CGRP-like Immunoreactivity Research Articles

Differential effects of olanzapine, haloperidol and risperidone on calcitonin gene-related peptide in the rat brain

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide which acts on central nervous system (CNS) neurons and is involved in activities related to dopamine. These effects of CGRP suggest that the peptide may have a role in pathophysiology and treatment of schizophrenia where dopaminergic system hypoactivity in the frontal cortex and hyperactivity in the subcortical structures have been demonstrated. In this study we measured by radioimmunoassay (RIA) the brain levels of CGRP-like immunoreactivity (CGRP-LI) in rats treated with either classical (haloperidol) or atypical (risperidone and olanzapine) antipsychotic drugs. Both haloperidol and risperidone decreased CGRP-LI in the striatum. Risperidone also decreased CGRP-LI in the occipital cortex. On the other hand, olanzapine increased CGRP-LI in the striatum, the frontal cortex and hypothalamus. The differential effects on CGRP could reflect a different profile of side effects and further suggest that CGRP is involved in CNS functions related to psychiatric disorders.

Modulation of Capsaicin-Sensitive Nerve Activation by Low pH Solutions in Guinea-Pig Lung

We have studied the stimulation of airways sensory nerves by low pH solutions and concomitantly induced bronchoconstriction. The effect of low pH buffer and lactic acid solutions at the same pH (5 and 6) were compared and the influence of low pH on the capsaicin effect was recorded. We have used the isolated guinea-pig perfused lung model taking the insufflation pressure as an indicator of bronchial smooth muscle tone while the calcitonin gene-related peptide-like immunoreactivity measured in the lung perfusate represented sensory nerves activation. Low pH buffer and lactic acid solution (3 and 4.1 mM) at the same pH of 5 and 6 induced pH-dependent bronchoconstriction and peptides release which were completely abolished after systemic pretreatment with capsaicin. Both responses were significantly inhibited after Ca2+-free infusion. Capsazepine (10(-6) M), a selective capsaicin antagonist, significantly reduced the calcitonin gene-related peptide-like immunoreactivity overflow evoked by all the solutions studied. Diclofenac (10(-5) M), a cyclooxygenase blocker, inhibited pH 5, pH 6 and lactic acid 3 mM (pH 6)-evoked peptide release, but not lactic acid 4.1 mM (pH 5). The functional response was not significantly modified after diclofenac while only the lactic acid 3 mM response was significantly reduced by capsazepine. There was a synergistic interaction between capsaicin and low pH buffer on calcitonin gene-related peptide-like immunoreactivity release and an additive effect on bronchoconstriction. It is concluded that in the isolated perfused guinea-pig lung, lactic acid and low pH buffer induced calcitonin gene-related peptide-like immunoreactivity release and bronchoconstriction by stimulation of capsaicin-sensitive C fibres via a pathway partly dependent of extracellular Ca2+. The mechanism of calcitonin gene-related peptide-like immunoreactivity release seems to be the same at pH 6, while differences are evident at pH 5 between low pH buffer and lactic acid. Our results also suggest that proton activity could exert a modulatory role on the capsaicin-sensitive sensory nerves by a mechanism which remains to be clarified.

Neurotrophic Effect of Hepatic Growth Factor (HGF) on Reinnervation of Perivascular Calcitonin Gene-Related Peptide (CGRP)-Containing Nerves Following Phenol-Induced Nerve Injury in the Rat Mesenteric Artery

Hepatic growth factor (HGF) has neurotrophic effects in the motor neurons and central nervous system. However, there has been no report about the neurotrophic action on perivascular nerves innervating the resistance artery. We investigated whether HGF can restore innervation or function of perivascular nerves, including neuropeptide Y (NPY)-containing sympathetic adrenergic nerves and calcitonin gene-related peptide (CGRP)-containing nerves, in rat mesenteric artery. To investigate HGF-mediated neurotrophic effects, Wistar rats under pentobarbital-Na anesthesia underwent in vivo perivascular denervation by topical application of phenol on the superior mesenteric artery, and then HGF or nerve growth factor (NGF) was administered for 7 days using an osmotic mini-pump after phenol-treatment. HGF significantly increased the density and number of CGRP-like immunoreactivity (LI)–containing nerve fibers compared with saline administration, while HGF did not affect the density of NPY-containing adrenergic nerve fibers. After 7-day treatment with HGF and phenol, the vascular response of vasodilation was recovered from nerve injury by phenol treatment, but vasoconstriction was not. HGF and NGF induced neurite outgrowth in rat cultured dorsal root ganglia (DRG). These results suggest that HGF has a specific neurotrophic action on reinnervation of vascular CGRP-LI–containing nerve fibers in the rat mesenteric artery and DRG.

