Transcription factors Sp1 and Sp4 regulate TRPV1 gene expression in rat sensory neurons

<p>Abstract</p> <p>Background</p> <p>The capsaicin receptor, transient receptor potential vanilloid type -1 (TRPV1) directs complex roles in signal transduction including the detection of noxious stimuli arising from cellular injury and inflammation. Under pathophysiologic conditions, TRPV1 mRNA and receptor protein expression are elevated in dorsal root ganglion (DRG) neurons for weeks to months and is associated with hyperalgesia. Building on our previous isolation of a promoter system for the rat TRPV1 gene, we investigated the proximal TRPV1 P2-promoter by first identifying candidate Sp1-like transcription factors bound <it>in vivo </it>to the P2-promoter using chromatin immunoprecipitation (ChIP) assay. We then performed deletion analysis of GC-box binding sites, and quantified promoter activity under conditions of Sp1 / Sp4 over-expression versus inhibition/knockdown. mRNA encoding Sp1, Sp4 and TRPV1 were quantified by qRT-PCR under conditions of Sp1/Sp4 over-expression or siRNA mediated knockdown in cultured DRG neurons.</p> <p>Results</p> <p>Using ChIP analysis of DRG tissue, we demonstrated that Sp1 and Sp4 are bound to the candidate GC-box site region within the endogenous TRPV1 P2-promoter. Deletion of GC-box "a" or "a + b" within the P2- promoter resulted in a complete loss of transcriptional activity indicating that GC-box "a" was the critical site for promoter activation. Co-transfection of Sp1 increased P2-promoter activity in cultured DRG neurons whereas mithramycin-a, an inhibitor of Sp1-like function, dose dependently blocked NGF and Sp1-dependent promoter activity in PC12 cells. Co-transfection of siRNA directed against Sp1 or Sp4 decreased promoter activity in DRG neurons and NGF treated PC12 cells. Finally, electroporation of Sp1 or Sp4 cDNA into cultures of DRG neurons directed an increase in Sp1/Sp4 mRNA and importantly an increase in TRPV1 mRNA. Conversely, combined si-RNA directed knockdown of Sp1/Sp4 resulted in a decrease in TRPV1 mRNA.</p> <p>Conclusion</p> <p>Based on these studies, we now propose a model of TRPV1 expression that is dependent on Sp1-like transcription factors with Sp4 playing a predominant role in activating TRPV1 RNA transcription in DRG neurons. Given that increases of TRPV1 expression have been implicated in a wide range of pathophysiologic states including persistent painful conditions, blockade of Sp1-like transcription factors represents a novel direction in therapeutic strategies.</p

Emergent Orthotopic Liver Transplantation for Hemorrhage from a Giant Cavernous Hepatic Hemangioma: Case Report and Review

IntroductionCavernous hemangiomas represent the most common benign primary hepatic neoplasm, often being incidentally detected. Although the majority of hepatic hemangiomas remain asymptomatic, symptomatic hepatic hemangiomas can present with abdominal pain, hemorrhage, biliary compression, or a consumptive coagulopathy. The optimal surgical management of symptomatic hepatic hemangiomas remains controversial, with resection, enucleation, and both deceased donor and living donor liver transplantation having been reported.Case reportWe report the case of a patient found to have a unique syndrome of multiorgan cavernous hemangiomatosis involving the liver, lung, omentum, and spleen without cutaneous involvement. Sixteen years following her initial diagnosis, the patient suffered from intra-abdominal hemorrhage due to her giant cavernous hepatic hemangioma. Evidence of continued bleeding, in the setting of Kasabach-Merritt Syndrome and worsening abdominal compartment syndrome, prompted MELD exemption listing. The patient subsequently underwent emergent liver transplantation without complication.ConclusionAlthough cavernous hemangiomas represent the most common benign primary hepatic neoplasm, hepatic hemangioma rupture remains a rare presentation in these patients. Management at a center with expertise in liver transplantation is warranted for those patients presenting with worsening DIC or hemorrhage, given the potential for rapid clinical decompensation

Functional Deactivation of the Major Neuronal Nicotinic Receptor Caused by Nicotine and a Protein Kinase C- Dependent Mechanism

