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Peter A Smith

Publications and source records attributed to Peter A Smith.

At least 19 recordsLinked to original sources

A phenotypic paradigm for cerebral palsy genetics.

Cerebral palsy (CP) represents a clinically and etiologically heterogeneous group of permanent but not unchanging disorders of movement, posture, and motor function resulting from non-progressive disturbances of the developing fetal or infant brain. Pathogenic variants in Mendelian disease-associated genes can be found in a subset of individuals with CP, with variants deemed causal of CP having been published for at least 515 genes. Currently, controversy exists as to whether to interpret such pathogenic variants as causing CP, whether the diagnosis instead should be "CP mimic," or whether a clinical diagnosis of CP should coexist with the molecular diagnosis of a Mendelian disease. Accordingly, there is no universally accepted model of the genetic architecture of CP. Here, we present a statistical approach that treats CP as a phenotypic feature for which some genetic disorders confer an increased risk. Based on comprehensive literature curation, we show that the null hypothesis of no CP association can be rejected for only 89 of the 515 genes. We applied these findings to the analysis of a cohort of 460 children diagnosed with CP in the Shriner Children's network who underwent genome sequencing. We identified pathogenic or likely pathogenic (P/LP) variants in 60 genes in 15.8% of the children. Only 16 of the 60 genes had significant evidence for CP association in our literature analysis. Our results suggest that a stratified approach to attributing causality to genetic variants in CP could support precision genomic medicine for affected individuals.

Humans↗

Spinal mechanisms of NPY analgesia.

We review previously published data, and present some new data, indicating that spinal application of neuropeptide Y (NPY) reduces behavioral and neurophysiological signs of acute and chronic pain. In models of acute pain, early behavioral studies showed that spinal (intrathecal) administration of NPY and Y2 receptor agonists decrease thermal nociception. Subsequent neurophysiological studies indicated that Y2-mediated inhibition of excitatory neurotransmitter release from primary afferent terminals in the substantia gelatinosa may contribute to the antinociceptive actions of NPY. As with acute pain, NPY reduced behavioral signs of inflammatory pain such as mechanical allodynia and thermal hyperalgesia; however, receptor antagonist studies indicate an important contribution of spinal Y1 rather than Y2 receptors. Interestingly, Y1 agonists suppress inhibitory synaptic events in dorsal horn neurons (indeed, well known mu-opioid analgesic drugs produce similar cellular actions). To resolve the behavioral and neurophysiological data, we propose that NPY/Y1 inhibits the spinal release of inhibitory neurotransmitters (GABA and glycine) onto inhibitory neurons, e.g. disinhibition of pain inhibition, resulting in hyporeflexia. The above mechanisms of Y1- and Y2-mediated analgesia may also operate in the setting of peripheral nerve injury, and new data indicate that NPY dose-dependently inhibits behavioral signs of neuropathic pain. Indeed, neurophysiological studies indicate that Y2-mediated inhibition of Ca(2+) channel currents in dorsal root ganglion neurons is actually increased after axotomy. We conclude that spinal delivery of Y1 agonists may be of use in the treatment of chronic inflammatory pain, and that the use of Y1 and Y2 agonists in neuropathic pain warrants further consideration.

Analgesics↗

Chain length dependence of the interactions of bisquaternary ligands with the Torpedo nicotinic acetylcholine receptor.

The interactions of a series of bisholine esters [(CH3)3N+CH(2)CH2OCO-(CH2)n-COOCH2CH2N+(CH3)3] with the Torpedo nicotinic acetylcholine receptor have been investigated. In equilibrium binding studies, [3H]-suberyldicholine (n=6) binds to an equivalent number of sites as [3H]-acetylcholine and with similar affinity (KD approximately 15 nM). In competition studies, all bischoline esters examined displaced both radioligands in an apparently simple competitive manner. Estimated dissociation constants (KI) showed clear chain length dependence. Short chain molecules (n 6) had high affinity similar to suberyldicholine. Functional responses were measured by either rapid flux techniques using Torpedo membrane vesicles or voltage-clamp analyses of recombinant receptors expressed in Xenopus oocytes. Both approaches revealed that suberyldicholine (EC50 approximately 3.4 microM) is 14-25-fold more potent than acetylcholine. However, suberyldicholine elicited only about 45% of the maximum response of the natural ligand, i.e., it is a partial agonist. The potency of this bischoline series increased with chain length. Whereas the shorter ligands (n or=4) had similar (or higher) potency to suberyldicholine. Ligand efficacy had an approximately bell-shaped dependence on chain length and compounds where n or=8 were very poor partial agonists. Based on estimates of interonium distances, we suggest that bisquaternary ligands can interact with multiple binding sites on the nAChR and, depending on the conformational state of the receptor, these sites are 15-20A apart.

