Ca2+ sensitization and PKC contribute to exercise training-enhanced contractility in porcine collateral-dependent coronary arteries

Exercise training enhances endothelium-dependent coronary vasodilatation, improving perfusion and contractile function of collateral-dependent myocardium. Paradoxically, studies from our laboratory have revealed increased Ca(2+)-dependent basal active tone in collateral-dependent arteries of exercise-trained pigs. In this study, we tested the hypothesis that exercise training enhances agonist-mediated contractile responses of collateral-dependent arteries by promoting Ca(2+) sensitization. Ameroid constrictors were surgically placed around the proximal left circumflex coronary (LCX) artery of female Yucatan miniature pigs. Eight weeks postoperatively, pigs were randomized into sedentary (pen confined) or exercise-training (treadmill run; 5 days/wk; 14 wk) groups. Arteries (～150 μm luminal diameter) were isolated from the collateral-dependent and nonoccluded (left anterior descending artery supplied) myocardial regions, and measures of contractile tension or simultaneous tension and intracellular free Ca(2+) concentration levels (fura-2) were completed. Exercise training enhanced contractile responses to endothelin-1 in collateral-dependent compared with nonoccluded arteries, an effect that was more pronounced in the presence of nitric oxide synthase inhibition (N(ω)-nitro-l-arginine methyl ester; 100 μM). Contractile responses to endothelin-1 were not altered by coronary occlusion alone. Exercise training produced increased tension at comparable levels of intracellular free Ca(2+) concentration in collateral-dependent compared with nonoccluded arteries, indicative of exercise training-enhanced Ca(2+) sensitization. Inhibition of PKC (calphostin C; 1 μM), but not Rho-kinase (Y-27632, 10 μM; or hydroxyfasudil, 30 μM), abolished the training-enhanced endothelin-1-mediated contractile response. Exercise training also increased sensitivity to the PKC activator phorbol 12,13-dibutyrate in collateral-dependent compared with nonoccluded arteries. Taken together, these data reveal that exercise training enhances endothelin-1-mediated contractile responses in collateral-dependent coronary arteries likely via increased PKC-mediated Ca(2+) sensitization.     

Altered composition of liver proteasome assemblies contributes to enhanced proteasome activity in the exceptionally long-lived naked mole-rat

The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life.     

Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries.

In contrast to its constrictor effects on peripheral arteries, 20-hydroxyeicosatetraenoic acid (20-HETE) is an endothelial-dependent dilator of pulmonary arteries (PAs). The present study examined the hypothesis that the vasodilator effects of 20-HETE in PAs are due to an elevation of intracellular calcium concentration ([Ca(2+)](i)) and the release of nitric oxide (NO) from bovine PA endothelial cells (BPAECs). BPAECs express cytochrome P-450 4A (CYP4A) protein and produce 20-HETE. 20-HETE dilated PAs preconstricted with U-46619 or norepinephrine and treated with the cytochrome P-450 inhibitor 17-octadecynoic acid and the cyclooxygenase inhibitor indomethacin. The dilator effect of 20-HETE was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) or by removal of endothelium. 20-HETE significantly increased [Ca(2+)](i) and NO production in BPAECs. 20-HETE-induced NO release was blunted by removal of extracellular calcium, as well as NO synthase inhibitors (L-NAME). These results suggest that 20-HETE dilates PAs at least in part by increasing [Ca(2+)](i) and NO release in BPAECs.     

Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.     

Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries.

