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Mu-opioid receptor desensitization: role of receptor phosphorylation, internalization, and representation.

It is generally accepted that the internalization and desensitization of mu-opioid receptor (MOR) involves receptor phosphorylation and beta-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of beta-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent beta-arrestin recruitment play important roles in the resensitization of internalized receptors.

Analgesics, Opioid↗

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling.

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.

Amino Acid Sequence↗

Agonist activated adrenocorticotropin receptor internalizes via a clathrin-mediated G protein receptor kinase dependent mechanism.

The physiological effects of the pituitary hormone, adrenocorticotropic hormone (ACTH) on the adrenal are mediated by the melanocortin 2 receptor (MC2R), a G protein coupled receptor (GPCR) that signals via adenylate cyclase to elevate intracellular cyclic AMP (cAMP) levels. The function and expression of the receptor is likely to be a major determinant of the response to ACTH. Following repeated stimulation, the cAMP signal is diminished or desensitized. Prolonged desensitization may involve internalization of the receptor. Internalization may occur by at least two mechanisms--receptor mediated endocytosis via clathrin-coated pits and by caveolae mediated internalization. The mode of internalization for the endogenous MC2R in Y1 cells was determined using radiolabelled ACTH. Treatment of Y1 cells with hypertonic sucrose or with concanavalin A, which inhibit clathrin-mediated endocytosis, blocked internalization. Filipin and nystatin, which inhibit caveolae formation, did not influence internalization. A dominant negative GRK2 inhibited internalization whilst the protein kinase A (PKA) consensus site mutant MC2R (S208A) internalized normally. However, dominant negative V53D beta-arrestin-1 did not inhibit ACTH internalization in Y1 cells. In conclusion, it appears that the MC2R in Y1 cells internalizes by a G protein coupled receptor kinase (GRK) dependent clathrin-coated pit mechanism.

Animals↗

Relationship between internal phase volume and emulsion stability: the cetyl alcohol/stearyl alcohol system.

The main objective of this study was to optimize the stability of cetyl alcohol/stearyl alcohol emulsions in terms of percentage of internal phase volume, emulsifier type and concentration, and amount of external phase (water). Creams (o/w emulsions) were prepared by phase inversion and physical properties as particle size of the internal phase, apparent viscosity, and sedimentation volume evaluated. Stability was performed at room temperature, 40 degrees C, 50 degrees C, and under stress conditions. High hydrophilic lipophilic balance (HLB) nonionic surfactants as tween 80, tween 20, Myrj 52, Brij 35, and low HLB span 60 were used as emulsifying agents. The percentage of internal phase components (cetyl alcohol and stearyl alcohol), percentage of emulsifying agents, and percentage of aqueous external phase were varied, and stability was investigated. As the level of emulsifier agent (tween 80 and span 60) increased from 3% to 15%, and the percent of the internal phase remained constant at 30%, the particle size of the internal phase decreased and the cream became more stable. Formulations of the same composition, but prepared using Myrj 53 and tween 20 as emulsifiers, showed a larger particle size than formulations prepared using tween 80 and Brij 35. As the level of the internal phase volume increased and consequently the amount of water decreased, emulsion viscosity increased. The best formulation containing 30% internal phase (50% cetyl alcohol, 35% stearyl alcohol), 15% emulsifying agents (tween 80/span 60 ratio of 3:1), and 70% water was selected, and effects of process temperature and cooling rate on emulsion stability investigated. This formulation was further investigated in terms of stability of a 1% hydrocortisone addition by varying the percentage (30%, 35%, 40%, and 45%) of internal phase and percentage of water (70%, 65%, 60%, and 55%). The best formulation contained 45% internal phase (22.5 g cetyl alcohol, 15.75 g stearyl alcohol, 15% emulsifying agent, which is equivalent to 5 g tween 80 and 1.7 g span 60), and 55% w/w water, was manufactured under different manufacturing processes. Emulsions prepared by homogenization at the beginning of the process of emulsification were stable with small internal phase particle diameter. This study demonstrates that at every cetyl alcohol/stearyl alcohol ratio there is a phase volume/emulsifier HLB ratio, which results in optimum stability.

Chemistry, Pharmaceutical↗

Adaptive prediction of internal target motion using external marker motion: a technical study.