SUMMARY The effect of nicotine on the major human neuronal nicotinic receptor (␣4␤2 subtype) was studied in permanently transfected HEK 293 cells. Prolonged exposure to low concentrations of nicotine (1 M) increased epibatidine binding but functionally deactivated the nicotinic receptor, abolishing Ca 2ϩ influx in response to an acute nicotine challenge. Deactivation could also be caused by down-regulating protein kinase C (PKC) activity with 0.5 M phorbol-12,13-dibutyrate or briefly incubating cells with the PKC inhibitor NPC-15437. Recovery from receptor deactivation caused by either nicotine treatment or PKC inhibition occurred slowly (4 -6 hr). Reversal of nicotineinduced deactivation was accelerated by the addition of inhibitors of protein phosphatases 2A and 2B. These data suggest a hypothetical mechanism of nicotine-induced deactivation that involves dephosphorylation of nicotinic receptors at PKC phosphorylation sites. The action of nicotine in the brain is mediated by a family of oligomeric ion channels whose opening is regulated by the binding of the neurotransmitter acetylcholine and drugs such as nicotine. Eleven different mammalian nAChR subunits (␣2-9 and ␤2-4) have been cloned (for reviews, see Refs. 1 and 2), and all the neuronal nicotinic receptors display a pronounced selectivity for Ca 2ϩ relative to Na ϩ (3-5). The most abundant receptor is composed of the ␣4 and ␤2 subunits with a stoichiometry of 2␣:3␤ (6) and is responsible for ϳ85% of the high affinity nicotine binding in the brain (6 -8). When nicotine is administered chronically to rats, a ϳ2-fold up-regulation of high affinity brain nicotinic receptors has been observed (9). This seems to be due to a dramatic decrease in the rate of degradation of the receptor in the cell membrane after nicotine treatment (10). The basis for this change in turnover is obscure. It has also been shown that chronic nicotine exposure leads to what has been termed &quot;functional deactivation&quot; of receptors to distinguish it from short term desensitization (11). Deactivation of neuronal nAChRs was first described and distinguished from receptor desensitization by Simasko et al. (12). The fact that chronic nicotine administration results in an increase in receptor number, coupled with a functional deactivation, suggests a mechanism for the addictive effects of nicotine (13). In this model, withdrawal of nicotine from an individual chronically exposed to the drug would result in reactivation of excess receptors, leading to craving, and prompting a further deactivating dose of the drug (13). Therefore, the relationship between nicotinic receptor number and intrinsic activity is a critical issue. In this report, we show that HEK 293 cells stably expressing the human ␣4␤2 nicotinic receptor subtype, after prolonged exposure to nicotine, display both a dramatic up-regulation of the receptor, together with a functional deactivation that is easily distinguished from simple short term desensitization. This functional deactivation can also be achieved by inhibition of PKC activity in the cells. Furthermore, phosphatase inhibitors increase the rate of recovery from nicotine-induced deactivation of the ␣4␤2 nicotinic receptor. Our data also indicate that the open state conformation per se, rather than calcium ion influx, directs formation of a deactivated receptor structure. Experimental Procedures Cloning human nicotinic receptor subunits. A human ␣4 subunit cDNA probe was generated by RT-PCR of total human brain RNA, with primers based on the rat ␣4 sequence. The resulting PCR fragment, which contained part of the human ␣4 sequence spanning nucleotides 755-985 (all numbering is from the ATG initiatio

Accuracy of carboxyhemoglobin detection by pulse CO-oximetry during hypoxemia.

BACKGROUND: Carbon monoxide poisoning is a significant problem in most countries, and a reliable method of quick diagnosis would greatly improve patient care. Until the recent introduction of a multiwavelength pulse CO-oximeter (Masimo Rainbow SET(®) Radical-7), obtaining carboxyhemoglobin (COHb) levels in blood required blood sampling and laboratory analysis. In this study, we sought to determine whether hypoxemia, which can accompany carbon monoxide poisoning, interferes with the accurate detection of COHb. METHODS: Twelve healthy, nonsmoking, adult volunteers were fitted with 2 standard pulse-oximeter finger probes and 2 Rainbow probes for COHb detection. A radial arterial catheter was placed for blood sampling during 3 interventions: (1) increasing hypoxemia in incremental steps with arterial oxygen saturations (SaO2) of 100% to 80%; (2) normoxia with incremental increases in %COHb to 12%; and (3) elevated COHb combined with hypoxemia with SaO2 of 100% to 80%. Pulse-oximeter (SpCO) readings were compared with simultaneous arterial blood values at the various increments of hypoxemia and carboxyhemoglobinemia (≈25 samples per subject). Pulse CO-oximeter performance was analyzed by calculating the mean bias (SpCO - %COHb), standard deviation of the bias (precision), and the root-mean-square error (A(rms)). RESULTS: The Radical-7 accurately detected hypoxemia with both normal and elevated levels of COHb (bias mean ± SD: 0.44% ± 1.69% at %COHb &lt;4%, and -0.29% ± 1.64% at %COHb ≥4%, P &lt; 0.0001, and A(rms) 1.74% vs 1.67%). COHb was accurately detected during normoxia and moderate hypoxia (bias mean ± SD: -0.98 ± 2.6 at SaO2 ≥95%, and -0.7 ± 4.0 at SaO2 &lt;95%, P = 0.60, and A(rms) 2.8% vs 4.0%), but when SaO2 decreased below approximately 85%, the pulse CO-oximeter always gave low signal quality errors and did not report SpCO values. CONCLUSIONS: In healthy volunteers, the Radical-7 pulse CO-oximeter accurately detects hypoxemia with both low and elevated COHb levels, and accurately detects COHb, but only reads SpCO when SaO2 is more than approximately 85%

Accuracy of carboxyhemoglobin detection by pulse CO-oximetry during hypoxemia.

