Spinal Opioid Tolerance Depends upon Platelet-Derived Growth Factor Receptor-ß Signaling, Not µ-Opioid Receptor Internalization.

Opioids are some of the most potent analgesics available. However, their effectiveness is limited by the development of analgesic tolerance. Traditionally, tolerance was thought to occur by termination of µ-opioid receptor (MOR) signaling via desensitization and internalization. Contradictory findings led to a more recent proposal that sustained MOR signaling caused analgesic tolerance. However, this view has also been called into question. We recently discovered that the platelet-derived growth factor receptor(PDGFR)-ß signaling system is both necessary and sufficient to cause opioid tolerance. We therefore propose a completely new hypothesis: that opioid tolerance is mediated by selective cellular signals and is independent of MOR internalization. To test this hypothesis, we developed an automated software-based method to perform unbiased analyses of opioid-induced MOR internalization in the rat substantia gelatinosa. We induced tolerance with either morphine, which did not cause MOR internalization, or fentanyl, which did. We also blocked tolerance by administering morphine or fentanyl with the PDGFR-ß inhibitor imatinib. We found that imatinib blocked tolerance without altering receptor internalization induced by either morphine or fentanyl. We also showed that imatinib blocked tolerance to other clinically used opioids. Our findings indicate that opioid tolerance is not dependent upon MOR internalization and support the novel hypothesis that opioid tolerance is mediated by intracellular signaling that can be selectively targeted. This suggests the exciting possibility that undesirable opioid side effects can be selectively eliminated, dramatically improving the safety and efficacy of opioids. SIGNIFICANCE STATEMENT: Classically, it was thought that analgesic tolerance to opioids was caused by desensitization and internalization of µ-opioid receptors (MORs). More recently, it was proposed that sustained, rather than reduced, MOR signaling caused tolerance. Here, we present conclusive evidence that opioid tolerance occurs independently of MOR internalization and that it is selectively mediated by platelet-derived growth factor receptor signaling. This novel hypothesis suggests that dangerous opioid side effects can be selectively targeted and blocked, improving the safety and efficacy of opioids.

Abused drugs modulate RGS4 mRNA levels in rat brain: comparison between acute drug treatment and a drug challenge after chronic treatment.

While different classes of abused drugs interact with distinct signaling substrates, it appears that all utilize receptors in the mesolimbic dopamine system to mediate their reinforcing effects. The regulator of G-protein signaling (RGS) proteins modulate G-protein coupled receptor (GPCR) signaling by increasing the rate of GTP hydrolysis of G proteins. This study was undertaken to determine whether morphine, cocaine, or amphetamine would modulate RGS4 mRNA levels in relevant brain regions. Acute administration of morphine and cocaine decreased levels of RGS4 mRNA in the reticulotegmental pontine nucleus (RtTg) and locus coeruleus (LC). Increases in RGS 4 mRNA levels were observed in the nucleus accumbens (NAc) and dorsal central gray (CGD). Acute drug challenge after chronic drug administration increased RGS4 mRNA in the CGD and decreased RGS4 levels in the red nucleus and RtTg. Interestingly, the LC exhibited biphasic modulation, with decreased RGS4 mRNA levels after acute administration and increased levels after chronic administration. These findings indicate that RGS4 mRNA levels are modulated in a similar manner by different drugs of abuse and imply that a common substrate could mediate some effects of abused drugs.

The biologic basis of fatigue.

In spite of many remarkable advances in our understanding of the mechanisms of cancer biology, very little attention has been directed toward mechanisms underlying cancer-induced symptoms. Although fatigue is a widely prevalent complication of cancer, there is a paucity of both basic and clinical research in this area. This article details our current knowledge of mechanisms causing cancer-related fatigue and briefly discusses currently available therapeutic options. A framework for addressing gaps in our knowledge and recommendations for future research directions are proposed.

Purification and characterization of human cell--cell adhesion molecule 1 (C-CAM1) expressed in insect cells.

The cell--cell adhesion molecule 1 (C-CAM1) plays an important role as a tumor suppressor for prostate cancer. Decreased expression of C-CAM1 was detected in prostate, breast, and colon carcinoma. Reexpression of C-CAM1 in prostate and breast cancer cell lines was able to suppress tumorigenicity in vivo. These observations suggest that C-CAM1 may be used as a marker for cancer detection or diagnosis. To generate monoclonal antibodies specific to C-CAM1, we have overexpressed full-length human C-CAM1 in Sf9 cells using a baculovirus expression system. The protein was purified 104-fold using nickel affinity chromatography. About 0.4 mg purified C-CAM1 was obtained from 200 mg of infected cells. When the purified protein was digested with peptidyl-N-glycosidase, the apparent mobility of the protein on SDS--PAGE changed from 90 to 58 kDa, which is close to the molecular weight predicted from the cloned cDNA sequence. This observation suggests that C-CAM1 was glycosylated on asparagine residues when expressed in Sf9 cells. Western blotting and internal protein sequencing analysis confirmed that the purified protein is human C-CAM1. Biochemical and functional assays indicate that this protein expressed in Sf9 cells displays characteristics similar to those of native protein, including adhesion function and glycosylation modification. Using this protocol, sufficient quantity of this protein can be produced with purity suitable for monoclonal antibody generation and biochemical study.

