Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Kentucky Bluegrass Response to Potassium and Nitrogen Fertilization

The response of Kentucky bluegrass (Poa pratensis L.) to potassium (K) fertilization has been inconsistent. The objective of this research was to determine the effects of K fertilization across varying nitrogen (N) rates and clipping management on Kentucky bluegrass clipping yields, quality, tissue K concentrations, apparent N recovery, and N use efficiency. A 2 x 4 x 4 factorial was arranged in a splitplot design and repeated across two years. Main plots were clipping treatments (returned vs. removed) and subplots were N rates (0, 98, 196, and 294 kg ha(-1) yr(-1)) in combination with K rates (0, 81, 162, and 243 kg ha(-1) yr(-1)). There was no positive effect of K on clipping yields and quality even though soil extractable K levels tested low. Higher K rates, however, increased N recovery and use efficiency for all but the highest N rate. Tissue K response to K fertilization was nonlinear. Yield and quality responses were not correlated to tissue K concentration. Nonexchangeable K levels were high in the native soil, and may have provided an additional source of K for bluegrass. The results suggest that extractable K values alone may not adequately predict available K to Kentucky bluegrass in this sandy loam soil.

Scaffolding of adenylyl cyclase by A Kinase Anchoring Proteins

Adenylyl cyclase (AC) converts ATP into cAMP, which activates protein kinase A (PKA). Activation of PKA leads to the phosphorylation of specific substrates. The mechanism of specificity of PKA phosphorylation baffled researchers for many years. The discovery of A Kinase Anchoring Proteins (AKAPs) has helped to unravel this mystery. AKAPs function to target PKA to specific regions within the cell. They also anchor other enzymes, receptors, or channels leading to tightly regulated signaling modules. Several studies have suggested an important role for activated PKA in these complexes, including the AKAPs yotiao and muscle AKAP (mAKAP). Yotiao, a plasma membrane AKAP, anchors PP1, NMDA receptors, IP3 receptors, and heart potassium channel subunit KCNQI. PKA phosphorylation of NMDA receptors as well as KCNQI leads to increased channel activity. Patients with mutations in KCNQI or yotiao that cause loss of targeting of KCNQI develop long QT syndrome, which can be fatal. mAKAP anchors several CAMP/PKA-regulated pathways to the nuclear envelope in cardiac myocytes. The necessity of activated PKA in these complexes led to the hypothesis that AC is also anchored. The results indicate that AC does associate with yotiao in brain and heart, specifically with AC types I-III, and IX. Co-expression of AC II or III with yotiao leads to inhibition of each isoform's activity. Binding assays revealed that yotiao binds to the N-terminus of AC II and that this region can reverse the inhibition of AC II, but not AC III, indicating unique binding sites on yotiao. AC II binds directly to as 808-957 of yotiao. Y808-957 acts as a dominant negative as the addition of it to rat brain membranes results in a ∼40% increase in AC activity. Additionally, AC was also found to associate with mAKAP in heart, specifically with AC types II and V. The binding site of AC was mapped to 275-340 of mAKAP, while mAKAP binds to the soluble domains of AC V as a complex. These results indicate that interactions between AC and AKAPs are specific and that AC plays an important role in AKAP-targeted signaling. ^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.

TRPV1 Channels Contribute to Behavioral Hypersensitivity and Spontaneous Activity in Nociceptors After Spinal Cord Injury