Influence of liquid diet feeding on calcitonin gene-related peptide-like immunoreactive nerve fibers in rat temporomandibular joints during growth period

Biomechanical factors of masticatory functions are related to the development of the temporomandibular joint (TMJ) components during growth. However, the effects on nerve fibers have not been fully clarified. We investigated the influence of masticatory muscle activity on nerve fibers in the TMJ during growth by changing the consistency of diet. Twenty 3-week-old male Wistar rats were divided into two groups. The experimental group was fed a liquid diet, while the control group had a normal hard diet. Rats were sacrificed at 6-, and 9-week-old. We investigated the expression of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers, which are most abundant in the TMJ and have a nociceptive function, using immunohistochemistry for CGRP. No significant difference in the number of CGRP-LI nerve fibers was detected in 6-week-old rats, but in 9-week-old ones the number in the experimental group was significantly greater than that in the control. We concluded that masticatory muscle activity in the TMJ closely correlates with peripheral sensory nerve fibers during growth.

Participation of the spinal TRPV1 receptors in formalin-evoked pain transduction: a study using a selective TRPV1 antagonist, iodo-resiniferatoxin

The involvement of spinal transient receptor potential vanilloid 1 (TRPV1) in formalin-evoked pain has remained unclear, because investigation of this kind of pain with selective antagonists has not been conducted. The purpose of this study is to investigate the participation of spinal TRPV1 in formalin-evoked pain with iodo-resiniferatoxin (I-RTX), a potent TRPV1-selective antagonist. I-RTX given intrathecally dose-dependently and significantly decreased the number of flinching responses in the formalin-evoked 1st and 2nd phase with ID50 values (drug dose producing 50% inhibition of response) of 1.0 and 3.8 microg, respectively, and concentration-dependently suppressed capsaicin-evoked calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) release from rat spinal cord slices with an IC50 value (drug concentration producing 50% inhibition of response) of 86 nM. Capsazepine, a classical non-selective TRPV1 antagonist, given intrathecally also inhibited formalin-evoked flinching in both the 1st and 2nd phase with ID50s of 420 and 200 microg, respectively, and CGRP-LI release from rat spinal cord slices with an IC50 of 7.8 microM. Ratios of in-vivo analgesic potencies of I-RTX and capsazepine well reflected their intrinsic in-vitro activity. These findings suggest that spinal TRPV1 participates in the transduction system of formalin-evoked pain.

Innervation and functional changes in mesenteric perivascular calcitonin gene-related peptide- and neuropeptide Y-containing nerves following topical phenol treatment

We have previously shown that age-related reduction of innervation and function in mesenteric perivascular calcitonin gene-related peptide-containing vasodilator nerves takes place in spontaneously hypertensive rats. The present study was performed to investigate innervation and functional changes in perivascular calcitonin gene-related peptide- and adrenergic neuropeptide Y-containing nerves after topical treatment with phenol, which damages nerve fibers, around the rat superior mesenteric artery. Under pentobarbital-Na anesthesia, 8-week-old Wistar rats underwent in vivo topical application of phenol (10% phenol in 90% ethanol) or saline (sham rats) to the superior mesenteric artery proximal to the bifurcation of the abdominal aorta. After the treatment, the animals were subjected to immunohistochemistry of the 3rd branch of small arteries proximal to the intestine and to vascular responsiveness testing on day 3 through day 14. The innervation levels of calcitonin gene-related peptide-like immunoreactivity containing fibers and neuropeptide Y-like immunoreactivity containing fibers were markedly reduced on day 3 to day 14 and on day 5 to day 14 after the treatment, compared with those in sham-operated rats, respectively. In perfused mesenteric vascular beds isolated from phenol-treated rats, adrenergic nerve-mediated vasoconstriction and calcitonin gene-related peptide nerve-mediated vasodilation in response to periarterial nerve stimulation (2–12 Hz) were significantly decreased on day 3 and day 7. Neurogenic release of norepinephrine in phenol-treated rats on day 7 was significantly smaller that that in sham-operated rats. Nerve growth factor content in the mesenteric arteries of phenol-treated rats was significantly lower than that in sham-operated rats. Administration of nerve growth factor using osmotic mini-pumps for 7 days after the phenol treatment resulted in greater density of calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity fibers than in phenol-treated rats and restored decreased vascular responses to periarterial nerve stimulation. These results suggest that topical phenol-treatment of the mesenteric artery effectively induces functional denervation of perivascular nerves, which can be prevented or reversed by nerve growth factor treatment.