Acetylcholine↗

Site-directed combinatorial construction of chimaeric genes: general method for optimizing assembly of gene fragments.

Site-directed construction of chimaeric genes by in vitro recombination "mixes-and-matches" precise building blocks from multiple parent proteins, generating libraries of hybrids to be tested for structure-function relationships and/or screened for favorable properties and novel enzymatic activities. A direct annealing and ligation method can construct chimaeric genes without requiring sequence identity between parents, except for the short (approximately 3 nt) sequences of the fragment overhangs used for specific ligation. Careful planning of the assembly process is necessary, though, in order to ensure effective construction of desired fragment assemblies and to avoid undesired assemblies (e.g., repetition of fragments, fragments out of order). We develop algorithms for specific planned ligation of short overhangs (SPLISO) that efficiently explore possible assembly plans, varying the fragment overhangs and the order of ligation steps in the assembly pathway. While there is a combinatorial explosion in the number of possible assembly plans as the number of breakpoints and parent genes increases, we employ a dynamic programming approach to find globally optimal ones in low-order polynomial time (in practice, taking only seconds for basic assembly plans). We demonstrate the effectiveness of our algorithms in planning the assembly of hybrid libraries, under a variety of experimental options and restrictions, including flexibility in the position and amino acid sequence of breakpoints. Our method promises to enable more effective application of site-directed recombination to protein investigation and engineering.

Algorithms↗

Sciatic chronic constriction injury produces cell-type-specific changes in the electrophysiological properties of rat substantia gelatinosa neurons.

Peripheral nerve injury increases spontaneous action potential discharge in spinal dorsal horn neurons and augments their response to peripheral stimulation. This "central hypersensitivity, " which relates to the onset and persistence of neuropathic pain, reflects spontaneous activity in primary afferent fibers as well as long-term changes in the intrinsic properties of the dorsal horn (centralization). To isolate and investigate cellular mechanisms underlying "centralization," sciatic nerves of 20-day-old rats were subjected to 13-25 days of chronic constriction injury (CCI; Mosconi-Kruger polyethylene cuff model). Spinal cord slices were then acutely prepared from sham-operated or CCI animals, and whole cell recording was used to compare the properties of five types of substantia gelatinosa neuron. These were defined as tonic, irregular, phasic, transient, or delay according to their discharge pattern in response to depolarizing current. CCI did not affect resting membrane potential, rheobase, or input resistance in any neuron type but increased the amplitude and frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) in delay, transient, and irregular cells. These changes involved alterations in the action potential-independent neurotransmitter release machinery and possible increases in the postsynaptic effectiveness of glutamate. By contrast, in tonic cells, CCI reduced the amplitude and frequency of spontaneous and miniature EPSCs. Such changes may relate to the putative role of tonic cells as inhibitory GABAergic interneurons, whereas increased synaptic drive to delay cells may relate to their putative role as the excitatory output neurons of the substantia gelatinosa. Complementary changes in synaptic excitation of inhibitory and excitatory neurons may thus contribute to pain centralization.

Animals↗

Substantia Gelatinosa neurons in defined-medium organotypic slice culture are similar to those in acute slices from young adult rats.

Peripheral nerve injury promotes an enduring increase in the excitability of the spinal dorsal horn. This change, that likely underlies the development of chronic pain, may be a consequence of prolonged exposure of dorsal horn neurons to mediators such as neurotrophins, cytokines, and neurotransmitters. The long-term effects of such mediators can be analyzed by applying them to spinal neurons in organotypic slice culture. To assess the validity of this approach, we established serum-free, defined-medium organotypic cultures (DMOTC) from E13-14 prenatal rats. Whole-cell recordings were made from neurons maintained in DMOTC for up to 42 days. These were compared with recordings from neurons of similar age in acute spinal cord slices from 15- to 45-day-old rats. Five cell types were defined in acute slices as 'Tonic', 'Irregular', 'Delay', 'Transient' or 'Phasic' according to their discharge patterns in response to depolarizing current. Although fewer 'Phasic' cells were found in cultures, the proportions of 'Tonic', 'Irregular', 'Delay', and 'Transient' were similar to those found in acute slices. GABAergic, glycinergic, and 'mixed' inhibition were observed in neurons in acute slices and DMOTC. Pure glycinergic inhibition was absent in 7d cultures but became more pronounced as cultures aged. This parallels the development of glycinergic inhibition in vivo. These and other findings suggest that fundamental developmental processes related to neurotransmitter phenotype and neuronal firing properties are preserved in DMOTC. This validates their use in evaluating the cellular mechanisms that may contribute to the development of chronic pain.