Association of mitral annular calcium with prior thromboembolic stroke in older White, African-American, and Hispanic men and women.

Prior thromboembolic stroke was present in 57 of 188 white men (30%) with mitral annular calcium (MAC) and in 62 of 303 white men (20%) without MAC, in 42 of 65 African-American men (65%) with MAC and in 50 of 123 African-American men (41%) without MAC, and in 13 of 27 Hispanic men (48%) with MAC and in 21 of 58 Hispanic (36%) without MAC. Prior thromboembolic stroke was present in 164 of 614 white women (27%) with MAC and in 85 of 516 white women (16%) without MAC, in 111 of 193 African-American women (58%) with MAC and in 77 of 225 African-American women (34%) without MAC, and in 36 of 69 Hispanic women (52%) with MAC, and in 17 of 58 Hispanic women (29%) without MAC.

Increased plasma homocysteine is an independent predictor of new atherothrombotic brain infarction in older persons.

A prospective study investigated the association of plasma homocysteine and other risk factors with the incidence of atherothrombotic brain infarction (ABI) at 31 +/- 9 month follow-up in 153 men and 347 women (mean age 81 +/- 9 years, median age 82). The stepwise Cox regression model showed that significant independent predictors of new ABI in older persons were age (risk ratio 1.060 for each 1-year increase of age), plasma homocysteine (risk ratio 1.079 for each 1 micromol/L increase), prior ABI infarction (risk ratio 3.282), current cigarette smoking (risk ratio 2.687), hypertension (risk ratio 2.965), and diabetes mellitus (risk ratio 2.015).

Use of transcutaneous electrical nerve stimulation in a young child with pain from open perineal lesions.

Transcutaneous electrical nerve stimulation (TENS) has been shown to be an effective treatment modality in adults experiencing pain associated with a variety of conditions. Therapeutic measures that are effective with adults can often be used with children. However, the benefit of TENS for children has not been well established since few research or clinical data have been published in the literature. This case report of a 4-year-old female with open perineal skin lesions who received TENS as an adjuvant therapy for painful dressing changes illustrates that TENS can be an effective treatment in children. In addition to the pain reduction seen in our patient, TENS therapy also had an opioid-sparing effect.

Association of left ventricular hypertrophy and chronic atrial fibrillation with the incidence of new thromboembolic stroke in 2,384 older persons.

In a prospective study of 2,384 persons, mean age 81 years, at 44-month follow-up, new thromboembolic stroke developed in 510 of 2,384 persons (21%). The Cox regression model showed that significant independent risk factors for new thromboembolic stroke were atrial fibrillation (risk ratio 3.2), left ventricular hypertrophy (risk ratio 2.8), prior stroke (risk ratio 2.2), and male gender (risk ratio 1.2).

Association of extracranial carotid arterial disease and chronic atrial fibrillation with the incidence of new thromboembolic stroke in 1,846 older persons.

In a prospective study of 1,846 persons, mean age 81 +/- 8 years, 281 persons (15%) had 40% to 100% extracranial carotid arterial disease and 253 persons (14%) had chronic atrial fibrillation. The Cox regression model showed that significant independent risk factors for new thromboembolic stroke were atrial fibrillation (p = 0.0001, risk ratio 3.3), 40% to 100% extracranial carotid arterial disease (p = 0.0001, risk ratio 2.5), prior stroke (p = 0.0001, risk ratio 2.1), and male gender (p = 0.045, risk ratio 1.2).

Risk factors for new atherothrombotic brain infarction in older African-American men and women.

Independent risk factors for new atherothrombotic brain infarction (ABI) in older African-American men were hypertension (risk ratio 4.381), diabetes mellitus (risk ratio 2.872), and previous ABI (risk ratio 1.904). Independent risk factors for new coronary events in older African-American women were cigarette smoking (risk ratio 2.754), hypertension (risk ratio 5.914), diabetes mellitus (risk ratio 3.464), serum total cholesterol (risk ratio 1.008), serum high-density lipoprotein cholesterol (inverse association) (risk ratio 0.958), age (risk ratio 1.026), and previous ABI (risk ratio 2.601).

