Calcitonin gene-related peptide: physiology and pathophysiology.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide. Discovered 30 years ago, it is produced as a consequence of alternative RNA processing of the calcitonin gene. CGRP has two major forms (α and ß). It belongs to a group of peptides that all act on an unusual receptor family. These receptors consist of calcitonin receptor-like receptor (CLR) linked to an essential receptor activity modifying protein (RAMP) that is necessary for full functionality. CGRP is a highly potent vasodilator and, partly as a consequence, possesses protective mechanisms that are important for physiological and pathological conditions involving the cardiovascular system and wound healing. CGRP is primarily released from sensory nerves and thus is implicated in pain pathways. The proven ability of CGRP antagonists to alleviate migraine has been of most interest in terms of drug development, and knowledge to date concerning this potential therapeutic area is discussed. Other areas covered, where there is less information known on CGRP, include arthritis, skin conditions, diabetes, and obesity. It is concluded that CGRP is an important peptide in mammalian biology, but it is too early at present to know if new medicines for disease treatment will emerge from our knowledge concerning this molecule.

Activation of PAR(2) receptors sensitizes primary afferents and causes leukocyte rolling and adherence in the rat knee joint.

BACKGROUND AND PURPOSE: The PAR(2) receptors are involved in chronic arthritis by mechanisms that are as yet unclear. Here, we examined PAR(2) activation in the rat knee joint. EXPERIMENTAL APPROACH: PAR(2) in rat knee joint dorsal root ganglia (DRG) cells at L3-L5, retrogradely labelled with Fluoro-gold (FG) were demonstrated immunohistochemically. Electrophysiological recordings from knee joint nerve fibres in urethane anaesthetized Wistar rats assessed the effects of stimulating joint PAR(2) with its activating peptide, 2-furoyl-LIGRLO-NH(2) (1-100 nmol·100 µL(-1) , via close intra-arterial injection). Fibre firing rate was recorded during joint rotations before and 15 min after administration of PAR(2) activating peptide or control peptide. Leukocyte kinetics in the synovial vasculature upon PAR(2) activation were followed by intravital microscopy for 60 min after perfusion of 2-furoyl-LIGRLO-NH(2) or control peptide. Roles for transient receptor potential vanilloid-1 (TRPV1) or neurokinin-1 (NK(1) ) receptors in the PAR(2) responses were assessed using the selective antagonists, SB366791 and RP67580 respectively. KEY RESULTS: PAR(2) were expressed in 59 ± 5% of FG-positive DRG cells; 100 nmol 2-furoyl-LIGRLO-NH(2) increased joint fibre firing rate during normal and noxious rotation, maximal at 3 min (normal; 110 ± 43%, noxious; 90 ± 31%). 2-Furoyl-LIGRLO-NH(2) also significantly increased leukocyte rolling and adhesion over 60 min. All these effects were blocked by pre-treatment with SB366791 and RP67580 (P < 0.05 compared with 2-furoyl-LIGRLO-NH(2) alone). CONCLUSIONS AND IMPLICATIONS: PAR(2) receptors play an acute inflammatory role in the knee joint via TRPV1- and NK(1) -dependent mechanisms involving both PAR(2) -mediated neuronal sensitization and leukocyte trafficking.

Proteinase activated receptor (PAR) involvement in mediating arthritis pain and inflammation.

Proteinase activated receptors (PARs) are a newly identified family of G-protein-coupled receptors that are activated by proteinases released into tissues during inflammation. Evidence has accumulated which shows that PARs can exhibit both anti- and pro-inflammatory properties in different organ systems. During arthritis, proteinases are known to be released into the joint where they orchestrate tissue remodelling and degeneration. By activating PARs, these proteinases have the potential to modulate joint inflammation and pain by a highly targeted and selective receptor pathway. The recent identification of PARs on synovial fibroblasts, neutrophils, macrophages and mast cells is further evidence that this intriguing family of receptors could play a role in the pathogenesis and symptoms of arthritis. This review article provides an overview of the current knowledge regarding PARs and their emerging role in arthritis.

Effects of unilateral cochlear removal on dendrites in the gerbil medial superior olivary nucleus.