Action Potentials↗

Mechanical properties of OI type III bone tissue measured by nanoindentation.

Nanoindentation was used to characterize the intrinsic mechanical properties of bone tissue from eight (8) children with type III Osteogenesis Imperfecta (OI). The bone samples were harvested from the cortex portion at the site of bowing (the mid 2/3 of the shaft of the tibia/femur). Unlike normal bone tissue, OI type III cortical bone exhibited more isotropic material properties. Young's modulus and hardness values measured in the longitudinal direction did not show significant differences from the transverse measurements. No differences were observed in modulus or hardness in an analysis of the cortical and trabecular samples. The deformation patterns of the OI type III bone during nanoindentation were found to be similar to those of normal adult bone in an analysis of the ratio of modulus to hardness. No correlation was found between nanoindentation measurement and age in an analysis of regression.

Anisotropy↗

Transretinal choroidal tumor biopsy with a 25-gauge vitrector.

OBJECTIVE: To describe and evaluate transretinal biopsy of choroidal tumors using 25-gauge vitrectomy instrumentation. DESIGN: Retrospective, consecutive, noncomparative case series. PARTICIPANTS: Fourteen patients undergoing choroidal tumor biopsy at an ocular oncology center. METHODS: The biopsies were performed under local or general anesthesia, alone or in combination with ruthenium plaque or tantalum marker insertion. Immunohistochemistry was performed on all samples, and some melanomas were also analyzed cytogenetically. RESULTS: Surgery was uneventful in all cases. A positive tissue diagnosis was made in 13 of 14 patients, albeit at the second attempt in 1 patient. The only failure occurred because the tumor was calcified. CONCLUSION: Transretinal choroidal biopsy with 25-gauge instrumentation yields a larger sample than fine-needle aspiration biopsy, usually producing sufficient tissue for cytogenetic studies. We did not identify safety concerns in this series of patients. Insufficient samples can occur in some patients, and further studies are needed to understand the reason for such failure.

Adenocarcinoma↗

Cytology PT.

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Clinical Competence↗

Opioid-like actions of neuropeptide Y in rat substantia gelatinosa: Y1 suppression of inhibition and Y2 suppression of excitation.

Neuropathic pain that results from injury to the peripheral or CNS responds poorly to opioid analgesics. Y1 and Y2 receptors for neuropeptide Y (NPY) may, however, serve as targets for analgesics that retain their effectiveness in neuropathic pain states. In substantia gelatinosa neurons in spinal cord slices from adult rats, we find that NPY acts via presynaptic Y2 receptors to attenuate excitatory postsynaptic currents (EPSCs) and predominantly on presynaptic Y1 receptors to attenuate glycinergic and GABAergic inhibitory postsynaptic currents (IPSCs). Because NPY attenuates the frequency of TTX-resistant miniature EPSCs and IPSCs, perturbation of the neurotransmitter release process contributes to its actions at both excitatory and inhibitory synapses. These effects, which are reminiscent of those produced by analgesic opioids, provide a cellular basis for previously documented spinal analgesic actions mediated via Y1 and Y2 receptors in neuropathic pain paradigms. They also underline the importance of suppression of inhibition in spinal analgesic mechanisms.

Analgesics, Opioid↗

Gait analysis in children and adolescents with spinal cord injuries.