Involvement of an increased spinal TRPV1 sensitization through its up-regulation in mechanical allodynia of CCI rats

Vanilloid receptor 1 (TRPV1) antagonists are known to attenuate the neuropathic pain symptoms in peripheral nerve injury models, but the mechanism(s) of their effect remains unclear. At the same time, the role of spinal TRPV1 in pain transduction system has not been fully understood. In this study, the role of spinal TRPV1 in mechanical allodynia in rat chronic constriction injury (CCI) model was investigated. Intrathecal administration of a selective TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropryazine-1(2H)-carbox-amide (BCTC) significantly attenuated mechanical allodynia in CCI rats at 100 and 300 nmol. In vitro, BCTC inhibited capsaicin (300 nM)-induced releases of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and substance P-like immunoreactivity (SP-LI) from the rat spinal cord slice preparations with IC 50s of 37.0 and 36.0 nM, respectively, confirming that BCTC potently inhibits TRPV1 function in the rat spinal cord. TRPV1 expression levels in the spinal cord following CCI were quantified in by Western blot analysis. TRPV1 protein levels were significantly increased in the ipsilateral side of the lumbar spinal cord at 7 and 14 days following CCI surgery, but not in the contralateral side. Furthermore, capsaicin (300 nM)-evoked release of CGRP-LI was significantly higher in the ipsilateral spinal cord of CCI rats (14 days after surgery) than that of sham-operated rats. These findings suggest that an increased sensitization of the spinal TRPV1 through its up-regulation is involved in the development and/or maintenance of mechanical allodynia in rat CCI model.

Diabetes Mellitus Influences the Degree of Colocalization of Calcitonin Gene-Related Peptide With Insulin and Somatostatin in the Rat Pancreas

Although calcitonin-gene related peptide (CGRP) (C162H262N50O50S2) has been shown to be present in pancreatic islet cells, no data have been reported on the pattern of its distribution in the islets of Langerhans of diabetic rats. The present study used immunohistochemical and immunofluorescence techniques to examine the pattern of distribution of CGRP-like immunoreactive (CGRP-LIR) cells and nerves in the pancreata of rats with streptozotocin-induced diabetes. The effect of CGRP on insulin secretion from rat pancreatic tissue fragments was also investigated using a radioimmunoassay technique. Numerous CGRP-LIR cells were observed in both the peripheral and central regions of the islets of Langerhans of normal pancreata where they colocalized with large and small subsets of insulin-LIR and somatostatin-LIR cells, respectively. By contrast, the islets of diabetic rat pancreata contained significantly (P < 0.0001) fewer CGRP-LIR cells compared with normal rats. In diabetic rat pancreata, CGRP was colocalized with larger and smaller subsets of somatostatin-LIR and insulin-LIR cells, respectively. CGRP-LIR nerve fibers were discerned in the perivascular and periacinar regions of the pancreata of both normal and diabetic rats. CGRP (10 M) induced a significant (P < 0.02) increase in insulin secretion from the pancreas of normal rat. CGRP is colocalized with insulin and somatostatin in the pancreata of normal and diabetic rats and may play an important role in the humoral and neural regulation of the endocrine pancreas.

A model of periodontitis in the rat: effect of lipopolysaccharide on bone resorption, osteoclast activity, and local peptidergic innervation.