The intracellular mechanisms underlying enhanced myogenic contraction (MC) in coronary resistance arteries (CRAs) from exercise-trained (EX) pigs have not been established. The purpose of this study was to test the hypothesis that exercise-induced alterations in protein kinase C (PKC) signaling underlie enhanced MC. Furthermore, we sought to determine whether modulation of intracellular Ca(2+) signaling by PKC underlies enhanced MC in EX animals. Male Yucatan miniature swine were treadmill trained (n = 7) at approximately 75% of maximal O(2) uptake for 16 wk (6 miles/h, 60 min) or remained sedentary (SED, n = 6). Diameter measurements in response to intraluminal pressure (60, 75, and 90 cmH(2)O) or 60 mM KCl were determined in single, cannulated CRAs ( approximately 100 microm ID) with and without the PKC inhibitor chelerythrine (CE, 1 microM). Confocal imaging of Ca(2+) signaling [myogenic Ca(2+) (Ca(m))] was also performed in CRAs of similar internal diameter after abluminal loading of the Ca(2+) indicator dye fluo 4 (1 microM, 37 degrees C, 30 min). We observed significantly greater MC in CRAs isolated from EX than from SED animals at 90 cmH(2)O, as well as greater reductions in MC after CE at all pressures studied. At intraluminal pressures of 75 and 90 cmH(2)O, CE produced greater decreases in Ca(m) in CRAs from EX than from SED animals (64% vs. 25%, P < 0.05). Inhibition of KCl constriction and Ca(m) by CE was also greater in EX animals (P < 0.05). Western blotting revealed significant increases in Ca(2+)-dependent PKC-alpha ( approximately 50%) but not Ca(2+)-independent PKC-epsilon levels in CRAs isolated from EX animals (P < 0.05). We also observed significant group differences in phosphorylated PKC-alpha levels. Finally, voltage-gated Ca(2+) current (VGCC) was effectively blocked by CE, bisindolylmaleimide, and staurosporine in isolated smooth muscle cells from CRAs, providing evidence for a mechanistic link between VGCCs and PKC in our experimental paradigm. These results suggest that enhanced MC in CRAs from EX animals involves PKC-dependent modulation of intracellular Ca(2+), including regulation of VGCCs.     

Mast cell contributes to cardiomyocyte apoptosis after coronary microembolization.

Coronary microembolization (CME) is associated with progressive myocardial dysfunction despite restoration of coronary flow reserve (CFR). The potential pathophysiological role of mast cells (MCs) remains unclear. Therefore, we induced CME in 18 miniswines and determined whether MC accumulation occurs and their effects on local cytokine secretion [interleukin (IL)-6, IL-8, tumor necrosis factor-alpha (TNF-alpha)]; cardiomyocyte apoptosis; and collagen formation at day 1 (D1), day 7 (D7), and day 30 (D30) after CME. Four sham-operated animals without CME (controls) and six animals treated with a MC stabilization agent (tranilast) for 30 days after CME were also studied. CFR decreased at D1 but returned to baseline level at D7 and D30. Coronary sinus levels of IL-6, IL-8, and TNF-alpha increased significantly at D1 and D7 (p<0.01 vs baseline). Levels of IL-6 and IL-8 at D30 returned to baseline level, but not those of TNF-alpha. The numbers of total and degranulating MCs, % apoptotic cardiomyocytes, and collagen volume fraction (CVF) over CME myocardium at D1, D7, and D30 were significantly higher than controls (p<0.01). Treatment with tranilast significantly reduced the serum level of TNF-alpha, numbers of total and degranulating MCs, % apoptotic cardiomyocytes, and CVF at D30 (all p<0.05). There was a significant positive correlation between the numbers of MCs with % apoptotic cardiomyocytes (r = 0.77, p<0.001) and CVF (r = 0.75, p<0.001) over the CME myocardium. Despite restoration of CFR, cardiomyocyte apoptosis persisted after CME and was positively correlated with the number of MCs but was prevented with tranilast treatment. These findings suggest that MCs contribute to cardiomyocyte apoptosis after CME.     

Endogenous prostaglandin release contributes directly to coronary artery tone.

An in vitro coronary artery preparation of beef heart was found to synthesize and release continuously large amounts of a prostaglandin of the E type. Inhibition of prostaglandin synthesis with aspirin, indomethacin, or eicosa-5,8,11,14-tetraynoic acid induced a sustained contraction of the coronary artery, and pretreatment with indomethacin diminished markedly the output of prostaglandin into the bathing medium. It appears that prostaglandin E1, generated from within the vessel wall itself, may act as an intrinsic regulator of coronary artery tone in the beef heart, and that blockade of this function leads to vasospasm.     

