Effects Of Procaine Research Articles

RfaH suppresses small RNA MicA inhibition of fimB expression in Escherichia coli K-12.

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE, and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.

Local anesthetics inhibit nitric oxide production and l-arginine uptake in cultured bovine aortic endothelial cells

Previous studies have shown that local anesthetics have various effects on nitric oxide (NO) production, but the mechanisms remain unclear. The purpose of this study was to evaluate the effects of local anesthetics on NO production and 2-amino-5-guanidinopentanoic acid (l-arginine) uptake in one cell line. Cultured bovine aortic endothelial cells (BAEC) were stimulated with bradykinin and/or acetylcholine to activate endothelial NO synthase (NOS). BAEC were also incubated with interleukin-1β and lipopolysaccharide to stimulate inducible NOS. NO production was measured with the rapid spectrophotometric method, and l-arginine uptake was measured with high performance liquid chromatography. To assess the effects of local anesthetics, NO production and l-arginine uptake were measured in the presence or absence of procaine or lidocaine. NO was produced in BAEC stimulated with bradykinin and acetylcholine or interleukin-1β and lipopolysaccharide, but NO production was not affected by the addition of superoxide dismutase. In the cells stimulated with bradykinin and acetylcholine, 10μM each of procaine and lidocaine significantly inhibited NO production by 35% and 20%, respectively. In the cells incubated with interleukin-1ß and lipopolysaccharide, the same quantities of procaine and lidocaine significantly inhibited NO production by 15% and 10%, respectively. Both procaine and lidocaine significantly suppressed l-arginine uptake in BAEC stimulated with either bradykinin/acetylcholine or interleukin-1β/lipopolysaccharide. It is suggested that inhibitory effects of procaine and lidocaine on NO production are partially due to suppression of l-arginine uptake.

The effects of lidocaine and procaine on microRNA expression of adipocyte-derived adult stem cells

BackgroundThe microRNA (miRNA) pathway has emerged as one of the biologic pathways implicated in stem cell regulation. miRNA is a noncoding, single-stranded RNA consisting of 20-25 nucleotides that inhibits the protein production at the step of translation. The molecular effects of lidocaine and procaine on adipose stem cells were investigated by examining RNA expression array.MethodsAdipose stem cells were isolated from a prior abdominal liposuction procedure. The human adipose stem cells were cultured and then added to a mixture of 1 ml of culture medium plus 1 ml of 2% lidocaine or 2% procaine for the duration of 30 minutes. The expression levels of miRNAs were estimated by using peptide nucleic acid (PNA)-miRNA array analysis throughout the denaturation and hybridization processes after the isolation of miRNA. The miRNAs detected by microarray that either decreased by half fold or increased by 1.5 fold from the control level were interpreted as significant.ResultsAccording to microarray analysis there were 61 miRNAs in total, and no miRNA had decreased expression levels. The stem cells treatment with lidocaine showed 4 alteration of expression with miR-9a* (1.53 fold), miR-29a (1.64 fold), miR-296-5p (1.64 fold) and miR-373 (1.94 fold). The stem cells treated with procaine showed 32 miRNAs that were significantly up-regulated with a range of 1.5 to 2.06 fold. They were stem cell differentiation-related miRNAs, apoptosis and cell cycle-associated miRNAs, immunity-associated miRNAs and hormonal response-related miRNAs.ConclusionsLidocaine and procaine affect the miRNA expression on adipose stem cells and the effect of procaine is more marked than that of lidocaine.