To establish and characterise a rat model of periodontitis that reiterates the features of human disease. Periodontal inflammation was induced by a single injection of 10 microg liposaccharide (LPS) (Salmonella typhimurium) in 1 microl saline into rat mandibular gingiva at the buccomesial aspect of the second molar. Animals were killed after 3, 7 and 10 days, mandibles dissected and sectioned for histological and immunocytochemical analysis. LPS injection resulted in a significant gingival and periodontal inflammation with inflammatory infiltrate, apical migration of the junctional epithelium, interdental bone loss, and activation of osteoclasts at the site of injection 7 and 10 days after injection. At 10 days post injection, there was a significant trend for bone loss on both sides of the mandible. Periodontal inflammation was associated with alteration in the levels of calcitonin gene-related peptide-like immunoreactivity in nerve terminals innervating the inflamed gingival papilla. Intragingival injection of LPS in the rat provides an easily induced reproducible experimental model of periodontal inflammation that reiterates features of human disease.

Distribution of calcitonin gene-related peptide at the neuromuscular junction of mdx mice.

In normal skeletal muscle, the protein dystrophin is associated with plasma membrane glycoproteins and may be involved in the stabilization of the sarcolemma. Mutant mdx mice are markedly deficient in dystrophin and show muscle fiber necrosis followed by regeneration. Changes in the distribution of acetylcholine receptors (AChRs) have been reported at the neuromuscular junction of mdx mice possibly as a result of alterations in the release or response to neural trophic factors. One such factor is calcitonin gene-related peptide (CGRP), which has been implicated in AChR synthesis and function. In this study, we used rhodamine-alpha-bungarotoxin and anti-CGRP IgG FITC to study AChR and CGRP distribution at the neuromuscular junction of mdx mice. Using laser scanning fluorescence confocal microscopy, it was possible to see that CGRP-like immunoreactivity had a presynaptic distribution, covering the AChRs. Thirty-four percent of dystrophic junctions were found to be labeled with CGRP compared to 80% of control endplates. Since CGRP-positive and -negative fibers showed similar changes in AChR distribution, it is suggested that CGRP is probably not directly involved in the altered pattern of AChR seen in dystrophin-deficient muscle fibers of mdx mice.

Plastic changes of calcitonin gene-related peptide in morphine tolerance: behavioral and immunohistochemical study in rats.

The present study was undertaken to investigate the plasticity of calcitonin gene-related peptide (CGRP) in antinociception after morphine tolerance in rats. The hindpaw withdrawal latencies (HWLs) to both thermal and mechanical stimulation increased significantly after intracerebroventricular injection of 2.5 nmol of CGRP in opioid-naive rats, indicating that CGRP produces an antinociceptive effect in the brain. Furthermore, there was an antinociceptive effect after intracerebroventricular injection of 2.5 nmol of CGRP in morphine-tolerant rats. Interestingly, the antinociceptive effect induced by intracerebroventricular injection of CGRP was lower in morphine-tolerant rats than that in opioid-naive rats at the same dose. At the same time, there was downregulation of CGRP-like immunoreactivity in both lateral septal nucleus and central nucleus of amygdala tested by immunohistochemical methods, whereas no significant changes were observed in arcuate nucleus of hypothalamus and periaqueductal gray after morphine treatment in rats. The present study demonstrates plastic changes in both CGRP-induced antinociception and CGRP-like immunoreactivity in rat brain after morphine tolerance, suggesting that CGRP may play an important role in morphine tolerance.

Novel distribution of calcitonin gene-related peptide in rodent subcommissural organs

For the first time we showed the presence of CGRP immunoreactivity in the SCO of golden hamsters, Wistar rats and Mongolian gerbils. In hamsters, the intense CGRP-like immunoreactivity was detected in the whole SCO, including Reissner's fiber- and granule-like structures at the ventricular surface of the SCO. The CGRP-positive hypendymal cells were frequently in contact with local blood vessels and some of them were situated intimately to the leptomeningeal space. In the SCOs of rats and gerbils, only the supranuclear area and apical cytoplasm of the ependymal cells were positive for CGRP, whereas the basal pole of the cells and the hypendyma were CGRP-negative. The existence of CGRP in rodents SCO was confirmed by the expression of CGRP mRNA in rat SCO by RT-PCR. The present results indicate that the SCOs of rodent species contain CGRP that may be in part synthesized by ependymocytes themselves. A species difference in the CGRP distributions of rodents SCOs may therefore imply its different synthesis rates or release pathways.

Spinal administration of lipoxygenase inhibitors suppresses behavioural and neurochemical manifestations of naloxone-precipitated opioid withdrawal.