Development of enhanced sensitivity to naloxone

The effects of naloxone were examined over a period of three and a half years in squirrel monkeys responding under a mult FR, FI schedule of food presentation. During the initial observation of naloxone's effects, monkeys were drug naive. At that time, doses of naloxone up to 3.0 mg/kg had very little effect on rates of responding under the multiple schedule. The effects of naloxone were then examined in combination with meperidine. Doses of naloxone between 0.3 and 3.0 mg/kg produced a dose-dependent antagonism of meperidine's effects. Monkeys were then exposed to ketocyclazocine and phencyclidine, alone and in combination with naloxone. When the naloxone dose-effect curve was redetermined subsequently, it had shifted to the left. Monkeys were then exposed to buprenorphine and diprenorphine, alone and in combination with naloxone. Redetermination of the naloxone dose-effect curve following this exposure revealed a further shift to the left. The effects of naloxone were then reexamined in combination with meperidine, and it was found that the leftward shift in the naloxone dose-effect curve was not accompanied by a decrease in the doses of naloxone which would reverse the effects of meperidine. Naloxone's effects were then examined in combination with either acute or chronic diazepam. The naloxone dose-effect curve determined during the chronic diazepam regimen was shifted to the right of that obtained prior to chronic diazepam.     

The role of the sodium-calcium exchanger for calcium extrusion in coronary arteries

Calcium ionophores, such as the A23187, cause endothelium-dependent relaxation of arterial strips with intact endothelium, whereas the effect of the ionophore should result from the combination of a relaxation caused by the endothelium-dependent factors and of a contraction of the smooth muscles. In addition, the application of a calcium ionophore to a strip of pig coronary arteries without endothelium does not change cytosolic free calcium concentration and force developed by the smooth muscle cells. To explain these paradoxes, the hypothesis that active calcium extrusion would match the entry of extracellular calcium caused by the ionophore was tested. We see that the sodium-calcium exchanger extrudes calcium that enters the smooth muscle cells in the absence of the ionophore. This exchanger is efficient enough to expel the increased influx of calcium created by the additional calcium carriers formed by the ionophore. This explains the inefficiency of calcium ionophores to increase cytosolic free calcium of smooth muscle cells and consequently, the fact that the ionophore does not cause a contraction of a strip without endothelium. This makes evident that a calcium ionophore fully relaxes, in an endothelium-dependent manner. an intact strip of porcine coronary artery.     

Enhancement of myofilament calcium sensitivity by acute hypoxia in rat distal pulmonary arteries

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 μM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 μM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.     

Enhanced sensitivity to calcium in Duchenne muscular dystrophy.

The ionophore A23187 causes an increase in the Ca content of human erythrocytes and a Ca-dependent increase in K efflux (Gardos effect). These changes are associated with a reduction in osmotic fragility and cell size. Treatment of erythrocytes from patients with Duchenne muscular dystrophy with A23187 results in ⁴⁵Ca uptake comparable to that of erythrocytes from control subjects. However, the reduction in osmotic fragility and K content observed in dystrophic erythrocytes is twofold greater than in control erythrocytes. These results indicate that an alteration in the regulation of erythrocyte membrane function by Ca occurs in Duchenne muscular dystrophy. This alteration may be responsible for other changes in erythrocyte membrane properties observed in Duchenne muscular dystrophy.     

Altered contractile sensitivity of isolated bronchial artery to phenylephrine in ovalbumin-sensitized rabbits.

We tested the hypothesis that atopy and/or allergic lung inflammation enhances alpha1-adrenoceptor-mediated contractions of the bronchial artery. Bronchial arterial resistance vessels were isolated from rabbits that had undergone either systemic ovalbumin (OVA) sensitization followed by saline aerosol challenge (OVA/saline rabbits), or OVA sensitization followed by OVA aerosol challenge (OVA/OVA rabbits), or no sensitization followed by saline aerosol challenge (control rabbits). In OVA/OVA rabbits, bronchoalveolar lavage and lung histology revealed lymphocytic and eosinophilic inflammation. Arterial rings were contracted with phenylephrine (PE). In endothelium-intact arteries isolated from OVA/saline and OVA/OVA rabbits, PE responsiveness was enhanced compared with that of arteries isolated from controls. The nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester increased the contractile response to PE in all three experimental groups to a similar degree, suggesting that depressed NOS activity was not involved in the enhanced PE responsiveness in OVA/saline and OVA/OVA rabbits. After endothelium removal, arteries from OVA/saline and control rabbits showed similar PE responsiveness, indicating that the enhancement of PE responsiveness was endothelium dependent, possibly due to an endothelial constricting factor. In OVA/OVA rabbits, endothelium-denuded arteries showed decreased PE responsiveness compared with the other two groups; this difference was abolished by NG-nitro-L-arginine methyl ester. We conclude that systemic sensitization with OVA per se enhances PE-induced contractions of isolated bronchial arteries in rabbits by an endothelium-dependent mechanism and that allergic lung inflammation attenuates this effect by increased nonendothelial NOS activity.     