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa

Reasons for low in vitro fertilisation rates in the horse include the difficulties in inducing capacitation and/or hyperactivation of stallion spermatozoa. The aim of this study was to analyse the effect of noncapacitating and capacitating modified Whitten's (MW) and modified Tyrode's medium (MT) and treatment with procaine (5 mmol), pentoxifylline (3.5 mmol) and trolox (120 mmol) on motility (CASA), capacitation, acrosomal status, viability and mitochondrial membrane potential of stallion spermatozoa (n = 4). While there was no influence of MW and MT on sperm motility, a significant increase in the percentage of viable-capacitated spermatozoa was observed after incubation in capacitating MW (P < 0.05). Pentoxifylline showed no significant effect on the motility pattern but increased the proportion of live-capacitated spermatozoa (P < 0.05). Trolox had no detectable effect on either capacitation or hyperactivation. Procaine was the only agent that induced hyperactivation in terms of a reduced proportion of progressively motile spermatozoa, straight line velocity, straightness, linearity and beat-cross frequency and an increase in the amplitude of lateral head displacement (P < 0.05). The combination of capacitating Whitten's medium and procaine showed the best results for the induction of capacitation and hyperactivation in stallion spermatozoa; this was possible even after short-term incubation.

Procaine Effect on Human Erythrocyte Membrane Explored by Atomic Force Microscopy

The procaine effect on human erythrocytes was investigated by atomic force microscopy (AFM) at three procaine concentrations, about 5 x 10(-7) M, 5 x 10(-5) M and 5 x 10(-4) M. The changes in surface morphology of erythrocyte membrane bring direct evidence on the procaine effect on the cell membrane at micro- and nanometer scale. AFM images of the control erythrocytes (without procaine) showed a well defined concave (donut) shape of cells. The structure of control erythrocytes membrane is featured by closely packed nanometer size intra-membranous particles. After the incubation of the fresh blood with increasing procaine concentrations, a progressive increase in both concave depth and surface roughness of erythrocyte membrane was observed. The particles (granules) of the membrane surface increased progressively with increasing procaine concentrations. The changes in the surface morphology of erythrocyte membrane can be associated with the enlargement of surface granules, due to the aggregation of membranous particles within the cell surface, and the domain structure formation induced by procaine. A large number of moderate elevations from 25 nm to almost 40 nm in lateral size were found to be rather uniformly distributed on the surface of whole erythrocytes at low and medium procaine concentrations, respectively. At the highest procaine concentration, the granules of about 80 nm to almost 90 nm lateral size were found to form rows rather well separated. These data are in substantial agreement with the published results obtained on membrane models in the presence of procaine.

Action Potential Bursts in Central Snail Neurons Elicited by Procaine: Roles of Ionic Currents

The role of ionic currents on procaine-elicited action potential bursts was studied in an identifiable RP1 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage clamp method. The RP1 neuron generated spontaneous action potentials and bath application of procaine at 10 mM reversibly elicited action potential bursts in a concentration-dependent manner. Voltage clamp studies revealed that procaine at 10 mM decreased [1] the Ca2+ current, [2] the Na+ current, [3] the delayed rectifying K+ current I(KD), and [4] the fast-inactivating K+ current (I(A)). Action potential bursts were not elicited by 4-aminopyridine (4-AP), an inhibitor of I(A), whereas they were seen after application of tetraethylammonium chloride (TEA), a blocker of the I(K)(Ca) and I(KD) currents, and tacrine, an inhibitor of I(KD). Pretreatment with U73122, a phospholipase C inhibitor, blocked the action potential bursts elicited by procaine. U73122 did not affect the I(KD) of the RP1 neuron; however, U73122 decreased the inhibitory effect of procaine on the I(KD). Tacrine decreased the TEA-sensitive I(KD) of RP1 neuron but did not significantly affect the I(A). Tacrine also successfully induced action potential bursts in the RP1 neuron. It is concluded that the inhibition on the I(KD) is responsible for the generation of action potential bursts in the central snail RP1 neuron. Further, phospholipase C activity is involved in the procaine-elicited I(KD) inhibition and action potential bursts.

