Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

Mechanisms of protease-activated receptor 2-evoked hyperexcitability of nociceptive neurons innervating the mouse colon

Agonists of protease-activated receptor 2 (PAR(2)) evoke hyperexcitability of dorsal root ganglia (DRG) neurons by unknown mechanisms. We examined the cellular mechanisms underlying PAR(2)-evoked hyperexcitability of mouse colonic DRG neurons to determine their potential role in pain syndromes such as visceral hyperalgesia. Colonic DRG neurons were identified by injecting Fast Blue and DiI retrograde tracers into the mouse colon. Using immunofluorescence, we found that DiI-labelled neurons contained PAR(2) immunoreactivity, confirming the presence of receptors on colonic neurons. Whole-cell current-clamp recordings of acutely dissociated neurons demonstrated that PAR(2) activation with a brief application (3 min) of PAR(2) agonists, SLIGRL-NH(2) and trypsin, evoked sustained depolarizations (up to 60 min) which were associated with increased input resistance and a marked reduction in rheobase (50% at 30 min). In voltage clamp, SLIGRL-NH(2) markedly suppressed delayed rectifier I(K) currents (55% at 10 min), but had no effect on the transient I(A) current or TTX-resistant Na(+) currents. In whole-cell current-clamp recordings, the sustained excitability evoked by PAR(2) activation was blocked by the PKC inhibitor, calphostin, and the ERK(1/2) inhibitor PD98059. Studies of ERK(1/2) phosphorylation using confocal microscopy demonstrated that SLIGRL-NH(2) increased levels of immunoreactive pERK(1/2) in DRG neurons, particularly in proximity to the plasma membrane. Thus, activation of PAR(2) receptors on colonic nociceptive neurons causes sustained hyperexcitability that is related, at least in part, to suppression of delayed rectifier I(K) currents. Both PKC and ERK(1/2) mediate the PAR(2)-induced hyperexcitability. These studies describe a novel mechanism of sensitization of colonic nociceptive neurons that may be implicated in conditions of visceral hyperalgesia such as irritable bowel syndrome.

Modelling the movement and survival of the root-feeding clover weevil, Sitona lepidus, in the root-zone of white clover

White clover (Trifolium repens) is an important pasture legume but is often difficult to sustain in a mixed sward because, among other things, of the damage to roots caused by the soil-dwelling larval stages of S. lepidus. Locating the root nodules on the white clover roots is crucial for the survival of the newly hatched larvae. This paper presents a numerical model to simulate the movement of newly hatched S. lepidus larvae towards the root nodules, guided by a chemical signal released by the nodules. The model is based on the diffusion-chemotaxis equation. Experimental observations showed that the average speed of the larvae remained approximately constant, so the diffusion-chernotaxis model was modified so that the larvae respond only to the gradient direction of the chemical signal but not its magnitude. An individual-based lattice Boltzmann method was used to simulate the movement of individual larvae, and the parameters required for the model were estimated from the measurement of larval movement towards nodules in soil scanned using X-ray microtomography. The model was used to investigate the effects of nodule density, the rate of release of chemical signal, the sensitivity of the larvae to the signal, and the random foraging of the larvae on the movement and subsequent survival of the larvae. The simulations showed that the most significant factors for larval survival were nodule density and the sensitivity of the larvae to the signal. The dependence of larval survival rate on nodule density was well fitted by the Michealis-Menten kinetics. (c) 2005 Elsevier B.V All rights reserved.

Influence of sodium chloride on seed germination and seedling root growth of cotton (Gossypium hirsutum L.)

Response of cotton (Gossypium hirsutum L. cv. NIAB-78) to salinity, in terms of seed germination, seedling root growth and root Na+ and K+ content was determined in a laboratory experiment. Cotton seeds were exposed to increasing salinity levels using germination water with Sodium chloride concentrations of 0, 50, 100, 150 and 200 mM, to provide different degrees of salt stress. Germinated seeds were counted and roots were harvested at 24, 48, 72 and 96 h after the start of the experiment. It appeared that seed germination was only slightly affected by an increase in salinity (in most cases the differences between treatment were non-significant), whereas root length, root growth rate, root fresh and dry weights were severely affected, generally highly significant differences in these variables were found for comparisons involving most combinations of salinity levels, in particular with increased incubation period. K+ contents decreased with increasing salinity levels, although differences in K+ content were only significant when comparing the control and the 4 salinity levels. Na+ content of the roots increased with increasing levels of NaCl in the germination water, suggesting an exchange of K+ for Na+. The ratio K+/Na+ strongly decreased with rising levels of salinity from around 4.5 for the control to similar to 1 at 200 mM NaCl.

