Profibrotic COVID-19 subphenotype exhibits enhanced localized ER-dependent HSP47+ expression in cardiac myofibroblasts in situ.

We recently described a subgroup of autopsied COVID-19 subjects (∼40%), termed 'profibrotic phenotype,' who exhibited clusters of myofibroblasts (Mfbs), which were positive for the collagen-specific chaperone heat shock protein 47 (HSP47+) in situ. This report identifies increased, localized (hot spot restricted) expression of αSMA, COLα1, POSTN and FAP supporting the identity of HSP47+ cells as myofibroblasts and characterizing a profibrotic extracellular matrix (ECM) phenotype. Coupled with increased GRP78 in COVID-19 subjects, these data could reflect induction of the unfolded protein response for mitigation of proteostasis (i.e., protein homeostasis) dysfunction in discrete clusters of cells. ECM shifts in selected COVID-19 subjects occur without significant increases in either global trichrome positive staining or myocardial injury based quantitively on standard H&E scoring. Our findings also suggest distinct mechanism(s) for ECM remodeling in the setting of SARS-CoV-2 infection. The ratio of CD163+/CD68+ cells is increased in hot spots of profibrotic hearts compared with either controls or outside of hot spots in COVID-19 subjects. In sum, matrix remodeling of human COVID-19 hearts in situ is characterized by site-restricted profibrotic mediated (e.g., HSP47+ Mfbs, CD163+ Mφs) modifications in ECM (i.e., COLα1, POSTN, FAP), with a strong correlation between COLα1 and HSP47+cells within hot spots. Given the established associations of viral infection (e.g., human immunodeficiency virus; HIV), myocardial fibrosis and sudden cardiac death, early screening tools (e.g., plasma biomarkers, noninvasive cardiac magnetic resonance imaging) for diagnosis, monitoring and treatment of fibrotic ECM remodeling are warranted for COVID-19 high-risk populations.

Effects of Aging on Cardiac Oxidative Stress and Transcriptional Changes in Pathways of Reactive Oxygen Species Generation and Clearance.

Background Age-related heart diseases are significant contributors to increased morbidity and mortality. Emerging evidence indicates that mitochondria within cardiomyocytes contribute to age-related increased reactive oxygen species (ROS) generation that plays an essential role in aging-associated cardiac diseases. Methods and Results The present study investigated differences between ROS production in cardiomyocytes isolated from adult (6 months) and aged (24 months) Fischer 344 rats, and in cardiac tissue of adult (18-65 years) and elderly (>65 years) patients with preserved cardiac function. Superoxide dismutase inhibitable ferricytochrome c reduction assay (1.32±0.63 versus 0.76±0.31 nMol/mg per minute; P=0.001) superoxide and H2O2 production, measured as dichlorofluorescein diacetate fluorescence (1646±428 versus 699±329, P=0.04), were significantly higher in the aged versus adult cardiomyocytes. Similarity in age-related alteration between rats and humans was identified in mitochondrial-electron transport chain-complex-I-associated increased oxidative-stress by MitoSOX fluorescence (53.66±18.58 versus 22.81±12.60; P=0.03) and in 4-HNE adduct levels (187.54±54.8 versus 47.83±16.7 ng/mg protein, P=0.0063), indicative of increased peroxidation in the elderly. These differences correlated with changes in functional enrichment of genes regulating ROS homeostasis pathways in aged human and rat hearts. Functional merged collective network and pathway enrichment analysis revealed common genes prioritized in human and rat aging-associated networks that underlay enriched functional terms of mitochondrial complex I and common pathways in the aging human and rat heart. Conclusions Aging sensitizes mitochondrial and extramitochondrial mechanisms of ROS buildup within the heart. Network analysis of the transcriptome highlights the critical elements involved with aging-related ROS homeostasis pathways common in rat and human hearts as targets.

Biphasic effect of metformin on human cardiac energetics.

Metformin is the first-line medication for treatment of type 2 diabetes and has been shown to reduce heart damage and death. However, mechanisms by which metformin protects human heart remain debated. The aim of the study was to evaluate the cardioprotective effect of metformin on cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) and mitochondria isolated from human cardiac tissue. At concentrations ≤2.5 mM, metformin significantly increased oxygen consumption rate (OCR) in the hiPSC-CMs by activating adenosine monophosphate activated protein kinase (AMPK)-dependent signaling and enhancing mitochondrial biogenesis. This effect was abrogated by compound C, an inhibitor of AMPK. At concentrations >5 mM, metformin inhibited the cellular OCR and triggered metabolic reprogramming by enhancing glycolysis and glutaminolysis in the cardiomyocytes. In isolated cardiac mitochondria, metformin did not increase the OCR at any concentrations but inhibited the OCR starting at 1 mM through direct inhibition of electron-transport chain complex I. This was associated with reduction of superoxide production and attenuation of Ca2+-induced mitochondrial permeability transition pore (mPTP) opening in the mitochondria. Thus, in human heart, metformin might improve cardioprotection due to its biphasic effect on mitochondria: at low concentrations, it activates mitochondrial biogenesis via AMPK signaling and increases the OCR; at high concentrations, it inhibits the respiration by directly affecting the activity of complex I, reduces oxidative stress and delays mPTP formation. Moreover, metformin at high concentrations causes metabolic reprogramming by enhancing glycolysis and glutaminolysis. These effects can be a beneficial adjunct to patients with impaired endogenous cardioprotective responses.