BACKGROUND/OBJECTIVE: Improving ambulation in patients with spinal cord injuries (SCIs) is a major challenge for the clinician. Physical examination can be supplemented with biomechanic information from the gait laboratory to comprehensively evaluate and improve walking. Gait analysis is an effective method to evaluate 3-dimensional dynamic joint range of motion (kinematics) and forces (kinetics) occurring at the hip, knee, and ankle/foot. Clinical gait analysis has proved to be essential for surgical and rehabilitative planning and posttreatment assessment in many neuromuscular disorders. Benefits of gait analysis also may apply to walking patients with SCIs. The goal of this study was to demonstrate the utility of gait analysis in children and adolescents with SCIs. METHODS: Motion analysis evaluations and physical examinations were performed on 33 children and adolescents with SCIs (16 males, 17 females). Mean age at time of gait analysis was 11.7 years, with an age range of 2.5 to 21 years. RESULTS: Abnormal kinematic patterns, which were repeatable over several years, were demonstrated in the patients with SCI. Gait analysis was beneficial in making educated treatment decisions about orthotic prescription, surgery, postsurgical evaluation, prescription of new therapy, evaluation of spasticity medications, and experimental treatments. Another advantage of gait analysis is recording and reviewing changes in gait over time. In our study, 22 of the 33 patients received a 1-year follow-up gait analysis. Patients followed over multiyear intervals have demonstrated characteristic changes in mobility patterns, resulting in changes in prescription of mobility aids. The benefits of gait analysis are demonstrated by case presentation. CONCLUSION: Improved ambulation for patients with SCI can be accomplished with physical therapy, orthotics, spasticity management, and surgery. These treatments, as well as innovative new areas such as functional electrical stimulation and robotic-assisted therapy, rely on gait analysis to provide a template of normal walking patterns and to measure function and document improvements.

Adolescent↗

Possible role of phosphatidylinositol 4,5 bisphosphate in luteinizing hormone releasing hormone-mediated M-current inhibition in bullfrog sympathetic neurons.

Luteinizing hormone releasing hormone (LHRH) is a physiological modulator of neuronal excitability in bullfrog sympathetic ganglia (BFSG). Actions of LHRH involve suppression of the noninactivating, voltage-dependent M-type K+ channel conductance (gM). We found, using whole-cell recordings from these neurons, that LHRH-induced suppression of gM was attenuated by the phospholipase C (PLC) inhibitor U73122 (10 microM) but not by the inactive isomer U73343 (10 microM). Buffering internal Ca2+ to 117 nM with intracellular 20 mM BAPTA + 8 mM Ca2+ or to < 10 nM with intracellular 20 mM BAPTA + 0.4 mM Ca2+ did not attenuate LHRH-induced gM suppression. Suppression of gM by LHRH was not antagonized by the inositol 1,4,5 trisphosphate (InsP3) receptor antagonist heparin (approximately 300 microM). Preventing phosphatidylinositol-4,5-bisphosphate (PIP2) synthesis by blocking phosphatidylinositol-4-kinase with wortmannin (10 microM) or with the nonhydrolysable ATP analogue AMP-PNP (3 mM) prolonged recovery of LHRH-induced gM suppression. This effect was not produced by blocking phosphatidyl inositol-3-kinase with LY294002 (10 microM). Rundown of gM was attenuated when cells were dialysed with 240 microM di-octanoyl PIP2 or 240 microM di-octanoyl phosphatidylinositol-3,4,5-trisphosphate (PIP3) but not with 240 microM di-octanoyl phosphatidylcholine. LHRH-induced gM suppression was competitively antagonized by dialysis with 240 microM di-octanoyl PIP2, but not with di-octanoyl phosphatidylcholine. These results would be expected if LHRH-induced gM suppression reflects a PLC-mediated decrease in plasma membrane PIP2 levels.

Animals↗

Neurotrophic regulation of calcium channels by the peptide neurotransmitter luteinizing hormone releasing hormone.

We exploited the simple organization of bullfrog paravertebral sympathetic ganglia (BFSG) to test whether the neurotransmitter peptide luteinizing hormone releasing hormone (LHRH), which generates the late slow EPSP, could also exert long-term neurotrophic control of ion channel expression. Whole-cell recordings from B-cells in BFSG showed that removal of all of the sources of ganglionic LHRH for 10 d by cutting preganglionic C-fibers in vivo caused a 28% reduction in Ca2+ current density. When BFSG B-neurons were dissociated from adult bullfrogs and maintained in a defined-medium, neuron-enriched, low-density, serum-free culture, the ICa density was increased by 49% after 6-7 d in the presence of 0.45 microm LHRH. This increase was not associated with alterations in the voltage dependence of Ca2+ current activation or inactivation and reflected a selective increase in N-type Ca2+ channel current. The increase in ICa density induced by LHRH was blocked by the transcription inhibitor actinomycin D. These results suggest that chronic exposure to a neurotransmitter that acts through G-protein-coupled receptors exerts long-term control of ion channel expression in a fully differentiated, adult sympathetic neuron in vitro or in vivo.