For most neurons, dendrites serve as the major pathway for incoming activity from other neurons. It might therefore be expected that dendrites are particularly sensitive to variations in the level of afferent input they receive. In the auditory brainstem, this expectation has been confirmed in neurons of the medial superior olivary nucleus (MSO). The MSO is uniquely suited to studies of afferent influences on dendrites, as lateral and medial dendrites of MSO neurons receive inputs almost exclusively from the ipsilateral and contralateral ears, respectively. Thus, the effects of unilateral afferent manipulations may be compared between defined dendrites on the same neurons. We have used unilateral deafening (by surgical destruction of the cochlea) in immature [postnatal day 18 (P18)] and adult gerbils to study the late maturation and effect of peripheral deafferentation on the dendrites of MSO neurons. In semi-thin, frontal sections from unoperated animals, we found a change between P18 and adulthood from a lateral to a medial bias in the symmetry of MSO dendrites. Cochlear removal in adulthood led to a reduction in the density of dendritic profiles on the side of the ablation in both MSOs. Cochlear removal at P18 led to a rapid (< 3 days) and sustained dendritic atrophy that was most marked in the caudal part of the nucleus. Electron microscope (EM) measurements in the sagittal plane on MSO dendrite profiles of animals unilaterally deafened at P18 showed a reduction in the number, but not in the area, of profiles on the side of the deafened ear. These results demonstrate a developmental change in the symmetry of MSO medial and lateral dendrites, and a rapid and long-lasting reduction in the number of distal dendrites produced by unilateral deafening either in infancy or adulthood.

Susceptibility of developing cochlear nucleus neurons to deafferentation-induced death abruptly ends just before the onset of hearing.

To investigate the ability of developing cochlear nucleus (CN) neurons to survive in the absence of afferent input, left cochlear removals were performed on gerbils at 2 day intervals from postnatal (P)3 to P11, and at P18 and P93. After a 3 month postsurgical survival period, Nissl-stained frontal sections through the brainstem were analyzed under the light microscope. CN volume, anteroventral cochlear nucleus (AVCN) neuron cross-sectional area, and total number of neurons in the CN were measured on both sides of the brain. Mean volume reduction of the deafferented CN relative to the intact CN ranged between 76% in the P3 group to 33% in the P11 group and did not differ significantly between P11 and P93. Cochlear removal at all ages reduced AVCN neuron cross-sectional area by approximately 40% in the deafferented CN relative to the intact CN, except for the P93 group where neuron atrophy was significantly less severe (23% mean reduction). Massive loss of CN neurons (>50% of the intact side) was observed following cochlear removal performed during the first postnatal week. However, between P7 and P9, neurons in all areas of the CN lose susceptibility to deafferentation-induced neuron death. No significant neuron loss was observed following cochlear removal after P7. This study shows that an abrupt transition in the ability of CN neurons to survive in the absence of afferent input is coincident with events leading to the onset of hearing.

Lateral superior olive projections to the inferior colliculus in normal and unilaterally deafened ferrets.

We have examined the projection from the lateral superior olive (LSO) to the inferior colliculus (IC) in the ferret, with particular interest in the laterality of the projection and in the effects of unilateral cochlear removal in infancy. Large or small deposits of the retrograde tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were made in the IC of anesthetized adult ferrets that either were normally hearing or had been unilaterally deafened in infancy (P5 or P25). After 2 days, the ferrets were perfused, and frontal sections of the brainstem were treated with tetramethyl benzidine. For large deposits of WGA-HRP, equal numbers of labelled neurons were found evenly spread through both LSOs. Smaller deposits of WGA-HRP produced four results that contrasted with previous reports on the cat. First, many more labelled neurons were found in the contralateral than in the ipsilateral LSO. Second, the relative number of labelled neurons in each LSO was independent of whether the deposits were in the ventral or in the dorsal IC. Third, the total number of labelled LSO neurons was independent of whether the deposits were in the ventral or in the dorsal IC. Fourth, the proportion of ipsilateral to contralateral labelled neurons was slightly higher in the medial LSO than in the lateral LSO. Ventral IC deposits resulted in more labelled neurons in the medial LSO, and dorsal IC deposits resulted in more labelled neurons in the lateral LSO, as expected. Neonatal cochlear removal did not change any of these results. We conclude that, in the ferret, the organization of the crossed and uncrossed projections from the LSO to the IC differs from that of the cat, and any similarity with the optic chiasm is not obvious.

Afferent reorganisation within the superior olivary complex of the gerbil: development and induction by neonatal, unilateral cochlear removal.

