| JOURNAL HOME | CME HOME | THIS MONTH | PAST ISSUES | ETOC | COLLECTIONS |
| AUTHORS | REVIEWERS | EDITORIAL BOARD | FEEDBACK | RSS | HELP |
| ||||||||||||||
| ||||||||||||||
|
|
|
|
||||||||||||
|
||||||||||||||
*Department of Anesthesiology, University Hospital, Erlangen;
Department of Anesthesiology and Critical Care Medicine Mannheim, University Heidelberg, Germany;
The Reference Laboratory, University Hospital Kopenhagen, Denmark; and
Department of Anesthesiology, LKH Klagenfurt, Austria
Address correspondence and reprint requests to James Blunk, Department of Physiology and Experimental Pathophysiology, University of Erlangen-Nuremberg, Universitätsstraße 17, D-91054 Erlangen, Germany. Address e-mail to blunk{at}physiologie1.uni-erlangen.de
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
IMPLICATIONS: Opioid effects on mast cells were assessed using intradermal microdialysis. Mast cell activation was seen with codeine and meperidine; no other opioid induced degranulation. Therefore, histamine release seen at large concentrations of potent µ agonists is caused by an unspecific effect rather than an activation of opioid receptors.
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the present study, using the technique of intradermal linear microdialysis, we assessed the ability of opioids to induce a dose-dependent degranulation of cutaneous mast cells and simultaneously measure the dermal effects of the mediators released from these mast cells. In vivo settings, in which intracutaneous injections of the respective drug have been made, offer only an indirect measurement of the degranulatory effects of mast cells, that is, the extent of the wheal and erythema formation. Using cutaneous microdialysis, the measurement of the released mediators and their vascular effects were assessed simultaneously in vivo (10). Besides using the potent mast cell degranulator codeine and morphine, a variety of semi-synthetic and synthetic opioid agonists and antagonists were investigated (meperidine, buprenorphine, fentanyl, alfentanil, sufentanil, remifentanil, and naloxone) to measure dose-response curves for protein extravasation, as well as for histamine and tryptase release. Moreover, laser Doppler imaging techniques with high spatial resolution were used to measure changes of local blood flow and blood flow changes arising from the induction of an axon reflex erythema.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The following drugs were used as stock solutions: Codeine phosphate (1 mg/mL; 3.34 mM), morphine hydrochloride (1 mg/mL; 3.11 mM, Merck, Whitehouse Station, NJ), meperidine hydrochloride (10 mg/mL; 40.4 mM, Dolantin®, Aventis, Bridgewater, NJ), buprenorphine (0.3 mg/mL; 0.64 mM, Temgesic®, Gruenenthal, Mitlodi, Switzerland), fentanyl (0.05 mg/mL; 0.15 mM, Bbraun, Sheffield, United Kingdom), alfentanil (0.5 mg/mL; 1.2 mM, Rapifen®, Janssen-Cilag, Neuss, Germany), sufentanil (5 µg/mL; 0.12 mM, Sufenta®, Janssen-Cilag), remifentanil (1 mg/mL; 2.65 mM, Ultiva®, Glaxo-Wellcome, Munich, Germany), and the opioid receptor antagonist naloxone (0.4 mg/mL; 1.22 mM, Narcanti®). Solutions of codeine, morphine, and meperidine were free of preservatives or other inactive components.
Intradermal microdialysis was used to administer the drugs intracutaneously without further injuring the skin. Five hollow plasmapheresis fibers with a diameter of 400 µm and a cutoff range of 3000 kD (Dermal Dialysis, Erlangen, Germany) were placed intracutaneously within the skin of the volar forearm using 25-gauge canula attached to the plasmapheresis fibers. Insertion length was approximately 15 mm, and the distance between each fiber was at least 30 mm oriented transversally to the axis of the forearm. The hollow fibers were perfused with Ringer’s solution (Ringerlösung, Bad Homburg, Germany) using a microdialysis pump (Pump 22, Harvard Apparatus, South Natick, MA) at a flow rate of 4 µL/min. Tygon® tubes (Cole-Parmer Instrument Company, Vernon Hills, IL) were used to connect the hollow fibers with the syringes (inner diameter, 0.38 mm). Insertion depth was controlled by ultrasound (Dermascan C, Cortex Technologies, Denmark); the average depth ranging between 0.5 and 0.9 mm. The dialysate was collected using glass capillaries tilted at an angle of 5 degrees to minimize outflow resistance. Dialysate was sampled every 15 min for 120 min, and frozen at -20°C in polyethylene cups for further analysis. After a baseline of 60 min, each of the five fibers was perfused with different concentrations of one opioid receptor agonist or antagonist for 30 min. This stimulation period was followed by a 30-min washout period. After waiting for the skin to adapt to the insertion trauma, the administration of the drugs involved no further injury of the skin, because the delivery of the drug was driven by diffusion from the dialysis fibers into the surrounding tissue.
