced with CGRP-immunopositive nerves. Scanning and immunoscanning electron microscopic images showed coalescence sites of different perivascular fibers before they intruded into smooth muscles. These results indicate that nicotine initially stimulates adrenergic nerves via nicotinic alpha(3)beta(4)-receptors to release protons and thereby induces CGRPergic nerve-mediated vasodilation via TRPV1.

Retrograde Neuron Tracing with Microspheres Reveals Projection of CGRP-Immunolabeled Vestibular Afferent Neurons to the Vestibular Efferent Nucleus in the Brainstem of Rats

Objective: A new retrograde neuron-tracing technique with microspheres was used to explore the possible innervation of calcitonin gene-related peptide (CGRP)-immunolabeled vestibular afferent neurons in the vestibular efferent immunolabeled nucleus in the brainstem. Methods: 0.1 µl of 5% microfluorospheres was injected into the area of the vestibular efferent nucleus, which is located lateral to the genu of the facial nerve. CGRP immunohistochemistry was processed in serial sections of the brainstem at the facial nerve genu level. Double-labeled neurons with both CGRP immunoreactivity and microfluorospheres were examined with fluorescence and confocal laser microscopy. Results: Three types of labeled neurons were observed: (1) neurons only retrogradely microfluorosphere-labeled that were mainly located in the medial vestibular nucleus, lateral vestibular nucleus, superior vestibular nucleus and parvicellular reticular nucleus on the ipsilateral side of the injection; (2) neurons that were both immunolabeled with CGRP and also retrogradedly labeled with microfluorospheres, indicating that they are CGRP cells projecting to the area of vestibular efferent nucleus, these cells were mainly distributed in the superior vestibular nucleus and dorsal vestibular nucleus, and (3) cells only immunolabeled for CGRP that were scattered extensively in the brainstem. Conclusion: The presented methodical contribution demonstrates the suitability of fluorescein-labeled microspheres for retrograde neuronal tracing. The vestibular nuclei contain numerous afferent neurons that send projections to the vestibular efferent nucleus, some of which are CGRP cells. This afferent innervation provides morphological evidence that the vestibular efferent neurons receive input from the vestibular afferent neurons including CGRP cells. These vestibular primary CGRP afferent neurons may have an influence on vestibular efferent neurons. CGRP acts as an important co-transmitter or modulator in the afferent-mediated activity of vestibular efferent neurons, which in turn affect afferents in the vestibular end organs.

Pregnancy increases perivascular trigeminal innervation of cerebral arteries without changing reactivity to calcitonin gene‐related peptide (CGRP)

This study investigated the influence of pregnancy and postpartum on trigeminal innervation of posterior cerebral arteries (PCA) and reactivity to CGRP. Third-order PCAs were dissected from nonpregnant (NP, n=35), late-pregnant (LP, day 19–20, n=32), or postpartum (PP, day 3–4, n=32) Sprague Dawley rats and either fixed for immunohistochemical staining of CGRP-reactive nerve fibers or mounted in an arteriograph chamber for concentration-response curves to CGRP (0.1–300nM). Nerve density was determined by Metamorph analysis as intersect points on a grid per area after confocal imaging. Pregnancy had a trophic influence on perivascular CGRP-containing nerve fibers, increasing nerve density from 0.6 ± 0.2 counts/μm2 in NP to 1.3 ± 0.3 counts/μm2 in LP (p<0.01). Nerve density significantly decreased in PP animals to 0.2 ± 0.1 counts/μm2 (p<0.01 vs. LP). The gestation-induced increase in CGRP innervation was not accompanied by a change in reactivity. Percent dilation from spontaneous tone at 300nM CGRP for NP, LP and PP animals was: 70±7%, 69±12% and 74±10% (p>0.05). These results demonstrate that trigeminal innervation of PCAs is increased during gestation that is not accompanied by changes in reactivity. It is possible that increased CGRP nerve density during pregnancy relates more to nociception and a predisposition to the neurologic symptoms of eclampsia than to changes in blood flow. Supported by RO1 NS045940 and AHA 0540081N.