Measuring root traits in barley (Hordeum vulgare ssp vulgare and ssp spontaneum) seedlings using gel chambers, soil sacs and X-ray microtomography

Root characteristics of seedlings of five different barley genotypes were analysed in 2D using gel chambers, and in 3D using soil sacs that were destructively harvested and pots of soil that were assessed non-invasively using X-ray microtomography. After 5 days, Chime produced the greatest number of root axes (similar to 6) and Mehola significantly less (similar to 4) in all growing methods. Total root length was longest in GSH01915 and shortest in Mehola for all methods, but both total length and average root diameter were significantly larger for plants grown in gel chambers than those grown in soil. The ranking of particular growth traits (root number, root angular spread) of plants grown in gel plates, soil sacs and X-ray pots was similar, but plants grown in the gel chambers had a different order of ranking for root length to the soil-grown plants. Analysis of angles in soil-grown plants showed that Tadmore had the most even spread of individual roots and Chime had a propensity for non-uniform distribution and root clumping. The roots of Mehola were less well spread than the barley cultivars supporting the suggestion that wild and landrace barleys tend to have a narrower angular spread than modern cultivars. The three dimensional analysis of root systems carried out in this study provides insights into the limitations of screening methods for root traits and useful data for modelling root architecture.

Host plant recognition by the root feeding clover weevil, Sitona lepidus (Coleoptera : Curculionidae)

This study investigated the ability of neonatal larvae of the root-feeding weevil, Sitona lepidus Gyllenhal, to locate white clover Trifolium repens L. (Fabaceae) roots growing in soil and to distinguish them from the roots of other species of clover and a co-occurring grass species. Choice experiments used a combination of invasive techniques and the novel technique of high resolution X-ray microtomography to non-invasively track larval movement in the soil towards plant roots. Burrowing distances towards roots of different plant species were also examined. Newly hatched S. lepidus recognized T. repens roots and moved preferentially towards them when given a choice of roots of subterranean clover, Trifolium subterraneum L. (Fabaceae), strawberry clover Trifolium fragiferum L. (Fabaceae), or perennial ryegrass Lolium perenne L. (Poaceae). Larvae recognized T. repens roots, whether released in groups of five or singly, when released 25 mm (meso-scale recognition) or 60 mm (macro-scale recognition) away from plant roots. There was no statistically significant difference in movement rates of larvae.

Effects of carbon dioxide on the searching behaviour of the root-feeding clover weevil Sitona lepidus (Coleoptera : Curculionidae)

The respiratory emission of CO2 from roots is frequently proposed as an attractant that allows soil-dwelling insects to locate host plant roots, but this role has recently become less certain. CO2 is emitted from many sources other than roots, so does not necessarily indicate the presence of host plants, and because of the high density of roots in the upper soil layers, spatial gradients may not always be perceptible by soil-dwelling insects. The role of CO2 in host location was investigated using the clover root weevil Sitona lepidus Gyllenhall and its host plant white clover (Trifolium repens L.) as a model system. Rhizochamber experiments showed that CO2 concentrations were approximately 1000 ppm around the roots of white clover, but significantly decreased with increasing distance from roots. In behavioural experiments, no evidence was found for any attraction by S. lepidus larvae to point emissions of CO2, regardless of emission rates. Fewer than 15% of larvae were attracted to point emissions of CO2, compared with a control response of 17%. However, fractal analysis of movement paths in constant CO2 concentrations demonstrated that searching by S. lepidus larvae significantly intensified when they experienced CO2 concentrations similar to those found around the roots of white clover (i.e. 1000 ppm). It is suggested that respiratory emissions of CO2 may act as a 'search trigger' for S. lepidus, whereby it induces larvae to search a smaller area more intensively, in order to detect location cues that are more specific to their host plant.

Modelling cereal root systems for water and nitrogen capture: Towards an economic optimum

A quantitative model of wheat root systems is developed that links the size and distribution of the root system to the capture of water and nitrogen (which are assumed to be evenly distributed with depth) during grain filling, and allows estimates of the economic consequences of this capture to be assessed. A particular feature of the model is its use of summarizing concepts, and reliance on only the minimum number of parameters (each with a clear biological meaning). The model is then used to provide an economic sensitivity analysis of possible target characteristics for manipulating root systems. These characteristics were: root distribution with depth, proportional dry matter partitioning to roots, resource capture coefficients, shoot dry weight at anthesis, specific root weight and water use efficiency. From the current estimates of parameters it is concluded that a larger investment by the crop in fine roots at depth in the soil, and less proliferation of roots in surface layers, would improve yields by accessing extra resources. The economic return on investment in roots for water capture was twice that of the same amount invested for nitrogen capture. (C) 2003 Annals of Botany Company.

