Ethanol stimulates epithelial sodium channels by elevating reactive oxygen species.

Alcohol affects total body sodium balance, but the molecular mechanism of its effect remains unclear. We used single-channel methods to examine how ethanol affects epithelial sodium channels (ENaC) in A6 distal nephron cells. The data showed that ethanol significantly increased both ENaC open probability (P(o)) and the number of active ENaC in patches (N). 1-Propanol and 1-butanol also increased ENaC activity, but iso-alcohols did not. The effects of ethanol were mimicked by acetaldehyde, the first metabolic product of ethanol, but not by acetone, the metabolic product of 2-propanol. Besides increasing open probability and apparent density of active channels, confocal microscopy and surface biotinylation showed that ethanol significantly increased α-ENaC protein in the apical membrane. The effects of ethanol on ENaC P(o) and N were abolished by a superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) and blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Consistent with an effect of ethanol-induced reactive oxygen species (ROS) on ENaC, primary alcohols and acetaldehyde elevated intracellular ROS, but secondary alcohols did not. Taken together with our previous finding that ROS stimulate ENaC, the current results suggest that ethanol stimulates ENaC by elevating intracellular ROS probably via its metabolic product acetaldehyde.

WNK4 kinase inhibits Maxi K channel activity by a kinase-dependent mechanism.

WNK [with no lysine (k)] kinase is a serine/threonine kinase subfamily. Mutations in two of the WNK kinases result in pseudohypoaldosteronism type II (PHA II) characterized by hypertension, hyperkalemia, and metabolic acidosis. Recent studies showed that both WNK1 and WNK4 inhibit ROMK activity. However, little is known about the effect of WNK kinases on Maxi K, a large-conductance Ca(2+) and voltage-activated potassium (K) channel. Here, we report that WNK4 wild-type (WT) significantly inhibits Maxi K channel activity in HEK αBK stable cell lines compared with the control group. However, a WNK4 dead-kinase mutant, D321A, has no inhibitory effect on Maxi K activity. We further found that WNK4 inhibits total and cell surface protein expression of Maxi K equally compared with control groups. A dominant-negative dynamin mutant, K44A, did not alter the WNK4-mediated inhibitory effect on Maxi K surface expression. Treatment with bafilomycin A1 (a proton pump inhibitor) and leupeptin (a lysosomal inhibitor) reversed WNK4 WT-mediated inhibition of Maxi K total protein expression. These findings suggest that WNK4 WT inhibits Maxi K activity by reducing Maxi K protein at the membrane, but that the inhibition is not due to an increase in clathrin-mediated endocytosis of Maxi K, but likely due to enhancing its lysosomal degradation. Also, WNK4's inhibitory effect on Maxi K activity is dependent on its kinase activity.

Protein-protein interaction between cPLA2 and splice variants of alpha-subunit of BK channels.

Altering the splice variant composition of large-conductance Ca(2+)-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca(2+) and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK alpha-subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A(2) (cPLA(2)). Here we used the Mammalian Matchmaker two-hybrid assay and two BK alpha-subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA(2) associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA(2) might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA(2) is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA(2) to the COOH terminus of the rSlo(0) alpha-subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.

ProTECT: a randomized clinical trial of progesterone for acute traumatic brain injury.

STUDY OBJECTIVE: Laboratory evidence indicates that progesterone has potent neuroprotective effects. We conducted a pilot clinical trial to assess the safety and potential benefit of administering progesterone to patients with acute traumatic brain injury. METHODS: This phase II, randomized, double-blind, placebo-controlled trial was conducted at an urban Level I trauma center. One hundred adult trauma patients who arrived within 11 hours of injury with a postresuscitation Glasgow Coma Scale score of 4 to 12 were enrolled with proxy consent. Subjects were randomized on a 4:1 basis to receive either intravenous progesterone or placebo. Blinded observers assessed patients daily for the occurrence of adverse events and signs of recovery. Neurologic outcome was assessed 30 days postinjury. The primary safety measures were differences in adverse event rates and 30-day mortality. The primary measure of benefit was the dichotomized Glasgow Outcome Scale-Extended 30 days postinjury. RESULTS: Seventy-seven patients received progesterone; 23 received placebo. The groups had similar demographic and clinical characteristics. Laboratory and physiologic characteristics were similar at enrollment and throughout treatment. No serious adverse events were attributed to progesterone. Adverse and serious adverse event rates were similar in both groups, except that patients randomized to progesterone had a lower 30-day mortality rate than controls (rate ratio 0.43; 95% confidence interval 0.18 to 0.99). Thirty days postinjury, the majority of severe traumatic brain injury survivors in both groups had relatively poor Glasgow Outcome Scale-Extended and Disability Rating Scale scores. However, moderate traumatic brain injury survivors who received progesterone were more likely to have a moderate to good outcome than those randomized to placebo. CONCLUSION: In this small study, progesterone caused no discernible harm and showed possible signs of benefit.

