Anesth Analg 2008; 106:480-485
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e318160648b
ANESTHETIC PHARMACOLOGY
Morphine Attenuates Microvascular Hyperpermeability via a Protein Kinase A-Dependent Pathway
Rudolph Puana, MD*,
Russell K. McAllister, MD*
,
Felicia A. Hunter, BS,
Julie Warden, MD*, and
Ed W. Childs, MD
From the Departments *Anesthesiology and Surgery, Scott and White Clinic and Memorial Hospital, Scott, Sherwood and Brindley Foundation, Texas A&M Health Science Center College of Medicine, Temple, Texas.
Address correspondence and reprint requests to Ed Childs, MD, Associate Professor of Surgery, Scott and White Memorial Hospital, 2401 S. 31st St., Temple, TX 76508. Address e-mail to echilds{at}swmail.sw.org.
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Abstract
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BACKGROUND: A recently published study from our laboratory demonstrated that morphine sulfate (MS) attenuates microvascular hyperpermeability after hemorrhagic shock in rats.
MS binds to the µ receptors located on the surface of endothelial cells. Activation of the endothelial cell µ receptors has been shown by several investigators to stimulate adenylate cyclase. We hypothesize that MS binding to the µ receptor on endothelial cells increases cyclic adenosine monophosphate via adenylate cyclase activation. Cyclic adenosine monophosphate inhibits the phosphoinositide/MAP kinase hyperpermeability pathway via the protein kinase A (PKA)-dependent inhibition of Raf-1.
METHODS: Studies were conducted in five groups of urethane-anesthetized Sprague-Dawley rats: Group 1—control group, Group 2—a non–receptor-blocking adenylate cyclase inhibitor: SQ22536, at 100 µg/kg (n = 5), Group 3—a PKA inhibitor: H89, at 10 µg/kg, Group 4—a morphine sulfate (10 µg/kg) and PKA inhibitor group, and Group 5—an adenylate cyclase inhibited and morphine (10 µg/kg) group. Intravital microscopy in mesenteric postcapillary venules and rat lung microvascular endothelial cell monolayers were used to measure permeability.
RESULTS: Adenylate cyclase and PKA inhibition resulted in vascular hyperpermeability.
CONCLUSION: Our data demonstrated an increase in vascular hyperpermeability after inhibition of adenylate cyclase via SQ22536, a nonreceptor inhibitor. This increase in hyperpermeability was attenuated when treated with MS. Morphine did not attenuate hyperpermeability after blockage following PKA with H89 suggesting the action of MS is upstream of PKA and PKA dependent.
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Introduction
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Morphine sulfate (MS) has been shown to attenuate microvascular hyperpermeability after hemorrhagic shock.1 Hemorrhagic shock has been demonstrated by several investigators to be associated with an increase in vascular permeability via the phosphoinositol/mitogen-activated (MAP)-kinase pathway.2–6 The phosphoinositol/MAP kinase pathway is regulated by the counterbalance between cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), with cAMP inhibiting hyperpermeability and cGMP stimulating hyperpermeability.6
Several investigators have shown that morphine binds to vascular endothelial cell µ receptors stimulating adenylate cyclase activity.7–9 Adenylate cyclase increases the production of cAMP from adenosine triphosphate. Cyclic AMP then binds to the R-group of protein kinase A (PKA). PKA then causes disassociation and inhibits Raf-1.6,10–12 The balance between the secondary messenger cGMP and cAMP is thought to be important in maintaining vascular permeability homeostasis via the MAP kinase pathway. In fact, He et al. have demonstrated that cAMP may be the dominant regulator of microvascular permeability.13 The purpose of this study was to determine whether morphine’s effect on vascular hyperpermeability is PKA dependent.
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METHODS
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Care of Animals
The surgical procedures and experimental protocols were conducted at Scott & White Hospital and Texas A&M Health Science Center College of Medicine after approval by the Animal Care and Use Committee and has been extensively described previously.1 The facility is approved by the American Association for Accreditation of Laboratory Animal Care in accordance with National Institutes of Health guidelines.