An adaptive prediction approach was developed to infer internal target position by external marker positions. First, a prediction model (or adaptive neural network) is developed to infer target position from its former positions. For both internal target and external marker motion, two networks with the same type are created. Next, a linear model is established to correlate the prediction errors of both neural networks. Based on this, the prediction error of an internal target position can be reconstructed by the linear combination of the prediction errors of the external markers. Finally, the next position of the internal target is estimated by the network and subsequently corrected by the reconstructed prediction error. In a similar way, future positions are inferred as their previous positions are predicted and corrected. This method was examined by clinical data. The results demonstrated that an improvement (10% on average) of correlation between predicted signal and real internal motion was achieved, in comparison with the correlation between external markers and internal target motion. Based on the clinical data (with correlation coefficient 0.75 on average) observed between external marker and internal target motions, a prediction error (23% on average) of internal target position was achieved. The preliminary results indicated that this method is helpful to improve the predictability of internal target motion with the additional information of external marker signals. A consistent correlation between external and internal signals is important for prediction accuracy.

Algorithms↗

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.

Adaptor Protein Complex 2↗

Plasticity of B cell receptor internalization upon conditional depletion of clathrin.

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by approximately 70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering G(M1) gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation.

Actins↗

Sleep and motor performance in on-call internal medicine residents.

STUDY OBJECTIVES: To compare vigilance and performance among internal medicine residents doing in-house call versus residents not doing in-house call. DESIGN: Prospective study of resident cohorts with repeated testing. SETTING: University Teaching Hospital. PARTICIPANTS: Internal medicine residents doing in-house call and residents not doing in-house call (pathology, endocrinology) (controls). MEASUREMENTS AND RESULTS: Subjective sleepiness scores (daily Stanford Sleepiness Scale and Epworth Sleepiness Scale at start and end of the test period), actigraphy, and daily sleep logs as well as regular psychomotor vigilance testing using a Palm version (Walter Reed Army Institute of Research) of the Psychomotor Vigilance Test (PVT). Subjects were enrolled for a period of 28 to 32 days, which included 4 to 6 on-call nights for the internal medicine residents. Controls took call from home. Participants were compensated for their time. RESULTS: Twenty residents were evaluated, 13 internal medicine and 7 controls. Overall median reaction time was slower in the internal medicine residents (264.7 +/- 102.9 vs 239.2 +/- 26.1 milliseconds; P < .001). Internal medicine residents showed no difference in reaction time postcall versus other periods (269.9 +/- 131.2 vs 263.6 +/- 95.6; P = .65). Actigraphic sleep time was shorter during on-call than noncall nights and in internal medicine residents as compared with controls (287.48 +/- 143.8 vs 453.49 +/- 178.5 and 476.08 +/- 71.9 minutes; P < .001). Internal medicine residents had significantly greater major and minor reaction-time lapses compared with controls (1.26 +/- 3.4 vs 0.53 +/- 1.1 & 2.4 +/- 7.4 vs 0.45 +/- 1.0; P < .001). They reported increased sleepiness on postcall days compared with the start of their call (Stanford Sleepiness Scale: 3.26 +/- 1.2 vs 2.22 +/- 0.8; P < .001) but had scores similar to those of controls by their next call (2.22 +/- 0.8 vs 2.07 +/- 0.8; P = .13). CONCLUSIONS: Internal medicine residents have impaired reaction time and reduced vigilance compared with controls. Despite subjective improvements in sleepiness postcall, there was no change in their objective performance across the study period, suggesting no recovery. Internal medicine residents did not get extra sleep on postcall nights in an attempt to recover their lost sleep time. Implications for residents' well-being and patient care remain unclear.

Adult↗

Do international medical graduates reduce rural physician shortages?

OBJECTIVES: The authors examined whether international medical graduates (IMGs) constitute a greater percentage of the US physician workforce in rural underserved areas than in rural non-underserved areas. Research findings could help policymakers determine whether the role of international medical graduates in compensating for local physician shortages counterbalances international medical graduates' potential for exacerbating a national oversupply. METHODS: This research was based on data from the American Medical Association Physician Masterfile and the Bureau of Health Professions' Area Resource File. The authors calculated the percentage international medical graduates of all US primary care physicians in rural areas, stratified by the Health Professional Shortage Area (HPSA) designation of underservice. RESULTS: The study showed that international medical graduates do constitute a greater percentage of US primary care physicians in rural areas with physician shortages than in rural areas without physician shortages. This finding held true at the national, Census region, and state scales of analysis, but to varying degrees. The finer the scale of analysis, the greater the variation in international medical graduates' practice in rural, underserved areas. There was substantial interstate variation in the extent to which international medical graduates practice in rural underserved areas. CONCLUSIONS: International medical graduates do help reduce rural physician shortages, but interstate variation points to the role of state policies in influencing international medical graduates' distribution in rural, underserved areas. Such variation also can come about from many different causes, so there is a need for further research to determine why international medical graduates help compensate for physician shortages more so in some states than in others.