Intravenous mononuclear marrow cells reverse neuropathic pain from experimental mononeuropathy.

BACKGROUND: Stem cells mediate neuroprotection in a variety of nervous system injury models. In this study, we evaluated a potential role for stem cells in pain therapies. Marrow mononuclear cells containing mixed stem cell populations were used because of wide experience with these cells in experimental and clinical transplantation. METHODS: After sciatic nerve chronic constriction injury (CCI), adult male Sprague Dawley rats were treated with freshly isolated marrow mononuclear cells (10(7) cells in 0.5 mL IV) from the same strain, or with carrier. The major end points of analysis were thermal and mechanical hypersensitivity using paw withdrawal latency (PWL) to a calibrated heat source and paw withdrawal response to von Frey filaments, evaluated by a blinded investigator. RESULTS: Marrow transplantation did not prevent pain, and 5 days after CCI all animals were equivalently lesioned. However, 10 days after CCI, rats that received marrow transplants demonstrated paw withdrawal response and PWL patterns indicating recovery from pain, whereas untreated rats continued to have significant pain behavior patterns. For example, PWL values for marrow-treated animals were similar to baseline pre-CCI values (P = 0.54) but significantly shorter latency to withdrawal indicative of continuing pain was seen in untreated rats compared with pre-CCI values (P < 0.001). CONCLUSIONS: These studies suggest that stem or progenitor cell-mediated therapies may be useful for the treatment of pain after nerve injury, and deserve further study to elucidate the mechanisms of analgesia.

Steady-state serum concentrations of progesterone following continuous intravenous infusion in patients with acute moderate to severe traumatic brain injury.

Progesterone (PG) has been shown to provide substantial neuroprotection after traumatic brain injury (TBI) in multiple animal models. As a first step in assessing applicability to humans, the authors examined the effects of acute TBI and extracranial trauma on the pharmacokinetics of PG given by intravenous infusion. Multiple blood samples were obtained from 11 female and 21 male trauma patients receiving PG and 1 female and 3 male patients receiving placebo infusions for 72 hours. Values for C(SS), CL, t(1/2), and Vd were obtained using AUC((0-72)) and postinfusion blood samples. C(SS) values were 337 +/- 135 ng/mL, which were significantly lower than the target concentration of 450 +/- 100 ng/mL. The lower C(SS) is attributed to the CL, which was higher than anticipated. In addition, t(1/2) was longer and V(d) was higher than anticipated. These results demonstrate that stable PG concentrations can be rapidly achieved following TBI.

Difference in analgesia following epidural blockade in patients with postoperative or chronic low back pain.

Subjective responses of continuous epidural analgesia with bupivacaine were compared in 30 patients with acute (postoperative) or chronic (low back) pain. In the acute pain patients, sensory block was 4 dermatomes at 9 h and 6 dermatomes at 64 h. Corresponding values in the chronic pain patients were 8 and 6 dermatomes respectively. Motor blockade of the lower limbs was more profound in the acute pain group. The acute pain patients had significantly better pain relief (VAS: 85-96% vs. 55-70%) and a significantly higher proportion of these patients reported a global score of 3 (excellent; 80% vs. 7%). The mean dosage of bupivacaine decreased in the acute pain group from 21.0 +/- 5.7 (mean +/- S.D.) mg/h at 9 h to 15.1 +/- 8.5 mg/h at 64 h. Corresponding values for the chronic pain group were 20.7 +/- 5.9 and 12.0 +/- 6.0 mg/h respectively. Mean plasma concentration of bupivacaine increased from 1.2 +/- 0.8 micrograms/ml at 9 h to 2.1 +/- 1.4 micrograms/ml at 64 h in the acute pain patients and was 0.8 +/- 0.3 micrograms/ml at 9 h to 1.0 +/- 1.0 micrograms/ml at 64 h in the chronic pain patients. The incidence of side effects was approximately the same in both groups. No signs of accumulation or toxic reactions to bupivacaine were seen.

Rapid local anesthesia in humans using minimally invasive microneedles.