1. This study investigated the role of spinal lipoxygenase (LOX) products in the induction and expression of opioid physical dependence using behavioural assessment of withdrawal and immunostaining for CGRP and Fos protein expression in the spinal cord. 2. Administration of escalating doses (5-50 mg kg-1; i.p.) of morphine for 5 days markedly elevated CGRP-like immunoreactivity in the dorsal horn of the rat spinal cord. Naloxone (2 mg kg-1; i.p.) challenge precipitated a robust withdrawal syndrome that depleted CGRP-like immunoreactivity and increased the number of Fos-like immunoreactive neurons in the dorsal horn. 3. Intrathecal administration of NDGA (10, 20 microg), a nonselective LOX inhibitor, AA-861 (1.5, 3 microg), a 5-LOX selective inhibitor, or baicalein (1.4, 2.8 microg), a 12-LOX selective inhibitor, concurrently with systemic morphine for 5 days or as a single injection immediately preceding naloxone challenge, blocked the depletion of CGRP-like immunoreactivity, prevented increase in the number of Fos-like immunoreactive neurons in the dorsal horn, and significantly attenuated the morphine withdrawal syndrome. 4. The results of this study suggest that activity of LOX products, at the spinal level, contributes to the expression of opioid physical dependence, and that this activity may be expressed through increased sensory neuropeptide release.

Chronic Angiotensin II Inhibition Increases Levels of Calcitonin Gene-Related Peptide mRNA of the Dorsal Root Ganglia in Spontaneously Hypertensive Rats.

We previously reported that the vasodilation mediated by calcitonin gene-related peptide (CGRP)-containing nerves and level of CGRP mRNA in the dorsal root ganglia (DRG) in spontaneously hypertensive rats (SHR) decreased with age, and that the reduced function of CGRP nerves was restored by chronic inhibition of angiotensin II. The present study was performed to investigate the effect of long-term treatment with angiotensin II type-1 receptor antagonists (L-158,809 and olmesartan), an angiotensin converting enzyme inhibitor (temocapril) and hydralazine on levels of CGRP mRNA in DRG of SHR and the contents of CGRP in the mesenteric artery and atrium. The level of CGRP mRNA and degree of CGRP-like immunoreactivities (CGRP-LI) were measured by Northern blot hybridization assay and enzyme-linked immunosorbent assay, respectively. Seven week-treatment of 8 week-old SHR with temocapril (0.005%), L-158,809 (0.001%), olmesartan (0.01%) or hydralazine (0.01%) administered in drinking water significantly lowered the systolic blood pressure of SHR. The level of CGRP mRNA in the DRG of control SHR was significantly lower than that in normotensive Wistar Kyoto rats (WKY), whereas the level of CGRP-LI in the mesenteric artery and atrium of SHR were significantly greater than those in WKY. Treatment of SHR with temocapril, L-158,809, or olmesartan, but not hydralazine, significantly elevated the levels of CGRP mRNA in DRG, markedly increased the level of CGRP-LI in the mesenteric artery, and slightly increased the CGRP-LI level in the atrium. These results suggest that long-term inhibition of angiotensin II restores the reduced expression of CGRP mRNA in DRG and may facilitate neurotransmission of CGRP-containing vasodilator nerves in SHR.

C-fiber modulation of the rat type I slowly adapting mechanoreceptor

Effects of C-fiber activation on type I slowly adapting mechanoreceptor responses were investigated in a rat in vitro nerve–skin preparation using controlled mechanical stimuli. Two changes in behavior were evoked by antidromic C-fiber stimulation: (1) The type I response to mechanical stimuli was modulated in a graded fashion by antidromic C-fiber activation. The average decrease in mechanoresponse from baseline discharge was 53% at 20-Hz, 51% at 5-Hz, and 30% at 1-Hz stimulation rate. The type I response recovered to baseline levels following termination of antidromic electrical stimulation. (2) Antidromic C-fiber activation generated a spontaneous ongoing activity in many skin units; this was independent of mechanical stimulation and outlasted electrical stimulation. The fact that neither antidromic electrical stimulation of the crushed nerve trunk nor selective A-fiber activation elicited these reactions suggests that they were mediated via action potentials of slowly conducting (C-fiber) axons. Immunohistochemical staining revealed both substance P- and calcitonin gene-related peptide-like immunoreactivity in small unmyelinated nerve fibers entering the touch dome. These results support the concepts that (1) the type I slowly adapting mechanoreceptor in rat receives input from nociceptive terminals within the touch dome. (2) The function of type I slowly adapting mechanoreceptors is modulated by axon reflex activation of nociceptor terminals, which may play a role in altering the type I response during states of mechanical allodynia and have paracrine and autocrine influences on maintenance of touch dome structure.