Each dialysate sample (60 µL) was analyzed for total protein (5 µL) and tryptase (30 µL) content. Protein was measured photometrically (MRX reader, Dynatech, Denkendorf, Germany) using Coomassie blue dye for the analysis, with bovine serum albumin as a standard. Tryptase was analyzed by fluorescent immunoassay (UniCAP 100, Pharmacia & Upjohn, Freiburg, Germany). Histamine was measured using a fiber-based spectrofluorometric assay (11).
Superficial blood flow of the forearm was measured at regular intervals by laser-Doppler imager (LDI, Moor Instruments Ltd, Devon, United Kingdom), scanning the injection sites within an area of 20 x 10 cm with a resolution of 16,380 pixels. Local vasodilation as well as the axon reflex vasodilation was measured every 15 min during the equilibration period and every 5 min during the stimulation period. During the washout period of 30 min, the scanning interval was changed back to 15 min. Images were processed by dedicated software (MoorLDI Version 3.0, Moor Instruments Ltd). Local vasodilation was determined within a 0.4-cm narrow and 1.5-cm long region of interest placed directly above the membranes. The mean flux of developing axon reflex vasodilation was determined by moving the region of interest previously placed above the membrane site exactly 1 cm proximally.
During the first minute after onset of stimulation, the subjects were asked to rate the maximum itch or pain sensation on a numeric rating scale (NRS) separately for each stimulation site. The end-points were defined as "no itch/pain" (NRS = 0) and "maximum itch/pain" (NRS = 10).
To assess whether the µ receptor antagonist naloxone is able to antagonize the effect of morphine (0.5 mg/mL), an agonist with a high-affinity to the µ receptor, of codeine (0.5 mg/mL), the prodrug to morphine with a low µ affinity, and of meperidine (1 mg/mL), showing low affinity to the µ receptor but high affinity to the 
-receptor, we co-injected these drugs with naloxone (0.2 mg/mL) intradermally. Blood flow changes were assessed by laser Doppler imaging, and the extent of the developing wheal reaction was measured 20 min after the injection. Moreover, the subjects were asked to rate the intensity of itch sensations using the NRS described above.
The in vivo delivery of fentanyl and buprenorphine was determined from the remaining dialysate. The delivery was calculated as the relative difference between the amount of opioid in the perfusate and the dialysate (percent decline in concentration) (12). Fentanyl and buprenorphine were analyzed using liquid chromatography mass-spectrometry (13).
Results are expressed as mean ± SD. Dose response curves for local vasodilation and for the axon reflex erythema represent the relative increase from baseline values during the entire stimulation period (six data points). These six data points were calculated as area under the curve. In some cases, this leads to negative values reflecting a further decrease during perfusion with the smallest concentrations. Potencies are given as concentration required for 50% of the maximum effect (ED50). For each subject, ED50s and effectiveness (maximum effect) of the respective opioid were obtained by nonlinear regression fitting to the logistic function y = ymin + (ymax-ymin)/(1 + 10( logx0-x)*p), in which p is the Hill coefficient, ymax represents efficacy, and x0 the concentration required for the ED50. Origin software (Microcal, Northampton, MA) was used for these calculations. Data were compared using one-way analysis of variance followed by Newman-Keuls post hoc tests. Significance levels were P < 0.05. The STATISTICA software package (Statsoft, Tulsa, OK) was used for statistical analysis.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Intradermal stimulation with morphine, codeine, and meperidine led to a massive dose-dependant increase of protein extravasation and vasodilatation (Fig. 1) resulting from mast cell activation with tryptase and histamine release (Fig. 2). In addition, this liberation of histamine provoked distinct itch sensations. Stimulation with remifentanil, fentanyl, sufentanil, alfentanil, buprenorphine, and naloxone did not induce histamine or tryptase release.
|
|
|
The joint injection of morphine (0.5 mg/mL), codeine (0.5 mg/mL), and meperidine (1 mg/mL) with naloxone (0.2 mg/mL) did not have any effect on the size and intensity of the developing axon-reflex flare, itch sensation, or on wheal formation mirroring protein extravasation (Table 1).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Under the conditions used in our experiments, the intradermal stimulation with the high-affinity µ receptor agonist morphine, with the low-affinity µ receptor agonist codeine, and with meperidine, which primarily shows -agonistic action, led to a dose-dependent increase of plasma extravasation, to the liberation of histamine and tryptase from cutaneous mast cells, to the subsequent induction of local vasodilation, to the formation of an axon-reflex erythema, and to distinct itch sensations. Systemic effects were not seen during the skin perfusion with even the largest concentrations of the most potent opioids. Considering the recovery experiments, in which recovery values between 25% and 35% were determined, approximately two-thirds of the concentration reached the skin of the subject, resulting in large local concentrations but only with a minor risk of systemic effects during the stimulation period.