OBJECTIVE: This study tested the hypothesis that minimally invasive microneedles cause less pain during injection of lidocaine, but induce local anesthesia in humans with the same rapid onset and efficacy as intradermal lidocaine injection using hypodermic needles. METHODS: This study was a randomized, single-blinded, within participants, controlled design. Hollow, 500-µm long microneedles were used to inject lidocaine to the forearm of 15 human participants. The associated pain was recorded using a visual analog (VAS) scale. The area and depth of numbness were determined at 0, 7.5, and 15 minutes after injection. Lidocaine was also injected to the dorsum of the hand near a vein, followed by placement of an intravenous catheter and measurement of associated pain. A 26-gauge intradermal bevel hypodermic needle similarly administered lidocaine on the opposite forearm/hand to serve as the positive control. RESULTS: VAS pain scores revealed that injection using microneedles was significantly less painful than hypodermic needles for both the forearm and dorsum of the hand injections. However, there was no significant difference in the area or depth of the resulting numbness between the 2 treatment methods at any time point (0, 7.5, and 15 min) indicating that microneedles had immediate onset and were as effective as hypodermic needles in inducing dermal anesthesia. Moreover, insertion of an intravenous catheter immediately after lidocaine injection on the dorsum of the hand led to comparable pain scores for the microneedle and hypodermic needle treated sites, further confirming efficacy of microneedles in inducing rapid local anesthesia. Lastly, 77% of the participants preferred microneedles and 80% indicated that they did not consider microneedles to be painful. DISCUSSION: This study demonstrates for the first time that microneedle-based lidocaine injection is as rapid and as effective as hypodermic injection in inducing local anesthesia while resulting in significantly less pain during injection.

Effect of microneedle design on pain in human volunteers.

OBJECTIVES: To design microneedles that minimize pain, this study tested the hypothesis that microneedles cause significantly less pain than a 26-gauge hypodermic needle, and that decreasing microneedle length and the number of microneedles reduces pain in normal human volunteers. METHODS: Single microneedles with lengths ranging from 480 to 1450 microm, widths from 160 to 465 microm, thicknesses from 30 to 100 microm, and tip angles from 20 to 90 degrees; and arrays containing 5 or 50 microneedles were inserted into the volar forearms of 10 healthy, human volunteers in a double-blinded, randomized study. Visual analog scale pain scores were recorded and compared with each other and to the pain from a 26-gauge hypodermic needle. RESULTS: All microneedles investigated were significantly less painful than the hypodermic needle with microneedle pain scores varying from 5% to 40% of the hypodermic needle. Microneedle length had the strongest effect on pain, where a 3-fold increase in length increased the pain score by 7-fold. The number of microneedles also affected the pain score, where a 10-fold increase in the number of microneedles increased pain just over 2-fold. Microneedle tip angle, thickness, and width did not significantly influence pain. DISCUSSION: Microneedles are significantly less painful than a 26-gauge hypodermic needle over the range of dimensions investigated. Decreasing microneedle length and number of microneedles reduces pain.

An objective evaluation of breast symmetry and shape differences using 3-dimensional images.

BACKGROUND: The concept of natural breast asymmetry is well known; however, actual documentation in the literature is limited. New technology is currently available which provides 3-dimensional surface images of the breast and the ability to qualitatively determine differences in breast size, shape, and contour. The purpose of this report is to objectively determine the extent to which this natural breast asymmetry exists. METHODS: Eighty-seven women without a history of breast cancer or previous breast surgery were included. Images were obtained using 3dMD technology. Data points queried included age, parity, body mass index (BMI), ethnicity, and bra size. Left/right images were superimposed and the distance between the 2 surfaces, and contour was calculated. The degree of asymmetry was determined and comparisons were made. Similar differences in nipple-to-notch measurements were calculated and compared. Subjective evaluations were included for clinical relevance. RESULTS: The average age was 49.6 years (range: 19-77), with an average BMI of 25 (range: 18.5-36.7). The average nipple to notch on the left was 24.3 cm and 23.8 cm on the right. The nipple-to-notch asymmetry was on average 3.2%, with the left breast measurement being greater the majority of the time (62%). The mean distance between each breast demonstrated consistent breast asymmetry, with an average measurement of +0.5 mm (left breast being larger than the right). The degree of breast asymmetry was documented by a root mean square value (RMS) of 5.93 mm. This was not related to age, parity, or ethnicity; however, it was significantly higher in those patients with larger BMI, cup size, and chest-wall circumference. Only 10% of women were found to have severe breast asymmetry on subjective evaluation, which correlated objectively with the RMS in that group being significantly higher at 9.8 mm (P < 0.05). There were no predictable patterns of asymmetry; however, the most common pattern was larger laterally and smaller medially, found in 32% of the women. CONCLUSION: Natural breast asymmetry does exist, demonstrated objectively using 3-dimensional surfaces images. The left breast is on average larger than the right, with differences in size and shape being consistent but fairly unpredictable. It is important that we know baseline differences in breast symmetry prior to objectively analyzing results following esthetic and reconstructive breast surgery.

Validating three-dimensional imaging of the breast.

