Biomedical subjects

Michael J Iadarola

Publications and source records attributed to Michael J Iadarola.

At least 19 recordsLinked to original sources

PubMed2006

Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1.

Transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel highly expressed in small-diameter sensory neurons, is activated by heat, protons, and capsaicin. The phosphorylation of TRPV1 provides a versatile regulation of intracellular calcium levels and is critical for TRPV1 function in responding to a pain stimulus. We have previously reported that cyclin-dependent kinase 5 (Cdk5) activity regulates nociceptive signaling. In this article we report that the Cdk5-mediated phosphorylation of TRPV1 at threonine-407 can modulate agonist-induced calcium influx. Inhibition of Cdk5 activity in cultured dorsal root ganglia neurons resulted in a significant reduction of TRPV1-mediated calcium influx, and this effect could be reversed by restoring Cdk5 activity. Primary nociceptor-specific Cdk5 conditional-knockout mice showed reduced TRPV1 phosphorylation, resulting in significant hypoalgesia. Thus, the present study indicates that Cdk5-mediated TRPV1 phosphorylation is important in the regulation of pain signaling.

Amino Acid Sequence↗

PubMed2006

Modulatory roles of the NPFF system in pain mechanisms at the spinal level.

The possible roles of the NPFF system in pain processing are summarized from the viewpoints of (1) biological activities of NPFF, (2) anatomical distribution of NPFF and its receptor(s) and (3) the regulation of NPFF and receptor(s) in animal models of pain. NPFF and NPFF analogues were found to have analgesic, pronociceptive and morphine modulating activities. Since the isolation of NPFF, several other RF-NH2 peptides have been identified and some of them were found to have nociceptive or morphine modulating activity. Depending on the pharmacological doses and locations of administration, NPFF may exhibit the biological activities of other structurally related RF-NH2 peptides thus complicating NPFF bioactivity studies and their interpretation. Acid sensing ion channels were found to respond to RF-NH2 peptides including NPFF, raising the possibility that interaction of NPFF and acid sensing ion channels can modulate nociceptive activity. NPFF and NPFF receptor mRNAs are highly expressed and localized in the superficial layers of the dorsal cord, the two genes are also in dorsal root ganglia though at much lower level. The spinal NPFF system is up-regulated by peripheral inflammation in the rat. Furthermore, immunohistochemically, NPFF receptor 2-protein was demonstrated to be increased in the primary afferents in the spinal cord of rats with peripheral inflammation. Regulation and localization of spinal NPFF systems, taken together with the analgesic bioactivity of intrathecally administered NPFF, strongly suggest involvement of spinal NPFF system in pain processing.

Amino Acid Sequence↗

PubMed2006

Cyclin-dependent kinase 5 activity regulates pain signaling.

Several molecules and cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase implicated in the development and disease of the mammalian nervous system. The precise role of this kinase in sensory pathways has not been well characterized. Here we report a molecular role for Cdk5 in nociception. We identified the expression of Cdk5 and its activator p35 in nociceptive neurons, which is modulated during a peripheral inflammatory response. Increased calpain activity in sensory neurons after inflammation resulted in the cleavage of p35 to p25, which forms a more stable complex with Cdk5 and, consequently, leads to elevation of Cdk5 activity. p35 knockout mice (p35(-/-)), which exhibit significantly decreased Cdk5 activity, showed delayed responses to painful thermal stimulation compared with WT controls. In contrast, mice overexpressing p35, which exhibit elevated levels of Cdk5 activity, were more sensitive to painful thermal stimuli than were controls. In conclusion, our data demonstrate a role for Cdk5/p35 activity in primary afferent nociceptive signaling, suggesting that Cdk5/p35 may be a target for the development of analgesic drugs.

Animals↗

PubMed2005

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography.

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.

Chromatography, High Pressure Liquid↗

PubMed2005

Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation.

BACKGROUND: Spinal cord N-methyl-D-aspartate (NMDA) receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury. RESULTS: Lumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation. CONCLUSION: These data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease.