Procaine in Cardioplegia: The Effect on EDHF-Mediated Function in Porcine Coronary Arteries

Hyperkalemia in cardioplegia impairs the endothelium-derived hyperpolarizing factor (EDHF)-mediated function. This study examined the effect of procaine in cardioplegia on the EDHF-mediated response in porcine coronary arteries. An isometric force study was performed in a myograph. Two rings taken from the same artery (diameter 200-450 microm) were incubated with Krebs solution (group I) or 20 mM K+ (group II) with/without procaine (1 mM) at 37 degrees C for 1 hour. The EDHF-mediated relaxation was induced by bradykinin (BK, -10 approximately -6.5 log M) after U46619 (-8 log M, in group I) or K+-precontraction (in group II) in the presence of indomethacin (7 microM), NG-nitro-L-arginine (300 microM), and hemoglobin (20 microM). The membrane potential of a single smooth muscle cell was measured by a microelectrode after superfusion with Krebs solution with/without procaine for 1 hour. The EDHF-mediated relaxation was increased by the treatment with procaine with the EC50 shifted leftward (97.3 +/- 0.6% vs. 83.0 +/- 5.1% at -7 log M and 99.4 +/- 0.6% vs. 96.7 +/- 1.6% at -6.5 log M, p < 0.05; EC50: -8.57 +/- 0.24 vs. -7.92 +/- 0.23 log M, p < 0.05). Procaine decreased the BK-induced hyperpolarization from -72.3 +/- 0.7 mV to -68.8 +/- 0.8 mV (-6.5 log M, p < 0.01). The EDHF-mediated relaxation in arteries exposed to 20 mM K+ was not altered by procaine (49.9 +/- 7.4% vs. 55.8 +/- 7.6%, p > 0.05). In the coronary arteries, procaine has a depolarizing effect but it enhances EDHF-mediated relaxation. Addition of procaine in cardioplegia did not change the EDHF-mediated endothelial function.

The effect of local anesthetics on the inhibition of adult muscle-type nicotinic acetylcholine receptors by nondepolarizing muscle relaxants

The primary action of local anesthetics is to inhibit voltage-gated Na(+) channels. However, local anesthetics also have an inhibitory effect on muscle-type nicotinic acetylcholine receptors. Because local anesthetics could increase the neuromuscular blockade produced by nondepolarizing muscle relaxants, we investigated the interaction of local anesthetics with nondepolarizing muscle relaxants at adult muscle-type nicotinic acetylcholine receptors. This study tested the effects of lidocaine and procaine, alone and in combination with vecuronium and cisatracurium, on adult muscle-type nicotinic acetylcholine receptors. The adult mouse muscle-type nicotinic acetylcholine receptor was expressed in HEK293 cells and activated with 10microM acetylcholine. Currents were recorded using the whole-cell voltage-clamp technique. Adult muscle-type nicotinic acetylcholine receptors were potently inhibited by all the tested compounds. Although the potencies of procaine and lidocaine were statistically significantly different at adult muscle-type nicotinic acetylcholine receptors (50% inhibitory concentration values of 45.5microM and 11.1microM, respectively), procaine and lidocaine enhanced the inhibitory effect of nondepolarizing muscle relaxants at adult muscle-type nicotinic acetylcholine receptors to the same extent. The increased adult muscle-type nicotinic acetylcholine receptor inhibition produced when local anesthetics are combined with nondepolarizing muscle relaxants may contribute to the clinical enhancement of neuromuscular blockade by local anesthetics.

Antioxidant effect of lidocaine and procaine on reactive oxygen species-induced endothelial dysfunction in the rabbit abdominal aorta

BackgroundReactive oxygen species (ROS) induce lipid peroxidation and tissue damage in the endothelium. We tested the antioxidant effect of lidocaine and procaine on ROS-induced endothelial damage in the rabbit aorta.MethodsAortic rings isolated from rabbits were suspended in an organ bath filled with Krebs-Henseleit (K-H) solution bubbled with 5% CO2 and 95% O2 at 37.5℃. After precontraction with phenylephrine (PE, 10-6 M), changes in tension were recorded following a cumulative administration of acetylcholine (ACh 3 × 10-8 to 10-6 M). Differences were measured as percentages of ACh-induced relaxation of aortic rings before and after exposure to ROS as generated by electrolysis of the K-H solution. The aortic rings were pretreated with lidocaine or procaine (10-5 M to 3 × 10-3 M) to compare their effects, as well as ROS scavengers, catalase, mannitol, sodium salicylate, and deferoxamine, and a catalase inhibitor, 3-amino-1,2,4-triazole (3AT).ResultsLidocaine and procaine dose-dependently maintained endothelium-dependent relaxation induced by ACh despite ROS activity (P < 0.05 vs control value). The 3AT pretreated procaine (3 × 10-3 M) group decreased more significantly than the un-pretreated procaine group (P < 0.05).ConclusionsThese findings suggest that lidocaine and procaine dose-dependently preserve endothelium-dependent vasorelaxation against ROS attack, potentially via hydrogen peroxide scavenging.