Chemicals and Solutions
The test solute for the permeability measurements was fluorescein isothiocyanate-bovine albumin (FITC-albumin; Sigma, St. Louis, MO). The test solution was prepared by dissolving 50 mg/kg of FITC in saline. Adenylate cyclase inhibitor (SQ22536; Calbiochem, San Diego, CA) and PKA inhibitor (H89; Calbiochem, San Diego, CA) were prepared by dissolving in dimethylsulfoxide 5 mg/mL. MS, 2 mg solution, was dissolved in 200 mL of 0.9% NaCl solution (Baxter HealthCare Corporation, Deerfield, IL).
Animal Surgery and Intravital Microscopy
Male Sprague-Dawley rats weighing 275–325 g were selected for study. The rats were fasted for 18 h and given water ad libitum before each experiment. The rats were anesthetized by an IM injection of 50% urethane (1.5 g/kg). Polyethylene cannulas (PE-50, 0.58 mm ID) were placed in the right internal jugular vein for IV fluid administration. Mean arterial blood pressure was measured continuously using a PE-50 cannula in the left femoral artery connected to a blood pressure analyzer (Dig-Med, BPA 400A; Micromed, Louisville, KY). The rats were placed in the lateral decubitus position on a temperature-controlled Plexiglas plate mounted on an intravital upright microscope (Nikon E 600, Tokyo, Japan). The rat’s temperature was maintained at 37°C. A midline laparotomy incision was made to expose a section of small bowel mesentery. The externalized segment of mesentery from the small intestine was draped over a temperature-controlled Plexiglas stage and used for microscopic examination. The mesentery was superfused with normal saline at 2 mL/h and covered with plastic wrap (Glad Wrap; Dow Chemical, Springfield, OH) to reduce evaporation. Venules with diameters of 20–35 µm were selected for study with a Nikon 20x flatfield objective, 0.45–2.16 mm working distance (Nikon Instruments, Inc., Natick, MA). Images were obtained with a Cascade CCD fluorescent camera (Photometrics, Tucson, AZ). A video time and date generator (WJ-8 10; Panasonic, Secaucus, NJ) provided onscreen time, date, and stopwatch functions. The image was projected onto a video monitor (Trinitron 20-inch monitor; Sony, New York, NY), captured digitally on computer, and stored on compact disk. Data were analyzed using MetaMorph 5.7 (Universal Imaging Corp., Downingtown, PA).
Experimental Protocols
Five groups of Sprague-Dawley rats consisting of five animals each were studied. Group 1—controls, Group 2—a non–receptor-blocking adenylate cyclase inhibitor: SQ22536, at 100 µg/kg (n = 5), Group 3—a PKA inhibitor: H89, at 10 µg/kg, Group 4—a MS (10 µg/kg) and PKA inhibitor group, and Group 5—an adenylate cyclase inhibited and morphine (10 µg/kg) group. The rats were allowed to recover from surgical manipulation for 30 min before the start of all experiments. During this period, animals were given FITC-albumin at 50 mg/kg for permeability determination and baseline integrated optical intensities were obtained intra- and extravascularly with predetermined areas. To determine permeability, images were taken at 5-min intervals during resuscitation after a 60-min shock or sham period. Exposure time of <15 s was performed to prevent quenching of the fluorescent indicators. Intensities were recorded at baseline and at 5-min intervals for 120 min after treatment.
Cell Culture Monolayer
Rat lung mesenteric endothelial cells (passes 7–12) were grown on gelatin-coated Costar Transwell membranes (VWR, Houston, TX). Monolayer permeability studies were performed on groups: Group 1, a non– receptor-blocking adenylate cyclase inhibitor: SQ22536 at 100 µg/kg (n = 5), Group 2, a PKA inhibitor: H89, at 10 µg/kg, Group 3, a MS (10 µg/kg) and PKA inhibitor group, and Group 4, an adenylate cyclase inhibited and MS (10 µg/kg) group. Permeability was determined by measuring FITC-albumin flux across the monolayer. FITC-albumin (10 µg/mL) was added to the luminal chamber for 60 min and samples were removed from both luminal and abluminal chambers for fluorometry analysis. The readings were converted with the use of a standard curve to albumin concentration. These concentrations were then used in the following equation to determine the permeability coefficient of albumin (Pa)
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where [A] is abluminal concentration; t is time in seconds; A is area of membrane in cm2; V is volume of abluminal chamber; and [L] is luminal concentration.