Foreign Medical Graduates↗

An update on internal mammary artery grafting for coronary artery disease.

PURPOSE OF REVIEW: To describe recent results regarding the use of the internal mammary artery for coronary artery bypass grafting with emphasis on bilateral internal mammary artery grafting, patency, resistance to atherosclerosis, skeletonisation, composite arterial grafts, flow measurement, vasodilatation, and non-invasive imaging techniques. RECENT FINDINGS: Coronary artery bypass grafting plays an important part in coronary revascularisation and seems to be associated with a survival benefit in comparison with percutaneous coronary intervention. After 10 years, internal mammary arteries demonstrate better patency than vein grafts except when grafting moderately stenosed right coronary arteries. Bilateral internal mammary artery grafting increases survival further, but carries a higher risk of sternal complications. Skeletonisation may reduce this risk. The internal mammary arteries are used increasingly as composite arterial grafts and this technical solution should no longer be considered experimental. Perioperative flow measurement by the transit-time method is recommended while postoperative echocardiography represents an accurate method for evaluation of flow in internal mammary artery grafts. Multi-detector computed tomography allows for accurate assessment of all types of bypass conduits and native coronary arteries. At present, magnetic resonance imaging of internal mammary artery patency and flow is possible. SUMMARY: Newer studies confirm earlier data with respect to improved long-term survival when using internal mammary artery grafting, and this survival benefit is superior to percutaneous coronary intervention treatment. Bilateral internal mammary artery grafting improves survival further. Skeletonisation of the internal mammary artery provides extra length of grafts for complete arterial revascularisation. Non-invasive imaging techniques are increasingly sophisticated and may change the referral pattern for patients with coronary artery disease to either percutaneous coronary intervention or coronary artery bypass grafting.

Coronary Artery Bypass↗

A comprehensive dose reconstruction methodology for former rocketdyne/atomics international radiation workers.

Incomplete radiation exposure histories, inadequate treatment of internally deposited radionuclides, and failure to account for neutron exposures can be important uncertainties in epidemiologic studies of radiation workers. Organ-specific doses from lifetime occupational exposures and radionuclide intakes were estimated for an epidemiologic study of 5,801 Rocketdyne/Atomics International (AI) radiation workers engaged in nuclear technologies between 1948 and 1999. The entire workforce of 46,970 Rocketdyne/AI employees was identified from 35,042 Kardex work histories cards, 26,136 electronic personnel listings, and 14,189 radiation folders containing individual exposure histories. To obtain prior and subsequent occupational exposure information, the roster of all workers was matched against nationwide dosimetry files from the Department of Energy, the Nuclear Regulatory Commission, the Landauer dosimetry company, the U.S. Army, and the U.S. Air Force. Dosimetry files of other worker studies were also accessed. Computation of organ doses from radionuclide intakes was complicated by the diversity of bioassay data collected over a 40-y period (urine and fecal samples, lung counts, whole-body counts, nasal smears, and wound and incident reports) and the variety of radionuclides with documented intake including isotopes of uranium, plutonium, americium, calcium, cesium, cerium, zirconium, thorium, polonium, promethium, iodine, zinc, strontium, and hydrogen (tritium). Over 30,000 individual bioassay measurements, recorded on 11 different bioassay forms, were abstracted. The bioassay data were evaluated using ICRP biokinetic models recommended in current or upcoming ICRP documents (modified for one inhaled material to reflect site-specific information) to estimate annual doses for 16 organs or tissues taking into account time of exposure, type of radionuclide, and excretion patterns. Detailed internal exposure scenarios were developed and annual internal doses were derived on a case-by-case basis for workers with committed equivalent doses indicated by screening criteria to be greater than 10 mSv to the organ with the highest internal dose. Overall, 5,801 workers were monitored for radiation at Rocketdyne/AI: 5,743 for external exposure and 2,232 for internal intakes of radionuclides; 41,169 workers were not monitored for radiation. The mean cumulative external dose based on Rocketdyne/AI records alone was 10.0 mSv, and the dose distribution was highly skewed with most workers experiencing low cumulative doses and only a few with high doses (maximum 500 mSv). Only 45 workers received greater than 200 mSv while employed at Rocketdyne/AI. However, nearly 32% (or 1,833) of the Rocketdyne/AI workers had been monitored for radiation at other nuclear facilities and incorporation of these doses increased the mean dose to 13.5 mSv (maximum 1,005 mSv) and the number of workers with >200 mSv to 69. For a small number of workers (n=292), lung doses from internal radionuclide intakes were relatively high (mean 106 mSv; maximum 3,560 mSv) and increased the overall population mean dose to 19.0 mSv and the number of workers with lung dose>200 mSv to 109. Nearly 10% of the radiation workers (584) were monitored for neutron exposures (mean 1.2 mSv) at Rocketdyne/AI, and another 2% were monitored for neutron exposures elsewhere. Interestingly, 1,477 workers not monitored for radiation at Rocketdyne/AI (3.6%) were found to have worn dosimeters at other nuclear facilities (mean external dose of 2.6 mSv, maximum 188 mSv). Without considering all sources of occupational exposure, an incorrect characterization of worker exposure would have occurred with the potential to bias epidemiologic results. For these pioneering workers in the nuclear industry, 26.5% of their total occupational dose (collective dose) was received at other facilities both prior to and after employment at Rocketdyne/AI. In addition, a small number of workers monitored for internal radionuclides contributed disproportionately to the number of workers with high lung doses. Although nearly 12% of radiation workers had been monitored for neutron exposures during their career, the cumulative dose levels were small in comparison with other external and internal exposure. Risk estimates based on nuclear worker data must be interpreted cautiously if internally deposited radionuclides and occupational doses received elsewhere are not considered.