Journal Article↗

PubMed2005

Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling.

Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.

Animals↗

PubMed2005

Peripheral targeting of the trigeminal ganglion via the infraorbital foramen as a therapeutic strategy.

A unique interventional approach for modulating sensory signaling involves targeting neurons in the sensory ganglia through use of pharmacological or gene therapies. This has previously been accomplished in the trigeminal system via stereotactic ganglionic microinjections. However, this procedure can be time consuming and requires expensive stereotactic equipment. The current paper describes a percutaneous approach for injecting the trigeminal ganglion with an electrical-stimulation needle inserted through the infraorbital foramen, infraorbital canal and foramen rotundum. The needle tip was positioned in the ganglion by eliciting a twitch in the ipsilateral masseter following electrical stimulation. The technique was imaged using computerized tomography and methylene blue dye to verify the site of the injection. We validated this technique by reproducing the results from our prior study that involved injection of resiniferatoxin (RTX) into the trigeminal ganglion using the stereotactic approach. Both techniques for intraganglionic injection of RTX produced a specific deletion of C-fiber neurons, as demonstrated by inhibition of neurogenic-induced plasma extravasation and behavioral assays. Thus, we propose a novel and simple technique for studying mechanisms of peripheral sensory modulation of orofacial pain via direct application of drugs, tracers or viral vectors around trigeminal sensory neuronal cell bodies. This technique minimizes trauma to brain structures that may have an impact on pain perception.

Animals↗

PubMed2005

Physiologic and antinociceptive effects of intrathecal resiniferatoxin in a canine bone cancer model.

BACKGROUND: Resiniferatoxin is a potent capsaicin analog. Intrathecal administration leads to selective, prolonged opening of the transient receptor potential V1 ion channel, which is localized mainly to C-fiber primary afferent nociceptive sensory neurons. Following work in laboratory animals, the authors explored the use of intrathecal resiniferatoxin to control spontaneous bone cancer pain in companion (pet) dogs. METHODS: Normal canine population: Behavioral testing was performed to establish baseline paw withdrawal latency; subsequently, general anesthesia was induced and resiniferatoxin was administered intrathecally while hemodynamic parameters were recorded. Behavior testing was repeated for 12 days after administration of resiniferatoxin. Clinical canine population: Twenty companion dogs with bone cancer pain were recruited. The animal's baseline level of discomfort and analgesic use were recorded. Resiniferatoxin was administered intrathecally and hemodynamic parameters were monitored while the dogs were under general anesthesia. Dogs were reevaluated up to 14 weeks after resiniferatoxin administration. RESULTS: Normal canine population: In the first minutes after resiniferatoxin injection, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate from baseline. Two days after injection, limb withdrawal latencies increased to the point of cutoff in the dogs that received at least 1.2 microg/kg resiniferatoxin. Clinical canine population: From baseline, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate after resiniferatoxin injection. Comfort scores were significantly improved at 2, 6, 10, and 14 weeks after resiniferatoxin administration (P < 0.0001). There was decreased or discontinued use of supplemental analgesics in 67% of the dogs 2 weeks after resiniferatoxin administration. CONCLUSIONS: Intrathecal resiniferatoxin elicits transient hemodynamic effects. In controls, a profound and sustained blockade of thermal stimuli is produced in a dose-dependent fashion. Similar administration in dogs with bone cancer produces a prolonged antinociceptive response.

Analgesics↗

PubMed2004

Vanilloid receptor 1 regulates multiple calcium compartments and contributes to Ca2+-induced Ca2+ release in sensory neurons.