Animals↗

Experiments to test the role of phosphatidylinositol 4,5-bisphosphate in neurotransmitter-induced M-channel closure in bullfrog sympathetic neurons.

Various neurotransmitters excite neurons by suppressing a ubiquitous, voltage-dependent, noninactivating K+ conductance called the M-conductance (gM). In bullfrog sympathetic ganglion neurons the suppression of gM by the P2Y agonist ATP involves phospholipase C (PLC). The present results are consistent with the involvement of the lipid and inositol phosphate cycles in the effects of both P2Y and muscarinic cholinergic agonists on gM. Impairment of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) with the phosphatidylinositol 4-kinase inhibitor wortmannin (10 microm) slowed or blocked the recovery of agonist-induced gM suppression. This effect could not be attributed to an action of wortmannin on myosin light chain kinase or on phosphatidylinositol 3-kinase. Inhibition of PIP2 synthesis at an earlier point in the lipid cycle by the use of R59022 (40 microm) to inhibit diacylglycerol kinase also slowed the rate of recovery of successive ATP responses. This effect required several applications of agonist to deplete levels of various phospholipid intermediates in the lipid cycle. PIP2 antibodies attenuated the suppression of gM by agonists. Intracellular application of 20 microm PIP2 slowed the rundown of KCNQ2/3 currents expressed in COS-1 or tsA-201 cells, and 100 microm PIP2 produced a small potentiation of native M-current bullfrog sympathetic neurons. These are the results that might be expected if agonist-induced activation of PLC and the concomitant depletion of PIP2 contribute to the excitatory action of neurotransmitters that suppress gM.

1-Phosphatidylinositol 4-Kinase↗

Effects and consequences of nerve injury on the electrical properties of sensory neurons.

Nociceptive pain alerts the body to potential or actual tissue damage. By contrast, neuropathic or "noninflammatory" pain, which results from injury to the nervous system, serves no useful purpose. It typically continues for years after the original injury has healed. Sciatic nerve lesions can invoke chronic neuropathic pain that is accompanied by persistent, spontaneous activity in primary afferent fibers. This activity, which reflects changes in the properties and functional expression of Na+, K+, and Ca2+ channels, initiates a further increase in the excitability of second-order sensory neurons in the dorsal horn. This change persists for many weeks. The source of origin of the pain thus moves from the peripheral to the central nervous system. We hypothesize that this centralization of pain involves the inappropriate release of peptidergic neuromodulators from primary afferent fibers. Peptides such as substance P, neuropeptide Y (NPY), calcitonin-gene-related peptide (CGRP), and brain-derived neurotrophic factor (BDNF) may promote enduring changes in excitability as a consequence of neurotrophic actions on ion channel expression in the dorsal horn. Findings that form the basis of this hypothesis are reviewed. Study of the neurotrophic control of ion channel expression by spinal peptides may thus provide new insights into the etiology of neuropathic pain.

Action Potentials↗

Morphine-3beta-D-glucuronide suppresses inhibitory synaptic transmission in rat substantia gelatinosa.

High doses of intrathecally applied morphine or morphine-3beta-D-glucuronide (M3G) produce allodynia and hyperalgesia. Whole-cell patch-clamp recordings were made from substantia gelatinosa neurons in transverse slices of adult rat lumbar spinal cord to compare the actions of M3G with those of the mu-opioid agonist, DAMGO ([D-Ala(2),N-Met-Phe(4),Gly-ol(5)]-enkephalin), and the ORL(1) agonist, nociceptin/orphanin FQ (N/OFQ). M3G (1-100 microM) had little or no effect on evoked excitatory postsynaptic currents (EPSC) and no effect on postsynaptic membrane conductance. In contrast, 1 microM DAMGO and 1 microM N/OFQ reduced the amplitude of evoked EPSCs and activated an inwardly rectifying K(+) conductance. M3G did not attenuate the effect of DAMGO or N/OFQ on evoked EPSC amplitude. However, 1 to 100 microM M3G reduced the amplitude of evoked GABAergic and glycinergic inhibitory postsynaptic current (IPSC) by up to 48%. This effect was naloxone-insensitive. The evoked IPSC was also attenuated by DAMGO, but not by N/OFQ. Because M3G reduced the frequency of tetrodotoxin-insensitive miniature IPSCs and increased paired-pulse facilitation, it appeared to act presynaptically to disinhibit substantia gelatinosa neurons. This effect, which does not appear to involve mu-opioid or ORL(1) receptors, may contribute to the allodynia and hyperalgesia observed after intrathecal application of high doses of morphine.