ENOS-dependent vascular interaction between nitric oxide and calcitonin gene-related peptide in mice: gender selectivity and effects on blood aggregation

The present study was performed to explore a possible vascular interplay between nitric oxide (NO) and calcitonin gene-related peptide (CGRP). We examined factors affecting CGRP release by the NO donor, nitroglycerin (NTG) and the potential involvement of endothelial NO synthase (eNOS) using eNOS knockout (−/−) vs. wild-type (+/+) mice. In the female eNOS (+/+) mice, but not in males, in vitro NTG (0.73 mM) induced significant increases in the release of CGRP-like immunoreactivity (CGRP-LI) from the aorta and the heart but not from the small intestine. In eNOS (−/−) mice, NTG incubation did not induce any CGRP-LI changes in either gender. These results suggest that NTG-induced CGRP release is eNOS-dependent and tissue- and gender-selective. The functional implication of this NO–CGRP interaction was further examined by testing the anti-aggregatory action of acetylcholine (Ach). Ach-induced platelet inhibition was significantly enhanced by the addition of aorta segments of either gender. However, the female aorta segments exhibited a greater platelet inhibitory effect, which could be reversed by the blockade of either CGRP or eNOS. Our study revealed a novel eNOS-dependent interaction between NO and CGRP, and the possible participation of regulatory peptides in affecting platelet function and possibly cardiovascular protection in females.

Calcitonin gene-related peptide immunoreactivity and afferent receptive properties of dorsal root ganglion neurones in guinea-pigs.

To establish the afferent receptive properties of lumbosacral dorsal root ganglion (DRG) neurones that express calcitonin gene-related peptide (CGRP), intracellular recordings were made with fluorescent dye-filled electrodes in deeply anaesthetised young guinea-pigs. After determination of neuronal functional properties, dye was injected into the soma. CGRP-like immunoreactivity (CGRP-LI) was examined on histological sections of dye-marked neurones. Fourteen of 34 C-fibre neurones showed CGRP-LI. These included 10/21 C-fibre nociceptive neurones. All C-polymodal nociceptors in glabrous (n = 4) but none in hairy skin (n = 4) were positive. Positive C-fibre high threshold mechanoreceptive (HTM) units had receptive fields in dermal or deeper tissue. Four (n = 6) unresponsive or unidentified C-fibre units were positive. Neither C-fibre cooling sensitive (n = 4) nor C-fibre low threshold mechanoreceptive (LTM) units (n = 3) had CGRP-LI. Six of 23 A-fibre nociceptive cells were positive including one Aalpha/beta unit. Three of these positive cells had epidermal and three had dermal/deep receptive fields. Three of 36 A-fibre LTM units exhibited CGRP-LI; all were Aalpha/beta-fibre G hair units. All glabrous skin and muscle spindle units and in hairy skin slowly adapting and field units, and some G-hair units lacked CGRP-LI. CGRP-LI stained fibres were found in tissues containing receptive fields of positive DRG neurones: glabrous skin, near hair follicles and in skeletal muscle. A few substance P-labelled neurones did not exhibit CGRP-LI and vice versa. Thus CGRP expression was detected in under half the nociceptive neurones, was not limited to nociceptive neurones and apart from receptive properties was also related to location/depth in the tissues of a DRG neurone's peripheral terminals.