Vanilloid receptor 1 belongs to the transient receptor potential ion channel family and transduces sensations of noxious heat and inflammatory hyperalgesia in nociceptive neurons. These neurons contain two vanilloid receptor pools, one in the plasma membrane and the other in the endoplasmic reticulum. The present experiments characterize these two pools and their functional significance using calcium imaging and 45Ca uptake in stably transfected cells or dorsal root ganglion neurons. The plasma membrane localized receptor is directly activated by vanilloids. The endoplasmic reticulum pool was demonstrated to be independently activated with 20 microm capsaicin or 1.6 microm resiniferatoxin using a bathing solution containing 10 microm Ruthenium Red (to selectively block plasma membrane-localized receptors) and 100 microm EGTA. We also demonstrate an overlap between the endoplasmic reticulum-localized vanilloid receptor regulated stores and thapsigargin-sensitive stores. Direct depletion of calcium via activation of endoplasmic reticulum-localized vanilloid receptor 1 triggered store operated calcium entry. Furthermore, we found that, in the presence of low extracellular calcium (10(-5) m), either 2 microm capsaicin or 0.1 nm-1.6 microm resiniferatoxin caused a pronounced calcium-induced calcium release in either vanilloid receptor-expressing neurons or heterologous expression systems. This phenomenon may allow new insight into how nociceptive neuron function in response to a variety of nociceptive stimuli both acutely and during prolonged nociceptive signaling.

Animals↗

PubMed2004

Comparison of experimental and acute clinical pain responses in humans as pain phenotypes.

UNLABELLED: This study evaluates the sensitivity of normal subjects (N = 617; 369 women, 248 men) to experimentally induced pain including thermal stimuli and the cold pressor test to delineate individual response patterns and pain phenotypes. A subset of subjects (n = 157; 99 women and 58 men) also underwent standardized oral surgery, and the responses to clinically induced acute inflammatory pain were evaluated. A wide range of pain responses was found in both the experimental and clinical situations. The latency for withdrawal in the cold pressor test exhibited a dichotomous distribution of short and long times. Women exhibited higher responses to cold (P < .001) and thermal stimuli (P < .05) than men. Ethnicity affected pain responses to thermal stimuli ranging from 43 degrees C to 47 degrees C (P < .05) and cold stimuli (P < .001). However, neither gender nor ethnicity affected pain responses to clinically induced acute inflammatory stimuli. Cross-modality comparisons of responses within experimental pain showed strong correlations (P < .01) but weaker relationships with clinical inflammatory pain. These data suggest that the background factors and characteristics of experimental pain responses differ from those of clinical pain; therefore, experimental pain ratings alone are not sufficient to predict responses to clinically induced acute pain. PERSPECTIVE: The findings of the present study suggest that investigations of pain phenotypes should take into consideration the subjects' gender and ethnicity and the pain-inducing stimuli. The predictive value of experimental pain for clinically induced pain is weak and not reliable.

Acute Disease↗

PubMed2004

Genetic influence on variability in human acute experimental pain sensitivity associated with gender, ethnicity and psychological temperament.

While a variety of cultural, psychological and physiological factors contribute to variability in both clinical and experimental contexts, the role of genetic factors in human pain sensitivity is increasingly recognized as an important element. This study was performed to evaluate genetic influences on variability in human pain sensitivity associated with gender, ethnicity and temperament. Pain sensitivity in response to experimental painful thermal and cold stimuli was measured with visual analogue scale ratings and temperament dimensions of personality were evaluated. Loci in the vanilloid receptor subtype 1 gene (TRPV1), delta opioid receptor subtype 1 gene (OPRD1) and catechol O-methyltransferase gene (COMT) were genotyped using 5' nuclease assays. A total of 500 normal participants (306 females and 194 males) were evaluated. The sample composition was 62.0% European American, 17.4% African American, 9.0% Asian American, and 8.6% Hispanic, and 3.0% individuals with mixed racial parentage. Female European Americans with the TRPV1 Val(585) Val allele and males with low harm avoidance showed longer cold withdrawal times based on the classification and regression tree (CART) analysis. CART identified gender, an OPRD1 polymorphism and temperament dimensions of personality as the primary determinants of heat pain sensitivity at 49 degrees C. Our observations demonstrate that gender, ethnicity and temperament contribute to individual variation in thermal and cold pain sensitivity by interactions with TRPV1 and OPRD1 single nucleotide polymorphisms.

Acute Disease↗

PubMed2004

Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control.