Statistical Analysis
The in vivo data were initially analyzed to determine significance among groups by the repeated measured analysis of variance. The procedure was followed by the Benferroni test for multiple comparisons. Comparisons were made regarding permeability versus sham-operated groups. The in vitro studies were analyzed by t-test and compared with controls. The difference was considered significant when P < 0.05.
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RESULTS
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Effect of Morphine on Vascular Permeability with Administration of Adenylate Cyclase Inhibitor
Figure 1 is a composite image (20x) of a rat’s mesenteric postcapillary venule demonstrating vascular hyperpermeability. The images labeled "sham" and "adenylate cyclase inhibitor" demonstrate an intense fluorescent signal inside the vessels injected with FITC-albumin. The image labeled "sham" shows minimal extravascular intensity of FITC albumin at 120 min. The image labeled "adenylate cyclase inhibitor" demonstrates an increase in FITC-albumin in the extravascular space at 120 min postshock indicative of increased vascular leak. The third image shows adenylate cyclase plus morphine attenuation of FITC albumin in the extravascular space at 120 min. Figure 2 demonstrates the effective relationship between control, adenylate cyclase inhibitor, and adenylate cyclase inhibitor plus morphine on vascular hyperpermeability. Hyperpermeability significantly increased starting at 30 min after treatment with SQ22536 and H89. Morphine 10 µg/kg pretreatment 10 min before administration of the adenylate cyclase inhibitor SQ22536 resulted in a significant decrease in the amount of perivascular FITC-albumin, representing a decrease in vascular leak (P < 0.05).
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Figure 1. Composite images (20x) at 120 min postdrug administration of a rat’s mesenteric postcapillary venule. The image labeled "sham" demonstrates an intense ITC fluorescent signal inside the vessels. The image labeled "adenylate cyclase inhibitor" illustrates an increase in vascular permeability indicated by the magnitude of FITC-albumin in the perivascular space. The third image shows adenylate cyclase plus morphine (10 µg/kg) and its attenuation of vascular hyperpermeability.
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Figure 2. This figure illustrates the effective relationship comparatively among all experimental groups [sham, SQ22536, SQ22536 and morphine sulfate (MS), protein kinase A inhibitor (H89), and H89 and morphine (H89 plus Ms)]. Please note morphine’s attenuation of increased vascular hyperpermeability due to the inhibition of adenylate cyclase by SQ22536 and morphine’s inability to attenuate vascular hyperpermeability after inhibition of PKA by H89.
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Effect of Morphine on Vascular Permeability with Administration of PKA Inhibitor
Figure 3 is a composite image (20x) of a rat’s mesenteric postcapillary venule demonstrating vascular permeability. The images labeled "sham" demonstrates an intense fluorescent signal inside the vessels injected with FITC-albumin. The image labeled PKA inhibitor (H89) illustrates an increase in vascular leak 120 min posttreatment, indicated by the magnitude of FITC-albumin in the perivascular space. The image labeled H89 plus morphine shows the presence of increased vascular hyperpermeability denoted by the increased FITC albumin in the extravascular space.
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Figure 3. Composite images (20x) at 120 min postdrug administration of a rat’s mesenteric postcapillary venule. The images labeled "sham" demonstrates an intense fluorescent signal inside the vessel. The image labeled "PKA inhibitor" illustrates an increase in vascular leak postdrug administration indicated by the magnitude of FITC-albumin in the perivascular space. The image labeled H89 plus morphine shows the presence of increased vascular permeability denoted by the increased FITC albumin in the extravascular space.
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Monolayer Results
Figure 4 shows a cumulative graph of a monolayer cultured cell in vitro experiments rat lung microvascular endothelial cell. Monolayer permeability increases with administration of adenylate cyclase inhibition SQ22536 and decreases with morphine administration 10 min before treatment. Monolayer permeability was significant (P < 0.05) at times between 60 and 120 min. These in vitro data correlate with our in vivo experiments.
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Figure 4. A cumulative graph of rat lung microvascular endothelial cell (RLMEC) monolayers cultured cell. RLMEC monolayer hyperpermeability increases with administration of adenylate cyclase inhibition (SQ22536) at 60 min posttreatment (P < 0.05). Morphine administration 10 min before dosing with SQ22536 attenuated this increase in monolayer hyperpermeability.