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion sperm

Effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion sperm

Long-Acting Local Anesthetics Attenuate FMLP-induced Acute Lung Injury in Rats

Endothelin-1 (ET-1) is a mediator of lung diseases and a potent pulmonary vasoconstrictor. In addition to thromboxane A2, it participates in the formation of lung edema. Both lidocaine and mepivacaine attenuate the increase of pulmonary arterial pressure (PAP) and lung edema development. We examined the effects of procaine, bupivacaine, and ropivacaine on experimentally evoked PAP increase and ET-1 release. PAP and lung weight were measured in isolated rat lungs during perfusion with Krebs-Henseleit hydroxyethyl starch buffer. Bupivacaine, ropivacaine, or procaine was added to the solution at concentrations of 10(-2)-10(-7) mg/kg. ET-1 levels were measured in the perfusate by enzyme-immunoassay, and thromboxane A2 levels were assayed by radioimmunoassay. N-formyl-L-leucine-methionyl-L-phenylalanine was used to activate human polymorphonuclear neutrophils. Bupivacaine, ropivacaine, and procaine significantly attenuated increases of PAP (P < 0.05) and resulted in a reduction of lung weight in these treatment groups compared with the sham group (P < 0.05). The long-acting anesthetics bupivacaine and ropivacaine (P < 0.05), but not procaine, reduced ET-1 levels, produced low inflammation rates, and did not affect lung structures at doses from 10(-3) to 10(-6) mg/kg. Bupivacaine and ropivacaine attenuated N-formyl-L-leucine-methionyl-L-phenylalanine-induced PAP, reduced lung edema, and diminished ET-1 release. Lidocaine and mepivacaine are more effective in reducing PAP and edema formation, but long-acting local anesthetics also inhibit ET-1 depletion and therefore have increased anti-inflammatory properties.

TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase

We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1–5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na +-channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of l-glutamate.

Procaine treatments for cognition and dementia

Procaine is a controversial substance which has been used for "antiageing" effects including cognitive improvement for more than 50 years.Preparations which contain procaine as a component are claimed to prevent, reverse and interrupt dementia. Several products are widely promoted and can be purchased "over the counter" outside the US and via the Internet. Procaine preparations are said to be readily available in over 70 countries and to be used by more than 100 million people. To assess the efficacy and adverse effects of procaine (and preparations containing procaine as a component) on cognitive function in the treatment of people with dementia as well as healthy elderly people. References regarding trials with people with dementia or cognitive impairment were identified from a search of the Specialized Register of the Cochrane Dementia and Cognitive Improvement Group on 8 September 2007 using the search terms: gerovital* or aslavital* or procain* or KH3 or novocain* or GH3 or trofibial or "Zell H3" or Vitacel* or GH7 or "Ultimate 9". The Cochrane Library, MEDLINE, EMBASE, PsycINFO, CINAHL and LILACS were searched on 18 September 2007 to find references to trials with healthy people.The CDCIG Specialized Register contains records from major health databases (including MEDLINE, EMBASE, CINAHL, PsycINFO, CENTRAL, LILACS) as well as many ongoing trial databases and grey literature sources. All human, unconfounded, randomized double-blind trials in which treatment with procaine was administered for its effects on cognitive function and behavioural symptoms in demented or healthy elderly participants in parallel group comparison with placebo. The two review authors independently selected trials, assessed quality, extracted data, and performed the data analysis. Pooling data from two studies showed a detrimental effect of procaine in terms of causing side effects (20/208 active versus 3/207 placebo, OR 7.30, 95% CI 2.13 to 25.02, P = 0.002). In patients with dementia, a single small study also suggested a detrimental effect. Two trials referring to healthy elderly persons suggested a positive effect of procaine preparation on cognitive function. Meta-analysis of beneficial outcomes was not appropriate due to the different preparations, durations and poor quality of trials. Most trials were performed before the 1990s and none reported any criteria for cognitive decline and dementia. This review suggests that the evidence for detrimental effects of procaine and its preparations is stronger than the evidence for benefit in preventing and/or treating dementia or cognitive impairment.There is some evidence from older studies that procaine preparations might improve memory in persons without cognitive impairment. However, the clear evidence of side effects suggests that the risks might outweigh the benefits. In the light of this, the strong marketing claims for procaine preparations should be withdrawn until trials of adequate size, duration and quality have been conducted.