A majority of persons who have sustained spinal cord injury (SCI) develop chronic pain. While most investigators have assumed that the critical mechanisms underlying neuropathic pain after SCI are restricted to the central nervous system (CNS), recent studies showed that contusive SCI results in a large increase in spontaneous activity in primary nociceptors, which is correlated significantly with mechanical allodynia and thermal hyperalgesia. Upregulation of ion channel transient receptor vanilloid 1 (TRPV1) has been observed in the dorsal horn of the spinal cord after SCI, and reduction of SCI-induced hyperalgesia by a TRPV1 antagonist has been claimed. However, the possibility that SCI enhances TRPV1 expression and function in nociceptors has not been tested. I produced contusive SCI at thoracic level T10 in adult, male rats and harvested lumbar (L4/L5) dorsal root ganglia (DRG) from sham-treated and SCI rats 3 days and 1 month after injury, as well as from age-matched naive control rats. Whole-cell patch clamp recordings were made from small (soma diameter <30 >μm) DRG neurons 18 hours after dissociation. Capsaicin-induced currents were significantly increased 1 month, but not 3 days, after SCI compared to neurons from control animals. In addition, Ca2+ transients imaged during capsaicin application were significantly greater 1 month after SCI. Western blot experiments indicated that expression of TRPV1 protein in DRG is also increased 1 month after SCI. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hindlimb withdrawal responses to heat and mechanical stimuli. Similar reversal of behavioral hypersensitivity was induced by intrathecal delivery of oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of spontaneous activity in dissociated nociceptors after SCI. Limited activation of TRPV1 was found to induce prolonged repetitive firing without accommodation or desensitization, and this effect was enhanced by SCI. These data suggest that SCI enhances TRPV1 expression and function in primary nociceptors, increasing the excitability and spontaneous activity of these neurons, thus contributing to chronic pain after SCI.

Modulation of high voltage -activated calcium channels by phosphorylation

High voltage-activated (HVA) calcium channels from rat brain and rabbit heart are expressed in Xenopus laevis oocytes and their modulation by protein kinases studied. A subtype of the HVA calcium current expressed by rat brain RNA is potentiated by the phospholipid- and calcium-dependent protein kinase (PKC). The calcium channel clone $\alpha\sb{\rm1C}$ from rabbit heart is modulated by the cAMP-dependent protein kinase (PKA), and another factor present in the cytoplasm.^ The HVA calcium channels from rat brain do not belong to the L-type subclass since they are insensensitive to dihydropyridine (DHP) agonists and antagonists. The expressed currents do contain a N-type fraction which is identified by inactivation at depolarized potentials, and a P-type fraction as defined by blockade by the venom of the funnel web spider Agelenopsis Aperta. A non N-type fraction of this current is potentiated, by using phorbol esters to activate PKC. This residual fraction of current resembles the newly described Q-type channel from cerebellar granule cells in its biophysical properties, and potentiation by activation of PKC.^ The $\alpha\sb{\rm1C}$ clone from rabbit heart is expressed in oocytes and single-channel currents are measured using the cell-attached and cell-excised patch clamp technique. The single-channel current runs down within two minutes after patch excision into normal saline bath solution. The catalytic subunit of PKA + MgATP is capable of reversing this rundown for over 15 minutes. There also appears to be an additional factor present in the cytoplasm necessary for channel activity as revealed in experiments where PKA failed to prevent rundown.^ These data are important in that these types of channels are involved in synaptic transmission at many different types of synapses. The mammalian synapse is not accessible for these types of studies, however, the oocyte expression system allows access to HVA calcium channels for the study of their modulation by phosphorylation. ^

Calcium-dependent mechanisms and long-term potentiation: Role of NMDA receptors, voltage -dependent calcium channels and persistent protein kinase activity