Incidence of new thromboembolic stroke in persons 62 years and older with chronic atrial fibrillation treated with warfarin versus aspirin.

OBJECTIVE: To investigate the incidence of new thromboembolic (TE) stroke in older persons with chronic atrial fibrillation treated with oral warfarin versus aspirin. DESIGN: In an observational study of 312 older persons with chronic atrial fibrillation, long-term aspirin 325 mg daily was administered to 187 persons, and oral warfarin, in a dose adjusted to maintain the international normalized ratio (INR) between 2.0 and 3.0, was administered to 115 persons. The incidence of new TE stroke was analyzed in persons treated with warfarin versus aspirin at 36 +/- 17 months (1 to 99 months) follow-up. SETTING: A large, long-term healthcare facility. PATIENTS: The patients included 208 women and 104 men, mean age 84 +/- 7 years (range 62 to 101 years). MEASUREMENTS AND MAIN RESULTS: Four of 125 persons (3%) on warfarin stopped taking warfarin compared with four of 187 persons (2%) on aspirin who stopped taking aspirin because of adverse effects (P not significant). In persons with prior stroke, the incidence of new TE stroke was 40% (27 of 67) in persons treated with warfarin versus 81% (56 of 69) in persons treated with aspirin (P < .001). In persons with no prior stroke, the incidence of new TE stroke was 22% (13 of 58) in persons treated with warfarin versus 56% (66 of 118) in persons treated with aspirin (P < .001). The incidence of new TE stroke in all subjects was 32% (40 of 125) in persons treated with warfarin versus 65% (122 of 187) in persons treated with aspirin (P < .001). Cox regression analysis showed that persons taking warfarin had a 76% less chance of developing a new TE stroke than those taking aspirin after controlling the confounding effects of other risk factors. CONCLUSION: In an observational study of older persons with chronic atrial fibrillation, persons treated with oral warfarin to maintain an INR between 2.0 and 3.0 had a significantly lower incidence of new TE stroke than persons treated with oral aspirin 325 mg daily.

Mu and kappa opioid receptors in periaqueductal gray and rostral ventromedial medulla.

The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) are important brain stem pain modulating regions. Recent evidence suggests that kappa opioids antagonize the effects of mu opioids in the RVM. However, the anatomical relationship between mu and kappa opioid receptors in PAG and RVM is not well characterized. This study examined relationships between mu and kappa opioid receptor immunoreactivity (IR) and mRNA in PAG and RVM. Brain slices were processed for either immunocytochemistry or in situ hybridization. We found considerable anatomical overlap of mu and kappa opioid IR and mRNA in the RVM and PAG. These results provide an anatomical basis for recent behavioral and electrophysiological findings in RVM, and suggest modulatory interactions between mu and kappa opioids in PAG.

Pattern of c-fos mRNA induction in rat brain by acute morphine.

Initially, opioid signaling had been thought to be mainly inhibitory in nature. However, it has been shown that opioids can activate specific signaling pathways and induce immediate early gene (IEG) transcription in brain. IEGs can then regulate the transcription of other genes, leading to changes in neuronal function in response to extracellular stimuli. This study was designed to identify brain regions that demonstrate specific induction of the IEG c-fos, a component of the AP-1 transcription factor, in response to acute morphine, and to contrast this induction with the stressful effects of the injection itself. Rats received either 10 mg/kg morphine or an equivalent volume of saline injected subcutaneously. Animals were then sacrificed 15, 30, or 60 min after injection. Specific induction of c-fos mRNA by morphine was seen in dorsomedial caudate-putamen, paraventricular nucleus of the thalamus, central and intralaminar thalamic nuclei, dorsal central grey, superior colliculus, lateral parabrachial nucleus, inferior olivary complex, and caudal nucleus tractus solitarius. These findings represent the first complete anatomical mapping of c-fos induction in rat brain, and show that acute morphine administration alters gene expression in several areas related to known functional properties of opioids. However, regions showing c-fos induction are not all classically associated with opioid receptors and opioid-mediated effects. These findings are considered in the context of the effects of opioids on neural circuitry as well as direct, receptor-mediated effects of morphine on neural cells.

Risk factors for new thromboembolic stroke in patients > or = 62 years of age with chronic atrial fibrillation.

In a prospective study of 312 older patients with chronic atrial fibrillation, at 36-month follow-up evaluation, new thromboembolic stroke developed in 162 of 312 patients (52%). Significant independent risk factors for new thromboembolic stroke were prior stroke (risk ratio = 1.6), rheumatic mitral stenosis (risk ratio = 2.0), left ventricular (LV) hypertrophy (risk ratio = 2.8), abnormal LV ejection fraction (risk ratio = 1.8), serum total cholesterol (risk ratio = 1.005), and serum high-density lipoprotein cholesterol (risk ratio = 0.96).