Effects of Sodium Azide, Barium Ion, d-Amphetamine and Procaine on Inward Rectifying Potassium Channel 6.2 Expressed in Xenopus Oocytes

Background/Purpose Inward rectifying potassium channel 6.2 (Kir6.2 Δ C26 channel) is closely related to ATP-sensitive potassium channels. Whether sodium azide, barium ion, d-amphetamine or procaine acts directly on the Kir6.2 Δ C26 channel remains unclear. We studied the effects of these compounds on Kir6.2 Δ C26 channel expressed in Xenopus oocytes. Methods The coding sequence of a truncated form of mouse Kir6.2 (GenBank accession number NP_034732.1), Kir6.2 1-364 (i.e. Kir6.2 Δ C26), was subcloned into the pET20b(+) vector. Plasmid containing the correct T7 promoter-Kir6.2 1-364 cDNA fragment [Kir6.2/pET20b(+)] was then subject to Not I digestion to generate the templates for in vitro run-off transcriptions. The channel was expressed in Xenopus laevis oocytes. Two-electrode voltage clamping was used to measure the effects of sodium azide, barium ion, d-amphetamine and procaine on Kir6.2 Δ C26 channel current. Results Sodium azide activated and barium ion and d-amphetamine inhibited the Kir6.2 Δ C26 channel. Procaine did not have any significant effect on the Kir6.2 Δ C26 channel. Conclusion Kir6.2 Δ C26 channel expressed in Xenopus oocytes can be used as a pharmacological tool for the study of inward rectifying potassium channels.

Using local anaesthetics to block neuronal activity and map specific learning tasks to the mushroom bodies of an insect brain

The formation of a stable olfactory memory requires activity within several brain regions. The honeybee provides a valuable model to map complex olfactory learning tasks onto certain brain areas. To this end, we used injections of the local anaesthetic procaine to reversibly block spike activity in a specific brain region, the mushroom body (MB). We first investigated the physiological effects of procaine on cultured MB neurons from adult honeybee brains. Using the whole-cell configuration of the patch-clamp technique, we show that procaine blocks voltage-gated Na+ and K+ currents in a dose-dependent manner between 0.1 and 10 mm. The effects are reversible within a few minutes of wash. Lidocaine acts similarly, but is less effective at the tested concentrations. We then studied the role of the MBs during reversal learning by blocking the neural activity within these structures by injecting procaine. During reversal learning bees learn to revert their responses to two odorants, one rewarded (A+) and one unrewarded (B-), if their contingencies are changed (A- vs B+). Injecting procaine into the MBs impaired reversal learning. Procaine treatment during acquisition left the later retention of the initial learning (A+ vs B-) intact. Similarly, a differential conditioning task involving novel odorants (C+ vs D-) was intact under procaine treatment. Our experiments show that local injections of procaine can be used to map learning tasks onto specific regions of the insect brain. We conclude that intact MB activity is required for the acquisition of reversal learning, but not for simple differential learning tasks.