Calcitonin gene-related peptide and calcitonin in the CSF of patients with dementia and depression: Possible disease markers

Cerebrospinal fluid (CSF) was obtained from 32 patients with dementia, 19 healthy controls that were age-matched with the dementia patients, and 29 DSM-IV major depression patients and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and calcitonin-like immunoreactivity (CT-LI) measured by RIA. CGRP-LI was lower in the dementia group compared to both the controls and depressed patients ( P<.01) after covarying out sex and age. CT-LI was decreased in the dementia and depressed patients ( P<.05) compared to the controls. A positive relationship between CGRP-LI and CT-LI was found in dementia. A logistic discriminant analysis with calcitonin gene-related peptide (CGRP) and log calcitonin (CT) predicting diagnosis (three classes) revealed a significant overall fit ( χ 2=18.08, P=.0011), with an effect test showing contributions of both independent variables: CGRP ( χ 2=10.03, P<.007), log CT ( χ 2=8.63, P=.013). In dementia, both CGRP-LI and CT-LI were decreased and their concentration ratio did not differ from that in controls, likely reflecting a general neuronal loss. Alternatively and more speculatively, but theoretically possible, expression of the α-CGRP/CT gene may be affected in dementia. In contrast, in depression, CT-LI but not CGRP-LI was decreased and the CGRP/CT concentration ratio was increased, which is consistent with a possibility of an altered splicing process favoring CGRP mRNA.

Improvement of preservation with cardioplegic solution by nitroglycerin-induced delayed preconditioning is mediated by calcitonin gene-related peptide

Improvement of preservation with cardioplegic solution by nitroglycerin-induced delayed preconditioning was studied in the isolated rat heart. The isolated rat heart was arrested using St. Thomas Hospital solution, and then reperfused with normothermic Krebs–Henseleit solution for 40 min after a 4-h hypothermic ischemic period. Heart rate, coronary flow, left ventricular pressure and the maxium value of the first derivatives of left ventricular pressure (±d p/d t max) were recorded, and plasma concentrations of CGRP-like immunoreactivity (CGRP-LI) and nitric oxide (NO), tumor necrosis factor-alpha (TNF-α) in myocardial tissues, and creatine kinase in coronary effluent were measured. Delayed preconditioning was induced by i.v. injection of nitroglycerin 24 h before the experiment. Nitroglycerin (60 μg/kg or 120 μg/kg) caused an improvement of cardiac function, a decrease in the release of creatine kinase in coronary effluent and a decrease in the content of TNF-α in myocardial tissues. Nitroglycerin significantly increased plasma concentrations of CGRP and NO. After pretreatment with capsaicin, which depletes neurotransmitters in sensory nerves, or methylene blue, a selective guanylate cyclase inhibitor, the protection and the elevated release of CGRP induced by nitroglycerin were abolished. The present study suggests that improvement of preservation with cardioplegic solution by nitroglycerin-induced delayed preconditioning is due to stimulation of CGRP release in the rat heart, and that the protection of CGRP-mediated nitroglycerin is related to inhibition of TNF-α production.

Localization of functional calcitonin gene-related peptide binding sites in a subpopulation of cultured dorsal root ganglion neurons

In this study we investigated whether cultured dorsal root ganglion (DRG) neurons from the adult rat express binding sites for calcitonin gene-related peptide (CGRP). These were identified on fixed cells by using CGRP labeled at the N-terminal site with 1.4-nm gold particles. After 1 day in culture, about 20% of small to medium-sized DRG neurons showed CGRP-gold binding. Binding of CGRP-gold was dose-dependently reduced by coadministration of CGRP. The calcium imaging technique in living cells revealed that the bath administration of CGRP evoked an increase of the intracellular calcium in up to 30% of the DRG neurons tested. Both depletion of intracellular calcium stores by thapsigargin or using a calcium-free medium blocked the CGRP-mediated increase of cytosolic calcium in most neurons. Thus intracellular and extracellular sources of calcium are relevant for the CGRP response. Using the whole-cell patch-clamp technique, about 30% of the neurons were found to exhibit an inward current and a depolarization upon administration of CGRP close to the neurons. Immunocytochemical double-labeling techniques showed that most of the CGRP-gold binding sites were expressed in unmyelinated (neurofilament 200-negative) DRG neurons. Most of the neurons with CGRP-gold binding sites also expressed the tyrosine kinase A receptor, and all of them showed CGRP-like immunoreactivity. This study shows, therefore, that a subpopulation of unmyelinated, peptidergic primary afferent neurons express CGRP binding sites that can be activated by CGRP in an excitatory direction. The binding sites may serve as autoreceptors because all of these neurons also synthesize CGRP. The activation of CGRP binding sites may sensitize primary afferent neurons and influence the release of mediators.