BackgroundCarbon monoxide poisoning is a significant problem in most countries, and a reliable method of quick diagnosis would greatly improve patient care. Until the recent introduction of a multiwavelength "pulse CO-oximeter" (Masimo Rainbow SET(®) Radical-7), obtaining carboxyhemoglobin (COHb) levels in blood required blood sampling and laboratory analysis. In this study, we sought to determine whether hypoxemia, which can accompany carbon monoxide poisoning, interferes with the accurate detection of COHb.MethodsTwelve healthy, nonsmoking, adult volunteers were fitted with 2 standard pulse-oximeter finger probes and 2 Rainbow probes for COHb detection. A radial arterial catheter was placed for blood sampling during 3 interventions: (1) increasing hypoxemia in incremental steps with arterial oxygen saturations (SaO2) of 100% to 80%; (2) normoxia with incremental increases in %COHb to 12%; and (3) elevated COHb combined with hypoxemia with SaO2 of 100% to 80%. Pulse-oximeter (SpCO) readings were compared with simultaneous arterial blood values at the various increments of hypoxemia and carboxyhemoglobinemia (≈25 samples per subject). Pulse CO-oximeter performance was analyzed by calculating the mean bias (SpCO - %COHb), standard deviation of the bias (precision), and the root-mean-square error (A(rms)).ResultsThe Radical-7 accurately detected hypoxemia with both normal and elevated levels of COHb (bias mean ± SD: 0.44% ± 1.69% at %COHb &lt;4%, and -0.29% ± 1.64% at %COHb ≥4%, P &lt; 0.0001, and A(rms) 1.74% vs 1.67%). COHb was accurately detected during normoxia and moderate hypoxia (bias mean ± SD: -0.98 ± 2.6 at SaO2 ≥95%, and -0.7 ± 4.0 at SaO2 &lt;95%, P = 0.60, and A(rms) 2.8% vs 4.0%), but when SaO2 decreased below approximately 85%, the pulse CO-oximeter always gave low signal quality errors and did not report SpCO values.ConclusionsIn healthy volunteers, the Radical-7 pulse CO-oximeter accurately detects hypoxemia with both low and elevated COHb levels, and accurately detects COHb, but only reads SpCO when SaO2 is more than approximately 85%

Pungent general anesthetics activate transient receptor potential-A1 to produce hyperalgesia and neurogenic bronchoconstriction

BACKGROUND: Volatile anesthetics such as isoflurane and halothane have been in clinical use for many years and represent the group of drugs most commonly used to maintain general anesthesia. However, despite their widespread use, the molecular mechanisms by which these drugs exert their effects are not completely understood. Recently, a seemingly paradoxical effect of general anesthetics has been identified: the activation of peripheral nociceptors by irritant anesthetics. This mechanism may explain the hyperalgesic actions of inhaled anesthetics and their adverse effects in the airways. METHODS: To test the hypothesis that irritant inhaled anesthetics activate the excitatory ion-channel transient receptor potential (TRP)-A1 and thereby contribute to hyperalgesia and irritant airway effects, we used the measurement of intracellular calcium concentration in isolated cells in culture. For our functional experiments, we used models of isolated guinea pig bronchi to measure bronchoconstriction and withdrawal threshold to mechanical stimulation with von Frey filaments in mice. RESULTS: Irritant inhaled anesthetics activate TRPA1 expressed in human embryonic kidney cells and in nociceptive neurons. Isoflurane induces mechanical hyperalgesia in mice by a TRPA1-dependent mechanism. Isoflurane also induces TRPA1-dependent constriction of isolated bronchi. Nonirritant anesthetics do not activate TRPA1 and fail to produce hyperalgesia and bronchial constriction. CONCLUSIONS: General anesthetics induce a reversible loss of consciousness and render the patient unresponsive to painful stimuli. However, they also produce excitatory effects such as airway irritation and they contribute to postoperative pain. Activation of TRPA1 may contribute to these adverse effects, a hypothesis that remains to be tested in the clinical setting