M. Brennan Harris

Ischemic but not thermal pain sensitivity varies across the menstrual cycle

Objective and Method Findings from both animal and human research suggest that pain sensitivity changes across the menstrual cycle; however, among humans the nature of these menstrual cycle effects remains unclear. The present study used a repeated-measures design to evaluate changes in thermal and ischemic pain responses during three phases of the menstrual cycle, midfollicular (postmenstrual), ovulatory, and mid-to-late luteal (premenstrual), in 11 healthy women. The cycle phase during which subjects began their participation was determined randomly. Plasma levels of estrogen, progesterone, luteinizing hormone (LH), testosterone, and beta-endorphin were determined at each experimental session. Participants also completed a daily diary of physical and emotional symptoms for two complete menstrual cycles before the experimental sessions. Results The results indicated that women showed less ischemic pain sensitivity during the midfollicular compared with the ovulatory and mid-to-late luteal phases, but thermal pain responses did not vary significantly across menstrual cycle phases. Physical and emotional symptoms were minimal and did not change significantly across the menstrual cycle. Conclusions These findings indicate greater ischemic but not thermal pain sensitivity among women after the midcycle LH surge. The practical relevance and potential mechanisms of these findings are discussed.

Relationship Between Pain Sensitivity and Resting Arterial Blood Pressure in Patients With Painful Temporomandibular Disorders

Objective Patients experiencing temporomandibular disorders (TMD) show greater sensitivity to painful stimuli than age- and gender-matched control subjects. This enhanced pain sensitivity may result, at least in part, from an alteration in pain regulatory systems that are influenced by resting arterial blood pressure. In this study, we examined the relationship between resting systolic blood pressure and pain perception in 64 female TMD and 23 age-matched pain-free female subjects. Method Resting arterial blood pressure and measures of thermal and ischemic pain threshold and tolerance were determined for each participant. Subjective ratings of thermal pain evoked by suprathreshold noxious thermal stimuli (45-49[degree sign]C) using a magnitude matching procedure were also obtained for both groups. Results TMD patients had lower thermal and ischemic pain thresholds and tolerances than pain-free subjects (ps<.05). Both groups provided equivalent intensity ratings to suprathreshold noxious thermal stimuli. A median split of each group based on resting systolic blood pressure revealed an influence of blood pressure on both thermal and ischemic pain perception for the Pain-Free group. The Pain-Free high resting blood pressure subgroup had higher thermal pain tolerances, higher ischemic pain thresholds, and provided lower magnitude estimates of the intensity of graded heat pulses compared with the Pain-Free low blood pressure subgroup. A trend toward a significant effect of blood pressure level on ischemic pain tolerance was also observed for the Pain-Free group. In contrast to the Pain-Free group, blood pressure level did not influence ischemic or thermal pain perception for TMD patients. Similar to the lack of effect of resting blood pressure on experimental pain perception in TMD patients, resting blood pressure was not related to measures of clinical orofacial pain in TMD patients. Conclusions These findings confirm our previous findings that TMD patients are more sensitive to noxious stimuli and suggest that painful TMD may result, at least in part, from an impairment in central pain regulatory systems that are influenced by resting arterial blood pressure.

Remodeling of the neuromuscular junction precedes sarcopenia related alterations in myofibers

Several mechanisms contributing to the etiology of sarcopenia (age-related loss of muscle size) have been postulated. One of these attributes the loss of muscle mass to a preceding age-related denervation of myofibers. The aim of this study was to determine if signs of denervation were apparent at the neuromuscular junction (NMJ) before fiber atrophy, or fiber type conversion could be documented, and to reveal if a muscles activity level impacts its sensitivity to age-related denervation. Plantaris and soleus muscles were obtained from young adult (10 months) and early aged (21 months) rats. Pre- and post-synaptic NMJ morphology was quantified with cytofluorescent staining of nerve terminal branches and endplate regions, respectively. Myofiber profiles (fiber size and fiber type composition) were assessed with histochemical procedures. Results show that in the lightly recruited plantaris, significant (P<0.05) signs of denervation were noted in aged rats, while the same muscles displayed no change in myofiber profile. In the heavily recruited soleus, however, there was little evidence of denervation, and again no alterations in myofiber profile. These results indicate that age-related denervation occurs before myofiber atrophy, and that high amounts of neuromuscular activity may delay the onset of age-related denervation and sarcopenia.

Pain sensitivity in patients with temporomandibular disorders: Relationship to clinical and psychosocial factors

OBJECTIVE We have previously reported that patients with temporomandibular disorders (TMD) exhibit enhanced sensitivity to experimentally evoked pain (1); however, the clinical relevance of this increased pain sensitivity remains unclear. The purpose of this study was to investigate the relationship of experimental pain sensitivity to clinical and psychosocial variables among patients with TMD. DESIGN Thirty-six TMD patients were studied, half of whom were pain sensitive (PS) and the other half pain tolerant (PT), based on their ability to tolerate an ischemic pain task. Responses to painful thermal and nonpainful visual stimuli as well as clinical/diagnostic symptoms and psychosocial variables were compared for the two groups (i.e., PS vs. PT). RESULTS Results indicated that, compared with PT patients, the PS group exhibited greater sensitivity to thermal pain and rated innocuous visual stimuli as more intense. PS patients also reported greater clinical pain, but in general the groups did not differ on diagnostic and psychosocial measures. CONCLUSIONS The results suggest that ischemic pain tolerance is a clinically relevant marker of pain sensitivity in TMD patients. These findings are consistent with the hypothesis that impairments in CNS inhibitory pathways may contribute to the pain associated with TMD.