Endothelin-1 sensitivity of porcine coronary arteries is reduced by exercise training and is gender dependent.

We tested the hypothesis that exercise training reduces the sensitivity of coronary smooth muscle to endothelin-1 (ET-1), with the adaptation being greater in male than in female miniature swine. The efficacy of training was similar in males and females. Cumulative ET-1 contractile responses of coronary branches and left circumflex artery were significantly shifted to the right in exercise-trained (Ex) males but not in Ex females. Analyses of the excitatory concentration causing a 50% response (EC(50)) showed a 1.7- to 2.2-fold shift in Ex males with no change in maximum tension. Nonselective blockade of K-channel activity with tetraethylammonium (TEA; 30-50 mM) significantly shifted the EC(50) to a lower concentration in both Ex males (1.25-fold) and Ex females (2.2-fold) but not in sedentary (Sed) groups. Females (combined Sed and Ex) exhibited a greater response to TEA than did combined Sed and Ex males. Changes in [(32)P]phosphatidic acid ([(32)P]PA) provided an indicator of ET-1-induced phospholipase activity. The magnitude of the [(32)P]PA response was reduced by Ex in both males and females without affecting the EC(50). It is concluded that the contractile sensitivity of coronary arteries to ET-1 is influenced by physical activity in a gender-dependent manner. It is unclear why the contractile sensitivity in females was not reduced by Ex as in the males, because Ex significantly affected responses to TEA and ET-1 stimulation of [(32)P]PA production in both males and females. A potential gender difference in K-channel function may contribute to this discrepancy.     

Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K(+) (K(max)) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca(2+) concentration at stretch ratios >2.0 in adult arteries, but increased Ca(2+) concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %K(max) values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 +/- 2.3%). Conversely, adult %K(max) values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 +/- 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.     

Prostacyclin increases cAMP in coronary arteries.

This study was designed to determine whether prostacyclin (PGI2)-induced relaxation in circular strips of coronary arteries might be mediated by cAMP. Partially depolarized circular strips of bovine coronary arteries were used and PGI2-induced changes in length were compared with tissue concentrations of cAMP and cGMP, measured by RIA. It was found that PGI2 produced significant and concentration dependent increases in cAMP levels which were closely associated with the relaxant effects produced by the same concentrations (0.3 - 26.7 muM). Cyclic GMP was not changed by these concentrations. The relaxant effects of PGI2 were not antagonized by propranolol. There was a significant linear correlation between log increases in cAMP and percent relaxation produced by PGI2 which was almost identical with similar correlations obtained with either isoprenaline or adenosine, indicating that the relaxant effects of PGI2 are in analogy to those of isoprenaline and adenosine likely to be mediated by cAMP.     

Improvements to enhanced horseradish peroxidase detection sensitivity

At very low horseradish peroxidase (HRP) concentrations, the enhanced chemiluminescence reaction is often characterized by a lag time between initiation of the reaction and beginning of light output. In this study, four treatments of luminol solution were examined in an effort to remove the lag time and to improve chemiluminescence light output. Addition of ammonium persulphate stimulated light output more than tenfold. Ultraviolet irradiation and photoactive dye pretreatment of luminol solution both increased light output fourfold. Luminol purity was the most important factor affecting detection sensitivity. Recrystallization of luminol from base improved the detection limit 13-fold although there was an improvement in the detection limit from 13 attomoles per millilitre to 5 attomoles per millilitre with highly purified luminol when photoactive dye pretreatment was utilized. The results are consistent with a simple interference mechanism whereby enhancer radicals produced by the enzyme are preferentially quenched by contaminants present in the luminol, in the enhancer and in the solvent used to dissolve the enhancer. Consumption of these interferences prior to light emission results in a lag time and a less favourable HRP detection limit.