Noninvasive biomarker-based risk stratification for development of new onset atrial fibrillation after coronary artery bypass surgery.

BACKGROUND: Postoperative atrial fibrillation (PoAF) is a common complication after cardiac surgery. A pre-existing atrial substrate appears to be important in postoperative development of dysrhythmia, but its preoperative estimation is challenging. We tested the hypothesis that a combination of clinical predictors, noninvasive surrogate markers for atrial fibrosis defining abnormal left atrial (LA) mechanics, and biomarkers of collagen turnover is superior to clinical predictors alone in identifying patients at-risk for PoAF. METHODS: In patients without prior AF undergoing coronary artery bypass grafting, concentrations of biomarkers reflecting collagen synthesis and degradation, extracellular matrix, and regulatory microRNA-29s were determined in serum from preoperative blood samples and correlated to atrial fibrosis extent, alteration in atrial deformation properties determined by 3D speckle-tracking echocardiography, and AF development. RESULTS: Of 90 patients without prior AF, 34 who developed PoAF were older than non-PoAF patients (72.04 ± 10.7 y; P = 0.043) with no significant difference in baseline comorbidities, LA size, or ventricular function. Global (P = 0.007) and regional longitudinal LA strain and ejection fraction (P = 0.01) were reduced in PoAF vs. non-PoAF patients. Preoperative amino-terminal-procollagen-III-peptide (PIIINP) (103.1 ± 39.7 vs. 35.1 ± 19.3; P = 0.041) and carboxy-terminal-procollagen-I-peptide levels were elevated in PoAF vs. non-PoAF patients with a reduction in miR-29 levels and correlated with atrial fibrosis extent. Combining age as the only significant clinical predictor with PIIINP and miR-29a provided a model that identified PoAF patients with higher predictive accuracy. CONCLUSIONS: In patients without a previous history of AF, using age and biomarkers of collagen synthesis and regulation, a noninvasive tool was developed to identify those at risk for new-onset PoAF.

Impact of statins on cellular respiration and de-differentiation of myofibroblasts in human failing hearts.

AIMS: Fibroblast to myofibroblast trans-differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de-differentiation could mitigate CF-related pathologies. We determined whether 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors-statins, commonly prescribed to patients at risk of heart failure (HF)-can de-differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients. METHODS AND RESULTS: Either in vitro statin treatment of differentiated myofibroblasts (n = 3-6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4-12). In vitro, hVFs were differentiated by transforming growth factor-ß1 (TGF-ß1) for 72 h (TGF-72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by α-smooth muscle actin (α-SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P < 0.05, considered significant. In vitro, statins concentration-dependently de-differentiated the myofibroblasts. The respective half-maximal effective concentrations were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 µmol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin). Mevalonic acid (300 µmol/L), the reduced product of HMG-CoA, prevented the statin-induced de-differentiation (α-SMA expression: 31.4 ± 10% vs. 58.6 ± 12%). Geranylgeranyl pyrophosphate (GGPP, 20 µmol/L), a cholesterol synthesis-independent HMG-CoA reductase pathway intermediate, completely prevented the statin-induced de-differentiation (α-SMA/GAPDH ratios: 0.89 ± 0.05 [TGF-72 h + 72 h], 0.63 ± 0.02 [TGF-72 h + simvastatin], and 1.2 ± 0.08 [TGF-72 h + simvastatin + GGPP]). Cellular metabolism involvement was observed when co-incubation of simvastatin (200 nmol/L) with glibenclamide (10 µmol/L), a KATP channel inhibitor, attenuated the simvastatin-induced de-differentiation (0.84 ± 0.05). Direct inhibition of mitochondrial respiration by oligomycin (1 ng/mL) also produced a de-differentiation effect (0.33 ± 0.02). OCR (pmol O2 /min/µg protein) was significantly decreased in the simvastatin-treated hVFs, including basal (P = 0.002), ATP-linked (P = 0.01), proton leak-linked (P = 0.01), and maximal (P < 0.001). The OCR inhibition was prevented by GGPP (basal OCR [P = 0.02], spare capacity OCR [P = 0.008], and maximal OCR [P = 0.003]). Congruently, hVFs from HF showed an increased population of myofibroblasts while HF + statin group showed significantly reduced cellular respiration (basal OCR [P = 0.021], ATP-linked OCR [P = 0.047], maximal OCR [P = 0.02], and spare capacity OCR [P = 0.025]) and myofibroblast differentiation (α-SMA/GAPDH: 1 ± 0.19 vs. 0.23 ± 0.06, P = 0.01). CONCLUSIONS: This study demonstrates the de-differentiating effect of statins, the underlying GGPP sensitivity, reduced OCR with potential activation of KATP channels, and their impact on the differentiation magnitude of hVFs in HF patients. This novel pleiotropic effect of statins may be exploited to reduce excessive CF in patients at risk of HF.