Association of mitral annular calcium with new thromboembolic stroke at 44-month follow-up of 2,148 persons, mean age 81 years.

We investigated in a prospective study of 2,148 persons (mean age 81 years), the association between mitral annular calcium and new thromboembolic stroke at 44-month follow-up. Independent risk factors for new thromboembolic stroke were prior stroke (risk ratio 2.4), mitral annular calcium (risk ratio 2.6), atrial fibrillation (risk ratio 3.0), and male gender (risk ratio 1.6).

Opioid effects on mitogen-activated protein kinase signaling cascades.

BACKGROUND: The molecular mechanisms underlying both beneficial and undesirable opioid actions are poorly understood. Recently, the three currently known mammalian mitogen-activated protein kinase (MAPK) signaling cascades (extracellular signal-related kinase [ERK], stress-activated protein kinase, and p38 kinase) were shown to play important roles in transducing receptor-mediated signaling processes. METHODS: To determine whether any of these kinase cascades were activated by opioids, mu, delta, or kappa opioid receptors were transiently introduced into COS-7 cells together with MAPKs tagged to allow recognition by specific antibodies, and then exposed to opioids. Mitogen-activated protein kinase activation was determined by an in vitro MAPK activation assay. In addition, C6 glioma cells with either mu, delta, or kappa receptors stably introduced were exposed to opioids and MAPK activation determined by in vitro activation assay or antibody detection of activated forms. RESULTS: Transient experiments in COS cells revealed potent stimulation of ERK by mu and delta receptor activation, weak stimulation of stress-activated protein kinase by all receptor types, and no activation of p38. In stably transfected C6 glioma cells, only ERK activation was observed. Extracellular signal-related kinase induction was rapid, peaking 5 min after stimulation, and its activation was receptor-type specific. Mu and delta receptor stimulation activated ERK, but kappa stimulation did not. CONCLUSIONS: These results show that acute opioid signaling is not only inhibitory, but can strongly activate an important signaling cascade. Extracellular signal-related kinase activation may contribute to desirable responses to opioids, such as analgesia and sedation, and also to undesirable adaptive responses, such as tolerance, physical dependence, and possibly addiction. Further study of this system could provide greater insight into the molecular mechanisms underlying these clinical problems.

Association of plasma renin activity and echocardiographic left ventricular hypertrophy with frequency of new coronary events and new atherothrombotic brain infarction in older persons with systemic hypertension.

In older hypertensive persons, male gender, prior coronary artery disease, prior atherothrombotic brain infarction (ABI), and echocardiographic left ventricular (LV) hypertrophy are independent risk factors for new coronary events; age, prior ABI, and echocardiographic LV hypertrophy are independent risk factors for new ABI. The data suggest that high plasma renin activity in hypertensive older persons is associated with a high risk of new coronary events and of new ABI through its association with echocardiographic LV hypertrophy.

Cancellation of pediatric outpatient surgery: economic and emotional implications for patients and their families.

STUDY OBJECTIVE: To determine the cause and timing of case cancellation in a pediatric outpatient surgical population, and to examine the economic and emotional impact of such cancellations on patients and their families. DESIGN: Questionnaire survey. SETTING: Outpatient surgery unit of a large university children's hospital. PARTICIPANTS: 127 parents of children whose elective outpatient surgery had been cancelled. INTERVENTIONS: A total of 200 questionnaires were mailed to the parents of children who had their outpatient surgery cancelled. MEASUREMENTS AND MAIN RESULTS: Of those children whose surgery had been cancelled, 34.6% were due to upper respiratory infections (URIs), 30.7% for other medical reasons, and the balance for scheduling errors, because the child had not fasted, or for difficulties with transportation. The majority of surgeries (58.3%) were cancelled prior to their scheduled surgery date. However, 18.9% were cancelled on the day of surgery prior to leaving for the hospital and 22.8% were cancelled on arrival at the outpatient surgery clinic. Of those patients whose surgeries were not cancelled until they arrived at the hospital, 38.5% of mothers and 50.0% of fathers missed a day of work and, of these, 53.3% and 42.1%, respectively, went unpaid for the work day missed. The mean number of miles driven (round trip) to the hospital for a cancelled operation was 158.8 miles (range 8 to 1,350 miles). Additional testing and new appointments were ordered in 25.2% of the cancelled cases. 45% of parents and 16% of children were disappointed by the cancellation; 16% of parents were frustrated by the cancellation and 3.3% were angry. CONCLUSIONS: This study suggests that last-minute cancellation of surgery has an important impact on patients and their families and suggests a need to review present protocols for screening patients prior to surgery.