Control of cancer, neuropathic, and postoperative pain is frequently inadequate or compromised by debilitating side effects. Inhibition or removal of certain nociceptive neurons, while retaining all other sensory modalities and motor function, would represent a new therapeutic approach to control severe pain. The enriched expression of transient receptor potential cation channel, subfamily V, member 1 (TRPV1; also known as the vanilloid receptor, VR1) in nociceptive neurons of the dorsal root and trigeminal ganglia allowed us to test this concept. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya induces calcium cytotoxicity by opening the TRPV1 ion channel and selectively ablates nociceptive neurons. This treatment blocks experimental inflammatory hyperalgesia and neurogenic inflammation in rats and naturally occurring cancer and debilitating arthritic pain in dogs. Sensations of touch, proprioception, and high-threshold mechanosensitive nociception, as well as locomotor function, remained intact in both species. In separate experiments directed at postoperative pain control, subcutaneous administration of RTX transiently disrupted nociceptive nerve endings, yielding reversible analgesia. In human dorsal root ganglion cultures, RTX induced a prolonged increase in intracellular calcium in vanilloid-sensitive neurons, while leaving other, adjacent neurons unaffected. The results suggest that nociceptive neuronal or nerve terminal deletion will be effective and broadly applicable as strategies for pain management.

Adult↗

PubMed2003

Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells.

Resiniferatoxin (RTX), a potent analog of capsaicin, was evaluated electrophysiologically in dorsal root ganglion (DRG) cells and cell lines ectopically expressing the vanilloid receptor type 1 (VR1) to determine if cell phenotype influenced RTXs neurotoxic properties. Furthermore, capsaicin and heat activation of VR1 were evaluated in these cells to determine if cellular damage was unique to RTX activation of the receptors. RTX application to DRG cells identified as type 1, 2 or 5, cell types known to express VR1, induced large inward currents. RTX did not induce currents in DRG cells that do not express the receptor (type 4 cells). In cell lines ectopically expressing VR1, RTX-induced similar currents. RTX produced no effect in non-transfected cells. After exposure to RTX both DRG cells and transfected cells failed to respond to subsequent applications of the agonist. In addition, whole cell capacitance was reduced up to 70%. The decrease in capacitance was associated with the loss of plasma membrane, as determined by confocal microscopy. Cell phenotype, other than VR1 expression, did not influence the response to RTX. Interestingly, capsaicin and heat activation of vanilloid receptors also decreased cell capacitance, but the loss of membrane was not as great as with RTX and responses to these stimuli were not lost after the initial exposure. The loss of cell membrane required elevated intracellular levels of Ca2+. From these data it was concluded that the loss of cell membrane was dependent on the presence of both VR1 and intracellular Ca2+ accumulation, but not on cell phenotype.

Animals↗

PubMed2003

Cystatin C as a cerebrospinal fluid biomarker for pain in humans.

Through a process of subtraction cloning and differential hybridization, we previously identified several new genes whose expression was induced by peripheral inflammation. One of these coded for cystatin C, a secreted cysteine protease inhibitor in the cystatin superfamily. We hypothesized that, concurrent with increased expression in dorsal horn, increased secretion would elevate the cystatin C content in cerebrospinal fluid (CSF) during active pain states. Alterations were assessed by immunoassay and by surface enhanced laser desorption ionization (SELDI) mass spectrometry with either reverse phase or immobilized anti-cystatin C antibody surfaces using CSF from ten age-matched obstetrical patients at term. Five control subjects were scheduled for an elective caesarian section and were not in pain. Another five subjects were in labor for 8.9+/-1h and were in severe pain as assessed with a visual analog scale and the McGill short form questionnaire. The level of cystatin C as measured by immunoassay in the non-pain patients was 2.77+/-0.75 microg/ml and in the pain patients 5.36+/-0.92 microg/ml (P<0.02). The elevation occurred without significant change in total CSF protein or beta-endorphin content. The cystatin C increase also was detectable by SELDI with either raw CSF or after antibody capture. These data are consistent with our previous animal study and the idea that persistent pain induces the synthesis and release of cystatin C in dorsal spinal cord, the surplus of which overflows into the CSF.