The Effects of the Local Anesthetics Lidocaine and Procaine on Glycine and γ-Aminobutyric Acid Receptors Expressed in Xenopus Oocytes

The voltage-dependent sodium channel is the primary site of action for local anesthetics (LAs). Although systemically administered low-dose LAs have been shown to exert antihyperalgesic effects, the molecular targets responsible for these effects are not fully known and their functional effects on inhibitory neurotransmitter receptors associated with antinociception have not been sufficiently studied. We examined the effects of lidocaine and procaine (0.1 microM to 3 or 10 mM) on recombinant human alpha1 glycine, alpha1beta2gamma2S gamma-aminobutyric acid type A (GABA(A)), and rho1 GABA(C) receptors expressed in Xenopus laevis oocytes, using a two-electrode voltage-clamp system. We also evaluated the effects of LAs on two mutant glycine receptors, alpha1(S267C) and alpha1(S267Q), in an effort to clarify the interaction between LAs and glycine receptors. Low concentrations of both lidocaine and procaine enhanced glycine receptor function, whereas high concentrations of lidocaine and procaine inhibited glycine receptor function. Lidocaine (10 microM) produced a significant leftward shift in the glycine concentration-response curve, indicating an increase in the apparent affinity for glycine. This enhancement was not altered in the mutant receptors. Both lidocaine and procaine at high concentrations inhibited GABA(A) receptor currents, whereas neither lidocaine nor procaine affected GABA(C) receptor function. Lidocaine and procaine enhanced glycine receptor function at low concentrations and inhibited the functions of glycine and GABA(A) receptors at high concentrations. The mechanism of the LA-induced enhancement of glycine receptor function probably differs from that of general anesthetics. These findings may explain the pharmacological effects of LAs, such as antinociception and convulsion.

Clinical signs and etiology of adverse reactions to procaine benzylpenicillin and sodium/potassium benzylpenicillin in horses

Case reports of 59 horses reacting adversely to procaine benzylpenicillin or to sodium or potassium benzylpenicillin in Sweden in 2003-2005 were obtained through contacts with horse-owners. For the assessment of the reports, various parameters were evaluated, such as the times to the reactions, information on previous penicillin treatment, the clinical signs and the actions taken in the reacting horses. Among the reports, two horses had received sodium or potassium benzylpenicillin intravenously, whereas the remaining 57 horses had been treated with procaine benzylpenicillin intramuscularly. Allergy may underlie the adverse reactions in the horses given sodium and potassium benzylpenicillin, and in a few of the horses given procaine benzylpenicillin. However, in most horses in the latter group, the clinical signs may be due to the toxic effects of procaine. In these horses, the dominating clinical signs were locomotor and behavioral changes. Some risk factors may enhance the probability that horses react to procaine. One is repeated injections, which increase the likelihood of intravascular administration and also may increase the sensitivity to procaine due to neuronal sensitization (kindling). Procaine is rapidly hydrolyzed by plasma esterases to nontoxic metabolites. When high amounts of procaine enter the circulation, the hydrolyzing capacity may be exceeded and toxicity occurs. Analyses of plasma esterases from reacting horses showed lower activity than in nonreacting control horses. Low esterase activity may increase the possibility of procaine toxicity and constitute another risk factor.

The role of Ca2+ influx and intracellular Ca2+ release in the muscarinic‐mediated contraction of mammalian urinary bladder smooth muscle