Adult↗

Cytochalasin-induced actin disruption of polarized enterocytes can augment internalization of bacteria.

Cytochalasin-induced actin disruption has often been associated with decreased bacterial internalization by cultured epithelial cells, although polarized enterocytes have not been systematically studied. In assays using confluent polarized HT-29 enterocytes, cytochalasin D appeared to increase internalization of wild-type Salmonella typhimurium, Proteus mirabilis, and Escherichia coli. HeLa and HEp-2 epithelial cells, as well as HT-29 and Caco-2 enterocytes, were used to clarify this unexpected observation. Resulting data showed that cytochalasin D was associated with increased internalization of S. typhimurium and P. mirabilis by both HT-29 and Caco-2 enterocytes and with increased internalization of E. coli by HT-29 enterocytes; with either HeLa or HEp-2 cells, cytochalasin was associated with no change or a decrease in internalization of these same bacterial strains. Cytochalasin caused decreased internalization of Listeria monocytogenes by HT-29, Caco-2, HeLa, and HEp-2 cells, indicating that cytochalasin did not consistently augment bacterial internalization by polarized enterocytes. Fluorescein-labeled phalloidin confirmed marked disruption of filamentous actin in cytochalasin-treated HT-29, Caco-2, HeLa, and HEp-2 cells. Cytochalasin had no noticeable effect on epithelial viability but caused distorted apical microvilli, cell rounding, and separation of adjacent enterocytes in confluent cultures (with a corresponding decrease in transepithelial electrical resistance). Scanning electron microscopy showed that cytochalasin-induced enhanced bacterial internalization was associated with preferential bacterial adherence on the exposed enterocyte lateral surface. Colchicine, used to disrupt microtubules, had no noticeable effect on bacterial internalization by HT-29 or Caco-2 enterocytes. These data indicated that for HT-29 and Caco-2 enterocytes, cytochalasin-induced disruption of filamentous actin might augment internalization of some bacterial species by a mechanism that appeared to involve exposure of the enterocyte lateral surface.

Actins↗

Characterization of the internal promoter of simian foamy viruses.

Simian and human foamy viruses (HFV and SFV), genetically related members of the spumavirus genus of retroviruses, have complex genome structures which encode the gag, pol, and env genes for virion proteins as well as additional open reading frames. One of these open reading frames is a viral transactivator, encoded by genes designated taf for SFV and bel-1 for HFV, which augments transcription directed by the long terminal repeat (LTR) through cis-acting targets in the U3 domain of the LTR. Recently, an internal transcriptional promoter has been identified in sequences within the 3' end of the HFV env gene (M. Lochelt, W. Muranyi, and R. M. Flugel, Proc. Natl. Acad. Sci. 90:7317-7321, 1993). We have demonstrated by using transient expression assays in several tissue culture cell lines and by analyzing viral transcripts in infected cells that SFV-1 from a rhesus macaque and SFV-3 from an African green monkey also encode an internal promoter in the env gene. Transcription directed by the internal promoters of SFV-1 and SFV-3 is activated by the taf-1 and taf-3 gene products, respectively, in several cell types. The importance of a TATA box for the SFV-1 internal promoter was established by site-specific mutagenesis, and the 5' ends of transcripts initiating in the internal promoter have been determined. cis-acting sequences in the SFV-1 env gene required for the response to taf-1 are contained within a 121-bp element located 5' to the TATA box in the internal promoter. This taf-1-responsive element in the internal promoter functions in a position- and orientation-independent fashion in a heterologous promoter and thus has the properties of an enhancer which depends on taf-1 activity. Alignments reveal that the SFV-1 internal promoter and the SFV-1 LTR have little sequence relatedness. Cross-transactivation studies show that the transactivators of SFV-1 and HFV function on the internal promoter and LTR of the homologous virus but not on the heterologous virus. In summary, the genomes of simian and human foamy viruses direct viral transcription through both the promoter in the LTR and an internal promoter within the env gene, and each promoter contains unique enhancer-like elements regulated by the viral transactivator.