Role of heat shock protein 90 in bradykinin-stimulated endothelial nitric oxide release

Previously we described ENAP-1, a 90-kDa protein that is tyrosine-phosphorylated in endothelial cells in response to bradykinin (BK) stimulation and is associated with endothelial nitric oxide synthase (eNOS). Subsequently, other investigators demonstrated that eNOS interacts with heat shock protein 90 (Hsp90) following stimulation of endothelial cells with vascular endothelial growth factor (VEGF), histamine, or fluid shear stress. Therefore, we tested the hypotheses that ENAP-1 and Hsp90 are the same protein and that BK activation of eNOS is dependent on Hsp90. Immunoblotting of immunoprecipitated Hsp90 with anti-phosphotyrosine antibody shows that Hsp90 is tyrosine-phosphorylated in response to BK stimulation of bovine aortic endothelial cells (BAECs). Coimmunoprecipitation of Hsp90 with anti-eNOS antibody reveals a Hsp90-eNOS complex in endothelial cells under basal conditions that is increased following BK stimulation. Taken together with the tyrosine phosphorylation data, these data suggest that ENAP-1 is Hsp90. BK-stimulated nitric oxide (NO) release is completely blocked by pretreatment with geldanamycin, a specific inhibitor of Hsp90, illustrating the importance of the Hsp90-eNOS interaction. In vitro binding assays with Hsp90-glutathione-S-transferase fusion proteins show direct binding of eNOS with the middle domain (residues 259-615) of Hsp90.

Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport

eNOS (endothelial nitric oxide synthase) catalyses the conversion of L-arginine into L-citrulline and NO. Evidence has been presented previously that eNOS is associated with the CAT (cationic amino acid transporter)-1 arginine transporter in endothelial caveolae, and it has been proposed that eNOS-CAT-1 association facilitates the delivery of extracellular L-arginine to eNOS. Definitive proof of a protein-protein interaction between eNOS and CAT-1 is lacking, however, and it is also unknown whether the two proteins interact directly or via an adaptor protein. In the present study, we raised a polyclonal antibody against CAT-1, and show using reciprocal co-immunoprecipitation protocols that eNOS and CAT-1 do indeed form a complex in BAECs (bovine aortic endothelial cells). In vitro binding assays with GST (glutathione S-transferase)-CAT-1 fusion proteins and eNOS show that the two proteins interact directly and that no single CAT-1 intracellular domain is sufficient to mediate the interaction. Overexpression of CAT-1 in BAECs by adenoviral-mediated gene transfer results in significant increases in both L-arginine uptake and NO production by the cells. However, whereas increased L-arginine transport is reversed completely by the CAT-1 inhibitor, L-lysine, increased NO release is unaltered, suggesting that NO production in this in vitro model is independent of CAT-1-mediated transport. Furthermore, eNOS enzymic activity is increased in lysates of CAT-1-overexpressing cells accompanied by increased phosphorylation of eNOS at Ser-1179 and Ser-635, and decreased association of eNOS with caveolin-1. Taken together, these data suggest that direct interaction of eNOS with CAT-1 enhances NO release by a mechanism not involving arginine transport.

Topical cooling (icing) delays recovery from eccentric exercise-induced muscle damage.

Abstract Tseng, C-Y, Lee, J-P, Tsai, Y-S, Lee, S-D, Kao, C-L, Liu, T-C, Lai, C-S, Harris, MB, and Kuo, C-H. Topical Cooling (Icing) Delays Recovery From Eccentric Exercise–Induced Muscle Damage. J Strength Cond Res 27(5): 1354–1361, 2013—It is generally thought that topical cooling can interfere with blood perfusion and may have positive effects on recovery from a traumatic challenge. This study examined the influence of topical cooling on muscle damage markers and hemodynamic changes during recovery from eccentric exercise. Eleven male subjects (age 20.2 ± 0.3 years) performed 6 sets of elbow extension at 85% maximum voluntary load and randomly assigned to topical cooling or sham groups during recovery in a randomized crossover fashion. Cold packs were applied to exercised muscle for 15 minutes at 0, 3, 24, 48, and 72 hours after exercise. The exercise significantly elevated circulating creatine kinase-MB isoform (CK-MB) and myoglobin levels. Unexpectedly, greater elevations in circulating CK-MB and myoglobin above the control level were noted in the cooling trial during 48–72 hours of the post-exercise recovery period. Subjective fatigue feeling was greater at 72 hours after topical cooling compared with controls. Removal of the cold pack also led to a protracted rebound in muscle hemoglobin concentration compared with controls. Measures of interleukin (IL)-8, IL-10, IL-1&bgr;, and muscle strength during recovery were not influenced by cooling. A peak shift in IL-12p70 was noted during recovery with topical cooling. These data suggest that topical cooling, a commonly used clinical intervention, seems to not improve but rather delay recovery from eccentric exercise–induced muscle damage.