Prolonged post-differentiation culture influences the expression and biophysics of Na+ and Ca2+ channels in induced pluripotent stem cell-derived ventricular-like cardiomyocytes.

Several studies have been reported in various domains from induction methods to utilities of somatic cell pluripotent reprogramming. However, one of the major struggles facing the research field of induced pluripotent stem cell (iPSC)-derived target cells is the lack of consistency in observations. This could be due to variety of reasons including varied culture periods post-differentiation. The cardiomyocytes (CMs) derived from iPSCs are commonly studied and proposed to be utilized in the comprehensive in vitro proarrhythmia initiative for drug safety screening. As the influence of varied culture periods on the electrophysiological properties of iPSC-CMs is not clearly known, using whole-cell patch clamp technique, we compared two groups of differentiated ventricular-like iPSC-CMs that are cultured for 10 to 15 days (D10-15) and more than 30 days (≥ D30) both under current and voltage clamps. The prolonged culture imparts increased excitability with high-frequency spontaneous action potentials, robust increase in the magnitude of peak Na+ current density, relatively shallow inactivation kinetics of Na+ channels, faster recovery from inactivation, and augmented Ca2+ current density. Quantitative real-time PCR studies of α-subunit transcripts showed enhanced mRNA expression of SCN1A, SCN5A Na+ channel subtypes, and CACNA1C, CACNA1G, and CACNA1I Ca2+ channel subtypes, in ≥ D30 group. Conclusively, the prolonged culture of differentiated iPSC-CMs affects the excitability, single-cell electrophysiological properties, and ion channel expressions. Therefore, following standard periods of culture across research studies while utilizing ventricular-like iPSC-CMs for in vitro health/disease modeling to study cellular functional mechanisms or test high-throughput drugs' efficacy and toxicity becomes crucial.

Aging-related increase in store-operated Ca2+ influx in human ventricular fibroblasts.

Senescence-related fibrosis contributes to cardiac dysfunction. Profibrotic processes are Ca2+ dependent. The effect of aging on the Ca2+ mobilization processes of human ventricular fibroblasts (hVFs) is unclear. Therefore, we tested whether aging altered intracellular Ca2+ release and store-operated Ca2+ entry (SOCE). Disease-free hVFs from 2- to 63-yr-old trauma victims were assessed for cytosolic Ca2+ dynamics with fluo 3/confocal imaging. Angiotensin II or thapsigargin was used to release endoplasmic reticulum Ca2+ in Ca2+-free solution; CaCl2 (2 mM) was then added to assess SOCE, which was normalized to ionomycin-induced maximal Ca2+. The angiotensin II experiments were repeated after phosphoenolpyruvate pretreatment to determine the role of energy status. The expression of genes encoding SOCE-related ion channel subunits was assessed by quantitative PCR, and protein expression was assessed by immunoblot analysis. Age groups of <50 and ≥50 yr were compared using unpaired t-test or regression analysis. Ca2+ release by angiotensin II or thapsigargin was not different between the groups, but SOCE was significantly elevated in the ≥50-yr group. Regression analysis showed an age-dependent phosphoenolpyruvate-sensitive increase in SOCE of hVFs. Aging did not alter the mRNA expression of SOCE-related genes. The profibrotic phenotype of hVFs was evident by sprouty1 downregulation with age. Thus, an age-associated increase in angiotensin II- and thapsigargin-induced SOCE occurs in hVFs, independent of receptor mechanisms or alterations of mRNA expression level of SOCE-related ion channel subunits but related to the cellular bioenergetics status. Elucidation of mechanisms underlying enhanced hVF SOCE with aging may refine SOCE targets to limit aging-related progression of Ca2+-dependent cardiac fibrosis. NEW & NOTEWORTHY Human ventricular fibroblasts exhibit an age-related increase in store-operated Ca2+ influx induced by angiotensin II, an endogenous vasoactive hormone, or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, independent of receptor mechanisms or genes encoding store-operated Ca2+ entry-related ion channel subunits. Selective inhibition of this augmented store-operated Ca2+ entry could therapeutically limit aging-related cardiac fibrosis.