Adrenomedullin facilitates reinnervation of phenol-injured perivascular nerves in the rat mesenteric resistance artery

Our previous report showed that innervation of calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-containing nerves in rat mesenteric resistance arteries was markedly reduced by topical application of phenol, and that nerve growth factor (NGF) facilitates the reinnervation of both nerves. We also demonstrated that a CGRP superfamily peptide, adrenomedullin, is distributed in perivascular nerves of rat mesenteric resistance arteries. In the present study, we investigated the influence of adrenomedullin on the reinnervation of mesenteric perivascular nerves following topical phenol treatment. Under pentobarbital-Na anesthesia, 8-week-old Wistar rats underwent in vivo topical application of phenol (10% phenol in 90% ethanol) to the superior mesenteric artery proximal to the bifurcation of the abdominal aorta. After the treatment, the animals were subjected to immunohistochemistry of the third branch of small arteries proximal to the intestine and to vascular responsiveness testing on day 7. Topical phenol treatment caused marked reduction of the density of NPY-like immunoreactive (LI)- and CGRP-LI nerve fibers in the arteries. Adrenomedullin (360 or 1000 ng/h) or NGF (250 ng/h), which was administered intraperitoneally for 7 days using an osmotic mini-pump immediately after topical phenol treatment, significantly increased the density of CGRP-LI- and NPY-LI nerve fibers compared with saline. Treatment with adrenomedullin (1000 ng/h) or NGF restored adrenergic nerve-mediated vasoconstriction and CGRP nerve-mediated vasodilation in the perfused mesenteric artery treated topically with phenol. These results suggest that adrenomedullin, like NGF, has a facilitatory effect on the reinnervation of perivascular nerves.

Increased Innervation and Ripening of the Prepartum Murine Cervix

Ripening of the cervix before birth is coincident with reduced collagen content and leukocyte immigration, characteristics that are analogous to a neurogenic inflammatory-like process. We sought to assess the morphologic relationship between innervation and remodeling of the peripartum cervix. Cervix was obtained from C3H/HeN mice on days 15 and 18 of pregnancy, 1 day postpartum, and from non-pregnant controls. Tissues were immersion-fixed, paraffin-embedded, and some sections stained with Picrosirius red to assess collagen content and complexity of organization. By image analysis of optical density, collagen content and structure were significantly decreased by the day before birth. Other sections were processed to visualize nerve fibers by immunohistochemistry with antibodies against neuron-specific epitopes, PGP9.5, peripherin, as well as brain nitric oxide synthase (bNOS), calcitonin gene-related peptide (CGRP), and other neuropeptides. Fiber density was assessed stereologically and normalized to cell density in non-pregnant cervix to correct for tissue hypertrophy due to reproductive status. In groups of non-pregnant, day 15 pregnant, and postpartum mice, cervix contained nerve fibers that were immunoreactive for the pan-neural markers PGP9.5 and peripherin. Punctate and beaded varicosities were sparsely distributed in stroma, subepithelium, and in proximity to vascular structures. By day 18 of pregnancy, 1 day before birth, fiber density was increased fourfold or more compared to other groups. bNOS fibers and, to a lesser extent, CGRP accounted for most of the increased innervation of the murine cervix by the day before birth, a period when macrophage numbers are enhanced. The findings suggest that increased bNOS and CGRP innervation contribute to early inflammatory-like processes that ripen the cervix before birth.

Potential role of the neuropeptide CGRP in the induction of differentiation of rat hepatic portal vein wall

The media of the rat hepatic portal vein is composed of an internal circular muscular layer (CL) and an external longitudinal muscular layer (LL). These two perpendicular layers differentiate progressively from mesenchymal cells within the first month after birth. In this paper, we studied the development of calcitonin gene-related peptide (CGRP) innervation during post-natal differentiation of the vessel. We show that CGRP innervation is already present around the vessel at birth in the future adventitia but far from the lumen of the vessel. Progressively, CGRP immunoreactive fibers reached first LL then CL. CL by itself become only innervated at day 14 after birth. This corresponds to the time at which thick filaments (myosin) are visible in electron microscopy and desmin visualisable by immunocytochemistry. Furthermore, we provide evidence by autoradiography, that binding sites for CGRP are transiently expressed on the portal vein media at day 1 and 14 after birth. Vascular smooth muscle cells were transfected with constructs containing promoters for desmin or smooth muscle myosin heavy chain (smMHC). CGRP treatment of the cells significantly increased the expression of smMHC. Overall these results suggest that CGRP can potentially influence the differentiation of smooth muscle cells from the vessel wall.