The potential to extrapolate accurate data from 3-dimensional (3D) images of the breast is enormous and will greatly improve our ability to qualitatively determine differences in shape, size, and contour. The validity of these calculated measurements is important and needs to be determined before any meaningful data can be evaluated. PART I: Premastectomy 3D images (3dMD patient) were obtained on 19 breasts (14 patients). The volume of the mastectomy specimen was determined intraoperatively using water displacement. Two independent raters then calculated breast volumes using the 3D images and software, and these were compared with the intraoperative volume. Inter- and intrarater reliability was determined. Part II: Surface measurements (nipple to notch) were then evaluated on 20 breasts (10 patients) by comparing the 3D image determined distance to the known measurements. PART I: The average breast volume was 500 mL, compared with 489 mL for rater 1 and 490 mL for rater 2. The relative difference between the measured volume and the calculated volume for rater 1 and rater 2 was about -2%, with a standard deviation of +/- 13% to 16%. The coefficient of reproducibility for each reader was excellent, at 0.80 for rater 1 and 0.92 for rater 2. The level of agreement between the readers was also high at 0.975. Part II: The average nipple to notch measurement for each patient was 27.1 cm, compared the calculated average of 25.1 cm for rater 1 and 26.1 cm for rater 2. The mean relative difference between the measured and calculated distances for raters 1 and 2 was about -6%, with a standard deviation of +/- 6% to 7%. The level of agreement between readers was high, at 0.975. The ability to objectively determine breast volume and surface measurements using 3D imaging technology is now available with consistent and reproducible accuracy. Measurements are typically underestimated, with more variability when calculating volumes. Although inherent subjectivity will always exist when evaluating breast measurements, 3D technology provides invaluable information, particularly in the longitudinal evaluation of results.

The effect of systemic zonisamide (Zonegran) on thermal hyperalgesia and mechanical allodynia in rats with an experimental mononeuropathy.

UNLABELLED: We studied the ability of zonisamide (Zonegran) to relieve thermal hyperalgesia and/or mechanical allodynia in the chronic constriction injury model of neuropathic pain. Zonisamide (25, 50, or 100 mg/kg) or saline was administered in a blinded, randomized manner by intraperitoneal injection on postoperative days (PODs) 4, 5, and 6. Paw withdrawal latency (PWL) to heat, paw withdrawal response to von Frey monofilaments, and pain scores based on weight-bearing were tested: before surgery; before and after zonisamide or saline (PODs 4, 5, and 6); and on POD 9. Systemic zonisamide relieved thermal hyperalgesia in a dose-dependent manner. All PWLs were significantly increased after zonisamide administration compared with pre-zonisamide measurements, except with the 100 mg/kg dose on POD 5. After zonisamide 100 mg/kg administration, there was a sustained increase in PWL on PODs 5 and 9, with significant carryover effect from the previous dose. However, zonisamide had little effect on mechanical allodynia, except at the 100 mg/kg dose, which was sedating in the rat. At the 100 mg/kg dose, paw withdrawal response was increased on PODs 4 and 5, whereas pain scores were reduced on PODs 4, 5, and 6. Pain scores were inconsistently reduced after 50 mg/kg or 25 mg/kg doses. IMPLICATIONS: Zonisamide causes a dose-related decrease in heat sensitivity in a rat model of neuropathic pain, but relieves mechanical sensitivity only in a dose that is sedating to the rat. Zonisamide may be useful in the treatment of some types of neuropathic pain.

Isoflurane induces dopamine transporter trafficking into the cell cytoplasm.

Previous investigations have shown that the binding of a selective hydrophilic positron emission tomography radiotracer for the dopamine transporter (DAT) (2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-18F-fluoroethyl)nortropane) decreased in monkey striatum during deep isoflurane anesthesia. Immunohistochemistry experiments suggested but did not prove that isoflurane induced a decrease in cell surface DAT. The present investigation was undertaken to demonstrate quantitatively the isoflurane-induced internalization of DAT using a rapid and relatively uncomplicated biochemical technique in human embryonic kidney (HEK-293) cells stably expressing the human DAT (h-DAT) protein. Biotinylation followed by Western blot analysis was used to determine the extent of change in cell surface expression of the DAT under control conditions and in the presence of a clinically relevant concentration of isoflurane. Isoflurane treatment for 30 min resulted in a highly significant decrease in the amount of h-DAT on the cell surface (21 +/- 15% of control; P < 0.01) (mean +/- SD; n = 4). These data are consistent with the hypothesis that isoflurane results in internalization of DAT from the cell membrane and further validate our qualitative results reported previously. In addition, the current results confirm the hypothesis that biotinylation can be used to quantitate the extent of disappearance of DAT from the cell surface making dose-response studies and comparisons of DAT internalization with other general anesthetics practical.