Long-term potentiation (LTP) is a rapidly induced and long lasting increase in synaptic strength and is the leading cellular model for learning and memory in the mammalian brain. LTP was first identified in the hippocampus, a structure implicated in memory formation. LTP induction is dependent on postsynaptic Ca2+ increases mediated by N-methyl-D-aspartate (NMDA) receptors. Activation of other postsynaptic routes of Ca2+ entry, such as voltage-dependent Ca2+ channels (VDCCs) have subsequently been shown to induce a long-lasting increase in synaptic strength. However, it is unknown if VDCC-induced LTP utilized similar cellular mechanisms as the classical NMDA receptor-dependent LTP and if these two forms of LTP display similar properties. This dissertation determines the similarities and differences in VDCC and NMDA receptor-dependent LTP in area CA1 of hippocampal slices and demonstrates that VDCCs and NMDA receptors activate similar cellular mechanisms, such as protein kinases, to induce LTP. However, VDCC and NMDA receptor activated LTP induction mechanisms are compartmentalized in the postsynaptic neuron, such that they do not interact. Consistent with activation properties of NMDA receptors and VDCCs, NMDA receptor and VDCC-dependent LTP have different induction properties. In contrast to NMDA-dependent LTP, VDCC-induced potentiation does not require evoked presynaptic stimulation or display input specificity. These results indicate that there are two different routes of postsynaptic Ca2+ which can induce LTP and the compartmentation of VDCCs and NMDA receptors and/or their resulting Ca2+ increases may account for the distinction between these LTP induction mechanisms.^ One of the molecular targets for postsynaptic Ca2+ that is required for the induction of LTP is protein kinases. Evidence for the role of protein kinase activity in LTP expression is either correlational or controversial. We have utilized a broad range and potent inhibitors of protein kinases to systematically examine the temporal requirement for protein kinases in the induction and expression of LTP. Our results indicate that there is a critical period of persistent protein kinase activity required for LTP induction activated by tetanic stimulation and extending until 20 min after HFS. In addition, our results suggest that protein kinase activity during and immediately after HFS is not sufficient for LTP induction. These results provide evidence for persistent and/or Ca2+ independent protein kinase activity involvement in LTP induction. ^

Broadcast and Band Phosphorus and Potassium Placement for Corn and Soybean Managed with Till or No Till

No-till minimizes the incorporation of crop residue and fertilizer with soil; resulting in wetter, colder soils and the accumulation of organic matter, phosphorus (P), and potassium (K) near the soil surface. Banding of P and Kcould be more effective than broadcast fertilization by counteracting stratification, applying nutrients in the root zone (starter effect), and minimizing reactions with the soil that may reduce their availability to plants. Therefore, a long-term study was established in 1994 to evaluate P and K fertilizer rates and placement methods for grain yield of corn and soybean managed with no-till and chiselplow/disk tillage.

Long-term Evaluation of Hybrid, Nitrogen, and Potassium Interactions in Continuous Corn

A long-term experiment was established in 2009 to study continuous corn responses to potassium (K), nitrogen (N), and hybrid rootworm resistance. Previous research suggested a need for this study. A long-term trial conducted until 2001 at the ISU Northern Research Farm showed that the maximum corn yield level and the N rate that maximized yield was higher when K was optimal or greater. In contrast, the relative yield response to N and the N rate that maximized yield were similar for soil-test phosphorus (P) levels ranging from very low to very high. Other studies have shown that rootworm resistance often increases yield compared with untreated susceptible hybrids. Also, that rootworm resistance does not consistently affect the K rate that maximizes yield, but increases K removal because of the higher yield levels. Therefore, this new study evaluates possible interactions between rootworm resistance and N and K fertilization in corn.

Phosphorus and Potassium Rates and Placement Methods for Corn and Soybean Managed with No-till or Tillage

No-till management for corn and soybean results in little or no incorporation of crop residues and fertilizer with soil. Subsurface banding phosphorus (P) and potassium (K) fertilizers with planter attachments could be more effective than broadcast fertilization, because in no-till with broadcast fertilizer, both nutrients accumulate at or near the soil surface. A long-term study was initiated in 1994 at the ISU Northwest Research Farm to evaluate P and K fertilizer placement for corn and soybean managed with no-till and chiselplow tillage.

Placement Methods of Phosphorus and Potassium for Corn and Soybean Managed with No-till and Chisel-plow/Disk Tillage

No-till management limits the incorporation of crop residue and fertilizer with soil resulting in wetter, colder soils and the accumulation of organic matter, phosphorus (P), and potassium (K) near the soil surface. Banding of P and K could be more effective than broadcast fertilization by counteracting stratification, applying nutrients in the root zone (starter effect), and minimizing reactions with the soil that may reduce their availability to plants. Therefore, this long-term study was established in 1994 to evaluate P and K fertilizer placement methods and grain yield of corn-soybean rotations managed with notill and chisel-plow/disk tillage.