Adult↗

PubMed2003

Peripherally induced resiniferatoxin analgesia.

Selective blockade of nociceptive pathways represents a mechanism-based approach that has attracted a large variety of pharmacological and molecular investigations. A potential site for selective intervention is the primary afferent nociceptive nerve terminal. Binding of resiniferatoxin (RTX) to the vanilloid-1 receptor (VR1) stimulates and then inactivates heat and vanilloid-responsive nerve endings involved in heat and inflammatory pain signaling which can progress to localized degeneration of the peripheral ending followed by regeneration. Application of RTX directly to peripheral nerve endings produces a long term, reversible attenuation of nociceptive transmission. Heat hyperalgesia and mechanical allodynia were assessed prior to injection of RTX into the hindpaw (baseline) and at acute (minutes-hours) and more chronic (days-weeks) times after injection. Acutely, an inverse dose-to-pain response (guarding, licking) for RTX (0.0625-2.0 microg) occurs, followed by selective attenuation of peripheral pain transmission. Thermal nociception was decreased in a concentration-dependent fashion and lasted up to 21 days, without impairing motor function. Administration of RTX blocked both inflammation-induced hyperalgesia and spinal c-Fos induction. The results demonstrate the efficacy and therapeutic potential of reversible, peripheral C-fiber 'inactivation' for intermediate duration pain control.

Analgesia↗

PubMed2003

Direct interstitial infusion of NK1-targeted neurotoxin into the spinal cord: a computational model.

Convection-enhanced delivery of substance P (SP) nocitoxins to the spinal cord interstitium is under consideration for the treatment of chronic pain. To characterize treatment protocols, a three-dimensional finite-element model of infusion into the human dorsal column was developed to predict the distribution of SP-diphtheria toxin fusion protein (SP-DT') within normal and target tissue. The model incorporated anisotropic convective and diffusive transport through the interstitial space, hydrolysis by peptidases, and intracellular trafficking. For constant SP-DT' infusion (0.1 microl/min), the distribution of cytotoxicity in NK1 receptor-expressing neurons was predicted to reach an asymptotic limit at 6-8 h in the transverse direction at the level of the infusion cannula tip ( approximately 60% ablation of target neurons in lamina I/II). Computations revealed that SP-DT' treatment was favored by a stable SP analog (half-life approximately 60 min), high infusate concentration (385 nM), and careful catheter placement (adjacent to target lamina I/II). Sensitivity of cytotoxic regions to NK1 receptor density and white matter protease activity was also established. These data suggest that intraparenchymal infusions can be useful for treatment of localized chronic pain.

Chronic Disease↗

PubMed2002

Measurement of resiniferatoxin in cerebrospinal fluid by high-performance liquid chromatography.

A sensitive and simple high-performance liquid chromatographic (HPLC) assay was developed for the quantification of resiniferatoxin (RTX) in canine cerebrospinal fluid (CSF). A reversed-phase C(18) column and acetonitrile in 0.02 M NaH(2)PO(4) as mobile phase provided satisfactory resolution for RTX analysis. Direct HPLC analysis of the CSF samples without sample extraction or preparation improves the accuracy and detection limits of this assay. This assay was applied to measure CSF RTX content to test this method for research and clinical applications related to studies examining its analgesia effects.

Animals↗

PubMed2002

Protein kinase C(alpha) is required for vanilloid receptor 1 activation. Evidence for multiple signaling pathways.

Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activated Ca(2+) uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu was antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist indicating that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulated PKC(alpha) in dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCbeta, -delta, -epsilon, and -zeta. Loss of PKC(alpha) correlated with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (i) direct ligand binding (anandamide, vanilloids) or (ii) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKC(alpha), but not PKC(epsilon). These studies suggest that PKC(alpha) in sensory neurons may elicit or enhance pain during inflammation or ischemia.

3T3 Cells↗