Animals↗

Changes in Na(+) channel currents of rat dorsal root ganglion neurons following axotomy and axotomy-induced autotomy.

Section of rat sciatic nerve (axotomy) increases the excitability of neurons in the L(4)-L(5) dorsal root ganglia (DRG). These changes are more pronounced in animals that exhibit a self-mutilatory behavior known as autotomy. We used whole cell recording to examine changes in the tetrodotoxin-sensitive (TTX-S) and the tetrodotoxin-resistant (TTX-R) components of sodium channel currents (I(Na)) that may contribute to axotomy-induced increases in excitability. Cells were initially divided on the basis of size into "large," "medium," and "small" groups. TTX-S I(Na) predominated in "large" cells, whereas TTX-R I(Na) predominated in some, but not all "small cells." "Small" cells were therefore subdivided into "small-slow" cells, which predominantly exhibited TTX-R I(Na) and "small fast" cells that exhibited more TTX-S I(Na). In contrast to results obtained in other laboratories, where slightly different experimental procedures were used, we found that axotomy increased TTX-R and/or TTX-S I(Na) and slowed inactivation. The effects were greatest in "small-slow" cells and least in "large" cells. The changes promoted by axotomy were expressed more clearly in animals that exhibited autotomy. Also, the presence of autotomy correlated with a shift in the properties of I(Na) in "large" rather than "small-slow," putative nociceptive cells. These trends parallel previous observations on axotomy-induced increases in excitability, spike height, and spike width that are also greatest in "small" cells and least in "large" cells. In addition, the presence of autotomy correlates with an increase in excitability of "large" rather than "small" cells. Increases in TTX-R and TTX-S I(Na) thus coincide with axotomy-induced increases in excitability and alterations in spike shape across the whole population of sensory neurons. Injury-induced changes of this type are likely associated with the onset of chronic pain in humans.

Animals↗

ATP-inhibition of M current in frog sympathetic neurons involves phospholipase C but not Ins P(3), Ca(2+), PKC, or Ras.

Suppression of the voltage-activated, noninactivating K(+) conductance (M conductance; g(M)) by muscarinic agonists, P(2Y) agonists or bradykinin increases neuronal excitability. All agonist effects are mediated, at least in part, via the Gq/(11) class of G protein. We found, using whole cell or perforated patch recording from bullfrog sympathetic B neurons that ATP-induced suppression of g(M) was attenuated by the phospholipase C (PLC) inhibitor, U73122 (IC(50) approximately 0.14 microM) but not by the inactive isomer, U73343. The ability of extracellularly applied U73122 to inhibit PLC was confirmed by its antagonism of ATP-induced elevation of intracellular Ca(2+) as measured by fura-2 photometry. ATP-induced g(M) suppression was not antagonized by the protein kinase C (PKC) inhibitor, chelerythrine (5 microM extracellular +10 microM intracellular), by the Ca(2+)-ATPase inhibitor, thapsigargin (5 microM), or by inositol trisphosphate (InsP(3)) receptor antagonists, heparin (approximaterly 300 microM) or xestospongin C (1.8 microM). The effect of ATP on g(M) was thus dependent on PLC yet independent of PKC and of InsP(3)-induced release of intracellular Ca(2+). We therefore tested the involvement of a PKC-independent action of diacylglycerol (DAG) that could occur via activation of Ras. This low-molecular-weight G protein is activated following DAG binding to Ras-GRP, a neuronal Ras-GTP exchange factor. However, impairment of Ras function by culturing neurons with isoprenylation inhibitors (perillic acid, 0.1 mM, or alpha-hydroxyfarnesyl-phosphonic acid, 10 microM) failed to affect ATP-induced g(M) suppression. Inhibition of MEK (mitogen-activated protein kinase), a downstream target of Ras, by using PD 98059 (10 microM) was also ineffective. The transduction mechanism used by ATP to suppress g(M) in frog sympathetic neurons therefore differs from the PLC-independent mechanism used by muscarine and from the PLC and Ca(2+)-dependent mechanism used by bradykinin and UTP in mammalian ganglia. The possibility remains that "lipid-signaling" mechanisms, perhaps involving PLC-induced depletion of phosphatidylinositol bisphosphate, are involved in PLC-mediated inhibition of g(M) by ATP in amphibian sympathetic neurons.

Adenosine Triphosphate↗