Effect of mycorrhizae and pH change at the root-soil interface on phosphorus uptake by Sorghum using a rhizocylinder technique

Sorghum (Sorghum bicolor) was grown for 40 days in. rhizocylinder (a growth container which permitted access to rh zosphere and nonrhizosphere soil), in two soils of low P status. Soils were fertilized with different rates of ammonium and nitrate and supplemented with 40 mg phosphorus (P) kg(-1) and inoculated with either Glomus mosseae (Nicol. and Gerd.) or nonmycorrhizal root inoculum.. N-serve (2 mg kg(-1)) was added to prevent nitrification. At harvest, soil from around the roots was collected at distances of 0-5, 5-10, and 10-20 mm from the root core which was 35 mm diameter. Sorghum plants, with and without mycorrhiza, grew larger with NH4+ than with NO3- application. After measuring soil pH, 4 3 suspensions of the same sample were titrated against 0.01 M HCl or 0.01 M NaOH until soil pH reached the nonplanted pH level. The acid or base requirement for each sample was calculated as mmol H+ or OFF kg(-1) soil. The magnitude of liberated acid or base depended on the form and rate of nitrogen and soil type. When the plant root was either uninfected or infected with mycorrhiza., soil pH changes extended up to 5 mm from the root core surface. In both soils, ammonium as an N source resulted in lower soil pH than nitrate. Mycorrhizal (VAM) inoculation did not enhance this difference. In mycorrhizal inoculated soil, P depletion extended tip to 20 mm from the root surface. In non-VAM inoculated soil P depletion extended up to 10 mm from the root surface and remained unchanged at greater distances. In the mycorrhizal inoculated soils, the contribution of the 0-5 mm soil zone to P uptake was greater than the core soil, which reflects the hyphal contribution to P supply. Nitrogen (N) applications that caused acidification increased P uptake because of increased demand; there is no direct evidence that the increased uptake was due to acidity increasing the solubility of P although this may have been a minor effect.

Distribution of nutrients in the root zone affects yield, quality and blossom end rot of tomato fruits

Tomato plants (Lycopersicon esculentum Mill. 'DRK') were grown hydroponically in two experiments to determine the effects of nutrient concentration and distribution in the root zone on yield, quality and blossom end rot (BER). The plants were grown in rockwool with their root systems divided into two portions. Each portion was irrigated with nutrient solutions with either the same or different electrical conductivity (EC) in the range 0 to 6 dS m(-1). In both experiments, fruit yields decreased as EC increased from moderate to high when solutions of equal concentration were applied to both portions of the root system. However, higher yields were obtained when a solution with high EC was applied to one portion of the root system and a solution of low EC to the other portion. For example, the fresh weight of mature fruits in the 6/6 treatment was only 20% that of the 3/3 treatment but the 6/0 treatment had a yield that was 40% higher. The reduction in yield in the high EC treatments was due to an increase in the number of fruits with BER and smaller fruit size. BER increased from 12% to 88% of total fruits as EC increased from 6/0 to 6/6 and fruit length decreased from 67 mm to 52 mm. Fruit quality (expressed as titratable acidity and soluble solids) increased as EC increased. In summary, high yields of high quality tomatoes with minimal incidence of BER were obtained when one portion of the root system was supplied with a solution of high EC and the other portion with a solution of moderate or zero EC.

Distribution of salinity in the root zone affects the yield, quality of tomato fruit

Tomato plants (Lycopersicon esculentum Mill. var. DRK) were grown with a split root system to determine the effect of an unequal distribution of salinity in the root zone on yield and quality. The roots of the plant were divided into two portions and each portion was irrigated with nutrient solutions differing in EC levels achieved by adding Na or K. The maximum yield was observed in treatments with unequal EC when one portion of the roots received only water and the lowest in the high EC treatments. The reduced yield in the high EC treatment was due to the incidence of blossom-end rot and reduced fruit size. Fruit size in the treatments receiving solutions of unequal EC was up to 12% greater than that in the control. No significant differences were found in soluble solids and acidity between control and all other unequal EC treatments. Ca concentration was significantly higher in the treatments where one portion of the root system received water. It was concluded that high salinity had positive effects on yield and quality provided that one portion of the root system were placed in low EC or only water.