Effect of N-2-mercaptopropionyl glycine on exercise-induced cardiac adaptations

The purpose of this study was to test the hypothesis that exercise-induced cardiac adaptations would be attenuated by the free radical scavenger N-2-mercaptopropionyl glycine (MPG). Male Sprague-Dawley rats were divided into four groups (n = 9-13 per group) for 3-4 wk: sedentary (S), S+MPG (100 mg/kg ip daily), exercised on a treadmill (E) (60 min/day, 5 days/wk, at a speed of 20 m/min up a 6° grade in a 6°C room), or E+MPG given 10 min prior to exercise. Additional rats (n = 55) were used to determine acute exercise effects on myocardial redox state [nonprotein nonglutathione sulfhydryls (NPNGSH)] and PI3K/Akt signaling pathway activation. Compared with S, NPNGSH levels were 48% lower in E (P < 0.05) and unchanged in E+MPG (P > 0.05). MPG also attenuated exercise-induced activation of the signaling proteins Akt and S6. Hearts from the 4-wk groups were weighed, and cardiac function was evaluated using an isolated perfused working heart preparation. Similar increases (P < 0.05) in both exercised groups were observed for heart weight and heart weight-to-body weight ratio. Cardiac function improved in E vs. S, as indicated by greater (P < 0.05) external work performed (cardiac output × systolic pressure) and efficiency of external work (work/Vo(2)). MPG prevented these exercise-induced functional improvements. Skeletal muscle mitochondria content increased to similar levels in E and E+MPG. This study provides evidence that free radicals do not play an essential role in the development of exercise-induced cardiac hypertrophy; however, they appear to be involved in functional cardiac adaptations, which may be mediated through the PI3K/Akt pathway.

Direct Interaction of the Cell Division Cycle 37 Homolog Inhibits Endothelial Nitric Oxide Synthase Activity

Endothelial NO synthase (eNOS) via the production of NO in the endothelium plays a key role in cardiovascular biology and is tightly regulated by co- and posttranslational mechanisms, phosphorylation, and protein–protein interactions. The cell division cycle 37 homolog (Cdc37) is a key heat shock protein 90 (Hsp90) cochaperone for protein kinase clients, and Akt/Hsp90 interaction is dependent on Cdc37. Because both Hsp90 and Akt are key eNOS regulatory proteins, we hypothesized that Cdc37 interacts with eNOS as part of the regulatory complex. In the present study, we demonstrate by coimmunoprecipitation and affinity purification in bovine aortic endothelial cells (BAECs) that Cdc37 is complexed with eNOS, Hsp90, and Akt. In addition, cell fractionation data indicate that Cdc37 is found in caveolae with eNOS. Further analysis by in vitro binding assays reveals a direct interaction between purified Cdc37 and eNOS. Incubation of purified Cdc37 with purified wild-type eNOS decreases eNOS activity in vitro. Overexpression of wild-type Cdc37 in BAECs inhibits eNOS activity and NO release, whereas overexpression of S13A-Cdc37 mutant in BAECs increases eNOS activity and NO release. Taken together, these data suggest that Cdc37 has a direct regulatory interaction with eNOS and may play an important role in mediating the eNOS protein complex formation as well as subsequent eNOS phosphorylation and activation.

Effect of resistance training on neuromuscular junctions of young and aged muscles featuring different recruitment patterns

To examine the effects of aging on neuromuscular adaptations to resistance training (i.e., weight lifting), young (9 months of age) and aged (20 months of age) male rats either participated in a 7‐week ladder climbing protocol with additional weight attached to their tails or served as controls (n = 10/group). At the conclusion, rats were euthanized and hindlimb muscles were quickly removed and frozen for later analysis. Longitudinal sections of the soleus and plantaris muscles were collected, and pre‐ and postsynaptic features of neuromuscular junctions (NMJs) were visualized with immunofluorescence staining procedures. Cross‐sections of the same muscles were histochemically stained to determine myofiber profiles (fiber type and size). Statistical analysis was by two‐way ANOVA (main effects of age and treatment) with significance set at P ≤ 0.05. Results revealed that training‐induced remodeling of NMJs was evident only at the postsynaptic endplate region of soleus fast‐twitch myofibers. In contrast, aging was associated with pre‐ and postsynaptic remodeling in fast‐ and slow‐twitch myofibers of the plantaris. Although both the soleus and the plantaris muscles failed to display either training or aging‐related alterations in myofiber size, aged plantaris muscles exhibited an increased expression of type I (slow‐twitch) myofibers in conjunction with a reduced percentage of type II (fast‐twitch) myofibers, suggesting early stages of sarcopenia. These data demonstrate the high degree of specificity of synaptic modifications made in response to exercise and aging and that the sparsely recruited plantaris is more vulnerable to the effects of aging than the more frequently recruited soleus muscle.