OBJECTIVE To study the involvement of extracellular Ca2+ and the properties of the intracellular Ca2+ ([Ca2+]i) stores on the carbachol-induced contraction of mammalian urinary bladder smooth muscle strips under polarized and depolarized conditions. Strips of bladder were suspended between platinum ring electrodes in a cylindrical organ bath (0.2 mL) and continuously superfused with Krebs' solution at 1 mL/min. The effect of nifedipine, cyclopiazonic acid (CPA), thapsigargin, procaine, ryanodine and caffeine before and during a 10-s application of 100 microm carbachol under polarized conditions were studied. The effect of these drugs was also assessed under depolarized conditions using a protocol that allowed a more detailed assessment of the role of [Ca2+]i stores, consisting of emptying the stores by exposure to Ca2+-free solution, rapidly refilling them by a 10-s application of 81.5 mm Ca2+ (priming), returning to the Ca2+-free solution for 3 min and then applying 100 microm carbachol (10 s) in Ca2+-free solution (store release). Under polarized conditions, nifedipine and Ca2+ removal almost completely inhibited the carbachol-induced contractions. CPA increased the amplitude and duration of both carbachol- and electrical field stimulation-induced contractions. Although ryanodine had no inhibitory effect, caffeine and procaine significantly inhibited the carbachol-induced contraction. Under depolarized conditions nifedipine blocked both priming and store release contractions. CPA, thapsigargin, procaine and ryanodine significantly increased the priming and inhibited the store release contractions. However, caffeine virtually abolished both priming and store release contractions. These results suggest that in guinea-pig urinary bladder smooth muscle the Ca2+ necessary for contraction enters the cell through voltage-dependent dihydropyridine-sensitive Ca2+ channels and is pumped into an intracellular store that is released by carbachol. Under polarized conditions, the blockade of sarco-endoplasmic reticulum calcium ATP-ase (SERCA) with CPA increases [Ca2+]i and carbachol-induced contractions. The effects of caffeine and procaine suggest that store release involves ryanodine receptors and calcium-induced calcium release. Under depolarized conditions, Ca2+ entry is blocked by nifedipine and the stores diminish. Stored Ca2+ is also greatly reduced by the blockade of SERCA with either CPA or thapsigargin. Procaine, ryanodine and caffeine blocked the store release contractions, suggesting that this involves ryanodine receptors and calcium-induced calcium release.

Local Anesthetics Suppress Nerve Growth Factor-Mediated Neurite Outgrowth by Inhibition of Tyrosine Kinase Activity of TrkA

Local anesthetics (LAs) suppress sympathetic sprouting, which correlates with neuropathic pain. However, the precise mechanism of the suppression is unknown. Nerve growth factor (NGF) contributes to the sympathetic sprouting, and NGF signaling starts with NGF-stimulated autophosphorylation of TrkA, which is a high affinity receptor of NGF. We examined the effects of lidocaine, bupivacaine, and procaine on NGF signaling under suppression of NGF-stimulated neurite outgrowth in PC12 cells, which is a cellular model of sympathetic sprouting. To investigate the effect of these LAs on NGF-mediated neurite outgrowth of PC12 cells, cells were incubated with 40, 400, and 4000 microM of each LA. The effect of LAs on NGF-stimulated TrkA activity was examined to analyze autophosphorylation of TrkA using immunoprecipitation and immunoblotting. Cytotoxic effects of LAs on PC12 cells were also assessed by lactate dehydrogenase release and by propidium iodide staining. Lidocaine (400 microM), bupivacaine (40 and 400 microM), or procaine (4000 microM) suppressed either neurite outgrowth or autophosphorylation significantly without cytotoxicity. The inhibition of NGF-stimulated tyrosine kinase activity of TrkA might be involved in the mechanisms of suppression of neurite outgrowth induced by LAs.

Imaging OmpR localization in Escherichia coli

We have used a fusion of GFP to the response regulator OmpR to image the spatial distribution of OmpR in live cells of Escherichia coli. We observed foci of increased OmpR-GFP fluorescence that appear to be due to interactions with the histidine kinase EnvZ. We also observed colocalization of OmpR-GFP with clusters of plasmids carrying OmpR binding sites, which enabled us to develop a simple method for imaging the binding of OmpR to DNA in live cells. We used the peak fluorescence intensity within cells to quantify the extent of OmpR-GFP localization either due to interactions with EnvZ or due to binding DNA. With these assays we compared the effects of osmolarity and procaine, both of which are believed to modulate EnvZ activity. Our results suggest that, at least under our growth conditions, procaine activates EnvZ-OmpR signalling whereas osmolarity has, at best, a weak effect on the EnvZ-OmpR system.