Animals↗

Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury.

Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Abeta fibers only, Abeta and Adelta fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Adelta or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20-24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Adelta fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32-36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Abeta fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.

Afferent Pathways↗

The influence of the Univent endotracheal tube on internal jugular vein cannulation.

This prospective clinical investigation assessed the effect of placement of a Univent tube on the anatomy of the internal jugular veins and the success of cannulation of the left internal jugular vein. After obtaining informed consent, 48 adult patients were enrolled. Of these, 42 patients were eligible and were divided into two groups: Univent tube (group U, n=21) and wire enforced endotracheal tube (group C, n=21). The Univent tube group were having a left thoracotomy. Using horizontal ultrasound scans just above the thyroid gland, the internal jugular vein was visualized and measured before and after Univent placement. The number of needle passes necessary to cannulate the left internal jugular vein in the two groups was also compared. Univent tubes were associated with lateral displacement of the right carotid artery and internal jugular vein on the convex side of the Univent tube, with compression of the right internal jugular vein by the artery, resulting in a kidney-shaped cross-section of the vein. On the left (concave side of the tube), the neck was indented, the sheath of the left carotid artery was displaced medially, and the left internal jugular vein distorted to an ellipse. There was a significant increase in the lateral diameter and a decrease in the cross-sectional area of the left internal jugular vein (t-test, P < 0.05). The first attempt at cannulation of the left internal jugular vein failed significantly more often in the Univent group (13/21 vs 5/21 in group C, Chi-square 6.22, P=0.025). Cannulation of the internal jugular vein before placement of the Univent tube, or placement with ultrasound guidance is suggested.

Carotid Arteries↗

Glenohumeral range of motion deficits and posterior shoulder tightness in throwers with pathologic internal impingement.

BACKGROUND: Alterations in glenohumeral range of motion, including increased posterior shoulder tightness and glenohumeral internal rotation deficit that exceeds the accompanying external rotation gain, are suggested contributors to throwing-related shoulder injuries such as pathologic internal impingement. Yet these contributors have not been identified in throwers with internal impingement. HYPOTHESIS: Throwers with pathologic internal impingement will exhibit significantly increased posterior shoulder tightness and glenohumeral internal rotation deficit without significantly increased external rotation gain. STUDY DESIGN: Case control study; Level of evidence, 3. METHODS: Eleven throwing athletes with pathologic internal impingement diagnosed using both clinical examination and a magnetic resonance arthrogram were demographically matched with 11 control throwers who had no history of upper extremity injury. Passive glenohumeral internal and external rotation were measured bilaterally with standard goniometry at 90 degrees of humeral abduction and elbow flexion. Bilateral differences in glenohumeral range of motion were used to calculate glenohumeral internal rotation deficit and external rotation gain. Posterior shoulder tightness was quantified as the bilateral difference in passive shoulder horizontal adduction with the scapula retracted and the shoulder at 90 degrees of elevation. Comparisons were made between groups with dependent t tests (P < .05). RESULTS: The throwing athletes with internal impingement demonstrated significantly greater glenohumeral internal rotation deficit (P = .03) and posterior shoulder tightness (P = .03) compared with the control subjects. No significant differences were observed in external rotation gain between groups (P = .16). CLINICAL RELEVANCE: These findings could indicate that a tightening of the posterior elements of the shoulder (capsule, rotator cuff) may contribute to impingement. The results suggest that management should include stretching to restore flexibility to the posterior shoulder.

Adult↗

Large-scale screening assay to measure epidermal growth factor internalization.

Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.

3T3 Cells↗

Agonist mediated internalization of M2 mAChR is beta-arrestin-dependent.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is beta-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of beta-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a beta-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous beta-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice that lack expression of either one or both isoforms of beta-arrestin (beta-arrestin 1 and 2). RESULTS: In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF beta-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either beta-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged beta-arrestin within endocytic structures in multiple cell lines; the compartment to which beta-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF beta-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (beta-arrestin 2 DeltaLIELD), AP-2 (beta-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of beta-arrestin 1 (319-418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells. CONCLUSION: In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles.

Journal Article↗