Cochlear removal in young animals has been shown to produce a variety of degenerative and generative effects within the auditory brainstem. A primary target for axons of neurons in the anteroventral cochlear nucleus (AVCN) is the superior olivary complex (SOC). Following unilateral cochlear removal in neonatal gerbils, AVCN neurons on the side of the removal die, and axons deriving from the AVCN on the unlesioned side produce new endings that innervate previously inappropriate target zones within the ipsilateral medial nucleus of the trapezoid body, both medial superior olives, and the contralateral lateral superior olive. In this study, we have used the anterograde transport of DiI and HRP from the AVCN to relate the formation of these endings to the time course of normal development in the gerbil brainstem. We have also examined the effects of cochlear removal at different ages, and survival to various ages after the removal, to define the time course for these generative phenomena. The results show that, while the major projection pathways from the AVCN to the SOC are in place at the time of birth, further and subtle development of AVCN terminal arbors occurs during the first postnatal week. This overlaps with the time during which cochlear removal produces the formation of exuberant afferents to the SOC from the intact AVCN. The exuberant afferents form through axon sprouting rather than through a suppression of normal, developmental regression. They appear to innervate tonotopically appropriate target regions within the SOC. The formation of the novel afferents begins within 3 days of the removal and appears to be complete within a further 5-7 days. By postnatal day (P) 10, both the normal development of the AVCN to SOC projection and the potential for alteration of that projection by removal of the contralateral cochlea appear to be over. These results suggest that the potential for forming novel projections in the gerbil auditory brainstem is lost before the onset of functional hearing (at P12) and is, therefore, unlikely to result from changes in auditory experience.

The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolites produced by thrombin-treated human platelets. II. Establishment of optimal assay conditions.

We have utilized HPLC to develop optimal conditions for assaying the transformation of arachidonic acid in thrombin-treated human platelets. In the presence of increasing amounts of albumin, the total amount of radioactivity released from thrombin-treated platelets pre-labeled with 3H-arachidonic acid is first enhanced and then inhibited. Maximal release, reflecting primarily enhanced amounts of free labeled arachidonic acid, occurs at a final albumin concentration of 0.5 mg/ml. Calcium promoted the release of all radiolabeled metabolites, but it specifically enhanced HETE formation and release. Magnesium was without effect. Cyclo-oxygenase derived products constituted the bulk of released label at short time intervals, but after ten minutes exposure to thrombin in the presence of albumin (0.5 mg/ml) and 3 mM calcium, radioactivity in the released products was equally distributed among cyclo-oxygenase derived products (TXB2 + PGD2 + HHT), HETE and free arachidonic acid.

The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolities produced by thrombin-treated human platelets. I. The validation of the technique.

We have developed a technique for the rapid separation and quantitative collection of thromboxane B2 (TXB2), PGE2, PGD2, PGF2 alpha, 12-hydroxy-5,8,10 heptadecatrienoic acid (HHT), 12-L-hydroxy-5,8,10,14 eicosatetraenoic acid (HETE), and arachidonic acid released from thrombin treated human platelets. Platelets were pre-labeled with 3H-arachidonic acid and then isolated by gel filtration. They were then exposed to thrombin for various intervals and separated by centrifugation. Aliquots of the cell-free medium were applied directly to a high pressure liquid chromatograph containing a fatty acid column as the stationary phase. A quarternary solvent system containing tetrahydrofuran (THF), acetonitrile (CH3CN), water and acetic acid (HOAC) resolved and eluted the arachidonic acid metabolites within 30 minutes. Since no sample preparation is required and since the solvent system does not quench the counting efficiency of a standard liquid scintillation fluor the technique permits rapid separation and quantitation of radiolabeled arachidonic acid and its metabolites.

Decision rules for hypertension screening: application in a poor community with endemic hypertension.

Data from a hypertension screening project involving 4,272 black residents of a rural southern community were analyzed to determine the effects of a set of admission-decision rules on the case load of a proposed hypertension clinic. Four decision rules were investigated: conjunctive (diastolic high or systolic high); disjunctive (diastolic and/or systolic high); additive (sum of diastolic and systolic high); and systolic only. Most information relevant to admission to treatment came from knowledge of systolic blood pressures, even though knowledge of the diastolic pressure is essential in individual diagnosis. Incremental increases in the minimum blood pressure necessary for admission to treatment from 140/90 mm Hg to 160/95 mm Hg resulted in a one-third reduction in the number of patients treated, a 24% reduction in personnel utilization per patient, and a 34% reduction in drug costs; but in an estimated 14% increase in the attack rate for morbid events in men.

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets.

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.