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DISCUSSION
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MS is a frequently administered analgesic, anxiolytic, and anesthetic in multiinjured patients requiring surgical intervention. MS is also a mediator of histamine release. Because of the potent vasoactive response of histamine on vascular permeability, morphine therapy would be expected to cause an increase in vascular permeability. However, the results of our previous study demonstrate that morphine attenuates vascular permeability after hemorrhagic shock.1 Histamine has a profound effect on vascular permeability5,6,14–18 through the phosphoinositol pathway. This pathway of increasing vascular permeability has been well described, beginning with the activation of receptor tyrosine kinase on the surface of endothelial cells (Fig. 5). This activation triggers phospholipase C along with calcium to stimulate the production of nitric oxide (NO) through endothelial-derived NO synthase. Endothelial-derived NO activates soluble guanylate cyclase, which converts guanine triphosphate to cGMP. Cyclic GMP, through protein kinase G, stimulates the MAP kinase pathway via Raf-1. Raf-1 stimulates mitogen-activated kinase (Mek1/2) and extracellular signal-regulated kinase (Erk1/2) to increase vascular permeability.4,14,16,19 This pathway is mediated via histamine along with other activators of the tyrosine kinase receptor, including vascular endothelial growth factor and tumor necrosis factor.3,6,14
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Figure 5. Proposed mechanism of action of morphine sulfate on the phosphoinositide/mitogen activated kinase pathway via the µ receptor located on endothelial cells.
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Morphine’s primary effect, analgesia, is mediated by µ receptors ubiquitously within the central nervous system. The purpose of endothelial µ receptors has been uncertain. Several investigators have shown that morphine binding to endothelial µ receptors stimulates adenylate cyclase activity.7–9 Adenylate cyclase increases the production of cAMP from adenosine triphosphate. Cyclic AMP then binds to the R-group of PKA. PKA then causes disassociation and inhibits Raf-1.6,10–12 The balance between the secondary messenger cGMP and cAMP is thought to be important in maintaining vascular permeability homeostasis via the MAP kinase pathway. In fact, He et al. have demonstrated that cAMP may be the dominant regulator of microvascular permeability.13 Our data demonstrated that morphine, through µ receptor activation, inhibits microvascular permeability in a PKA-dependent manner.
The effect of µ receptor activation on adenylate cyclase activity has been shown to vary according to the adenylate cyclase class.8,9,20 Investigators have shown different cells which account for decreases and/or increases of cAMP with the activation of µ receptors.8,9 Of the nine classes of adenylate cyclase, morphine’s µ activity was found to couple to adenylate cyclase 2 and 7 at GI 1-3, Go1-2, and Gz sites to increase levels of cAMP.20 In our experiment, a non–receptor-dependent inhibitor of adenylate cyclase resulted in increases in vascular permeability.21 This was attributed to the exhausted supply of cAMP caused by this inhibition.3,7 The administration of morphine to an inhibited, but still active, adenylate cyclase model decreased vascular permeability. Through the use of a non–receptor-dependent inhibitor of adenylate cyclase, it is postulated that activation of the µ receptor by morphine on adenylate cyclase is still possible. This stimulation results in increased cAMP with subsequent stimulation of PKA and the inhibition of Raf-1. We demonstrated that morphine acted directly on adenylate cyclase through PKA by the administration of a PKA inhibitor. After administration of the PKA inhibitor, vascular permeability increased as expected. Morphine was then given in conjunction with the PKA inhibitor and no decrease in vascular permeability was observed. These findings illustrate that MS’s attenuation of vascular permeability is through the action of adenylate cyclase stimulation and is PKA dependent.
Morphine is the standard of care for many disease states, including chronic cancer pain22–24 and acute myocardial infarction.23 Our study indicates that morphine may also play a vital role in the treatment of acute shock due to its inhibition of microvascular hyperpermeability. Understanding the relationship of MS to hyperpermeability may explain the favorable outcomes in patients with adequate pain control.21,22,25,26 The use of morphine in acute settings may provide therapeutic benefit in the management of hyperpermeability after hemorrhagic shock.
In conclusion, this study demonstrates that MS, through a novel PKA-dependent pathway decreases vascular permeability in the microcirculation of the mesenteric venule. We also demonstrated is the dominance of this decrease in vascular permeability over the effects of histamine release known to occur with morphine administration.
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Footnotes
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Accepted for publication October 15, 2007.
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Abstract
Introduction