Itch sensations experienced by the subjects ranged slightly more than the "scratch threshold" of 3 on the NRS of 0 to 10. This correlated well with the mast cell activation and the subsequent release of histamine and tryptase from the cytosolic granules (15). No other opioid induced any itch sensations. This correlated well with mast cell mediator levels ranging within control levels.
Similar to the observations of our previous study, in which the mast cell degranulatory effect of muscle relaxants was investigated, morphine, a high-affinity µ receptor agonist, codeine, the low-affinity µ receptor agonist, and meperidine all showed a very similar pattern of mast cell degranulation with a subsequent activation of C-nociceptive itch or pain fibers (16), vasodilation, and protein leakage from the vessels causing plasma extravasation (17). Again, none of the highly potent µ receptor agonists, the partial agonist buprenorphine, and the antagonist naloxone induced any of the effects related to neurogenic inflammation. Only the largest concentration of, for example, remifentanil (2.65 mM) provoked a slight increase of the local vasodilatory effect on the vessels located within the direct vicinity of the intradermal microdialysis membrane. Because no other effects, such as plasma extravasation, were seen, this was probably because of a direct action on the vessel walls, without increasing the permeability of the endothelial cell layer.
When intracutaneously administered, uncommonly large concentrations of, for example, fentanyl resulted in the formation of an axon-reflex erythema (5). In this study, the largest fentanyl concentration (0.3 mM) was double the normal clinically used concentration of 0.15 mM. All other opioids were administered in the widely used clinical concentrations, as described in Methods. Not even after the administration of the largest concentration of fentanyl did we see the induction of an axon-reflex flare or the formation of vessel leakage with subsequent plasma extravasation. In our study, the drug was continuously administered after an equilibration period in which the skin was allowed to recover. The flow rate of the stimulation period was slow, excluding effects of rapid injection into the tissue.
Naloxone did not induce mediator release from skin mast cells or provoke neurogenic inflammation. In case of an involvement of µ receptors on either the skin mast cells or directly upon the vasculature, it would be expected that naloxone would inhibit the induction of mediator release from mast cells and the subsequent formation of an axon reflex erythema and plasma extravasation. Because mediator release and plasma extravasation did not occur during skin perfusion with highly potent µ receptor agonists, only the opioids morphine, codeine, and meperidine were co-administered with naloxone. Contrary to earlier publications postulating the involvement of µ receptors on the histamine release from mast cells leading to an axon-reflex flare (5), naloxone did not attenuate erythema, protein extravasation, or itch induced by the co-administered morphine, codeine, or meperidine.
Mast cells can be activated by immunological and by nonimmunological pathways. Both pathways lead to the liberation of preformed mediators such as histamine and mast cell tryptase with only minor differences in the release of prostaglandins or leukotrienes (18). In contrast to the immunoglobulin (Ig)E-mediated immunological activation, a concentration-dependent direct activation of pertussis-toxin-sensitive heterotrimeric G-proteins (gi2, gi3) by cationic drugs amidated at the C-terminus is the most likely mechanism of this nonimmunological pathway (19,20).
In conclusion, large concentrations of morphine and other opioids with lower affinity to the µ receptor induce the liberation of the mast cell mediators histamine and tryptase. In contrast, the potent µ agonists did not activate cutaneous mast cells in vivo or induce neurogenic inflammation and the formation of an axon-reflex flare.
These effects seen together indicate that the activation of skin mast cells by morphine, codeine, and meperidine does not depend on µ receptors but, more likely, on the direct activation of G-proteins of mast cells. Thus, rather than using µ-opioid antagonists, the inhibition of stimulated G-protein activity, for example, by cromolyn (20), seems to be the more promising approach. This could be tested by intradermally co-injecting or co-perfusing the skin with, for example, codeine and cromolyn.
|
Acknowledgments |
|---|
The authors would like to thank Dr. Katharina Rentsch, University of Zürich, for her assistance in measuring the drug concentrations in the recovery experiments.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. Roosterman, T. Goerge, S. W. Schneider, N. W. Bunnett, and M. Steinhoff Neuronal control of skin function: the skin as a neuroimmunoendocrine organ. Physiol Rev, October 1, 2006; 86(4): 1309 - 1379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. T. Whiteside, J. M. Boulet, and K. Walker The Role of Central and Peripheral {micro} Opioid Receptors in Inflammatory Pain and Edema: A Study Using Morphine and DiPOA ([8-(3,3-Diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic Acid) J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1234 - 1240. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