Long-Term Inhibition of Angiotensin Prevents Reduction of Periarterial Innervation of Calcitonin Gene-Related Peptide (CGRP)-Containing Nerves in Spontaneously Hypertensive Rats

The aim of this study was to investigate age-related changes in the density of calcitonin gene-related peptide (CGRP)-containing nerve fibers in spontaneously hypertensive rats (SHR) and the effects of long-term inhibition of the renin-angiotensin system on these changes. The density of immunocytochemically stained nerve fibers in the mesenteric artery was quantified by computer-assisted image processing. An age-related decrease in the density of CGRP-like immunoreactive (LI)-containing nerve fivers but not neuropeptide Y (NPY)-LI-containing sympathetic nerve fibers was found in the mesenteric artery of SHR but not Wistar Kyoto rats (WKY). The density of NPY-LI-containing sympathetic nerve fibers was significantly greater in SHR than in WKY. SHR were treated for 7 weeks with angiotensin converting enzyme inhibitor (0.005% temocapril), angiotensin II type-1 (AT1) receptor antagonist (0.025% losartan) or vasodilator (0.01% hydralazine) in their drinking water. Each drug treatment significantly lowered the systolic blood pressure measured by tail-cuff method. Long-term treatment of SHR with temocapril and losartan significantly increased the density of CGRP-LI-containing nerve fibers in mesenteric arteries. However, the density after hydralazine treatment was similar to the level in non-treated SHR. The density of NPY-LI-containing nerve fibers was not increased by any of the drug treatments. These results suggest that long-term inhibition of the renin-angiotensin system in SHR prevents remodeling of CGRPergic nerve fibers and prevents the reduction of CGRPergic nerve function.

Distribution and regional variation of pituitary adenylate cyclase activating polypeptide and other neuropeptides in the rat urinary bladder and ureter: effects of age.

The distribution and regional variation of nerves immunoreactive for the neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP), calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal polypeptide were investigated in the urinary bladder and distal ureter of young adult (3 months) and aged (24 months) male Wistar rats by indirect immunohistochemistry. Semi-quantitative estimations of nerve densities were made of peptidergic fibres innervating the dome, body and base of the urinary bladder and distal ureter. Sensory innervation of the dome was very sparse and the overall density of innervation increased progressively towards the base of the bladder. The density of innervation in the aged rats was closely comparable to that in the young adults, with the exception of slight reductions in CGRP and SP innervation of the muscle layer. Moreover, there was a marked reduction in the density of PACAP innervation of the subepithelial plexus and of the muscle layer of the bladder base. However, radioimmunoassay showed no significant difference (P>0.05) in PACAP contents between young and aged rat urinary bladder. In the distal ureter of aged rats the densities of innervation by fibres immunoreactive for SP and PACAP but not CGRP were reduced. These findings suggest that the level of sensory innervation of the bladder and distal ureter are reduced in old age and that the afferent limb of voiding reflexes may in consequence be perturbed.

Adrenomedullin inhibits neurotransmission of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves in rat mesenteric resistance arteries

We have reported that the rat mesenteric resistance artery has innervation of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRPergic nerves). We also demonstrated that adrenomedullin (AM) causes mesenteric vasodilation through activation of CGRP receptors. The present study was designed to examine the effect of AM on neurotransmission of CGRPergic nerves in rat mesenteric arteries. In preconstricted preparations without endothelium, periarterial nerve stimulation (PNS, 1 and 2 Hz) induced a frequency-dependent vasodilation. A bolus injection of CGRP (10 pmol) into the perfusate also caused a vasodilation. AM (0.1 to 10 nM) concentration-dependently caused 40% to 60% inhibition of the PNS-induced vasodilation, but AM did not attenuate vasodilation induced by exogenous CGRP injection. The inhibitory effect of AM (10 nM) on PNS-induced vasodilation was further potentiated by CGRP [8–37] (CGRP receptor antagonist, 50 nM), which attenuated the vasodilator response to the CGRP injection. Combined perfusion of AM [22–52] (AM receptor antagonist, 10 to 100 nM) resulted in further inhibition of PNS-induced neurogenic vasodilation without affecting the vasodilator response to the CGRP injection. CGRP [8–37] but not AM [22–52] antagonized vasodilation induced by AM perfusion. These findings suggest that AM presynaptically inhibits neurotransmission of CGRPergic nerves, probably decreasing CGRP release, via receptors different from CGRP receptors.