Enhanced store-operated Ca2+ influx and ORAI1 expression in ventricular fibroblasts from human failing heart.

Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/106 cells vs 58.5±8.8 µg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective ICRAC inhibitor, for 48 h (66.78±5.87 µg/106 cells vs 55.81±7.09 µg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.

Sepiapterin ameliorates chemically induced murine colitis and azoxymethane-induced colon cancer.

The effects of modulating tetrahydrobiopterin (BH4) levels with a metabolic precursor, sepiapterin (SP), on dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)-induced colorectal cancer were studied. SP in the drinking water blocks DSS-induced colitis measured as decreased disease activity index (DAI), morphologic criteria, and recovery of Ca(2+)-induced contractility responses lost as a consequence of DSS treatment. SP reduces inflammatory responses measured as the decreased number of infiltrating inflammatory macrophages and neutrophils and decreased expression of proinflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and IL-17A. High-performance liquid chromatography analyses of colonic BH4 and its oxidized derivative 7,8-dihydrobiopterin (BH2) are inconclusive although there was a trend for lower BH4:BH2 with DSS treatment that was reversed with SP. Reduction of colonic cGMP levels by DSS was reversed with SP by a mechanism sensitive to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). ODQ abrogates the protective effects of SP on colitis. This plus the finding that SP reduces DSS-enhanced protein Tyr nitration are consistent with DSS-induced uncoupling of NOS. The results agree with previous studies that demonstrated inactivation of sGC in DSS-treated animals as being important in recruitment of inflammatory cells and in altered cholinergic signaling and colon motility. SP also reduces the number of colon tumors in AOM/DSS-treated mice from 7 to 1 per unit colon length. Thus, pharmacologic modulation of BH4 with currently available drugs may provide a mechanism for alleviating some forms of colitis and potentially minimizing the potential for colorectal cancer in patients with colitis.

Evidence for the putative cannabinoid receptor (GPR55)-mediated inhibitory effects on intestinal contractility in mice.

BACKGROUND: Cannabinoids inhibit intestinal motility via presynaptic cannabinoid receptor type I (CB1) in enteric neurons while cannabinoid receptor type II (CB2) receptors are located mainly in immune cells. The recently de-orphanized G-protein-coupled receptor, GPR55, has been proposed to be the 'third' cannabinoid receptor. Although gene expression of GPR55 is evident in the gut, functional evidence for GPR55 in the gut is unknown. In this study, we tested the hypothesis that GPR55 activation inhibits neurogenic contractions in the gut. METHODS: We assessed the inhibitory effect of the atypical cannabinoid O-1602, a GPR55 agonist, in mouse colon. Isometric tension recordings in colonic tissue strips were used from either wild-type, GPR55(-/-) or CB1(-/-)/CB2(-/-) knockout mice. RESULTS: O-1602 inhibited the electrical field- induced contractions in the colon strips from wild-type and CB1(-/-)/CB2(-/-) in a concentration-dependent manner, suggesting a non-CB1/CB2 receptor-mediated prejunctional effect. The concentration-dependent response of O-1602 was significantly inhibited in GPR55(-/-) mice. O-1602 did not relax colonic strips precontracted with high K(+) (80 mmol/l), indicating no involvement of Ca(2+) channel blockade in O-1602-induced relaxation. However, 10 µmol/l O-1602 partially inhibited the exogenous acetylcholine (10 µmol/l)-induced contractions. Moreover, we also assessed the inhibitory effects of JWH015, a CB2/GPR55 agonist on neurogenic contractions of mouse ileum. Surprisingly, the effects of JWH015 were independent of the known cannabinoid receptors. CONCLUSION: Taken together, these findings suggest that activation of GPR55 leads to inhibition of neurogenic contractions in the gut and are predominantly prejunctional.

Opioid-induced hypernociception is associated with hyperexcitability and altered tetrodotoxin-resistant Na+ channel function of dorsal root ganglia.

Opiates are potent analgesics for moderate to severe pain. Paradoxically, patients under chronic opiates have reported hypernociception, the mechanisms of which are unknown. Using standard patch-clamp technique, we examined the excitability, biophysical properties of tetrodotoxin-resistant (TTX-R) Na(+) and transient receptor potential vanilloid 1 (TRPV1) channels of dorsal root ganglia neurons (DRG) (L(5)-S(1)) from mice pelleted with morphine (75 mg) or placebo (7 days). Hypernociception was confirmed by acetic acid-writhing test following 7-day morphine. Chronic morphine enhanced the neuronal excitability, since the rheobase for action potential (AP) firing was significantly (P < 0.01) lower (38 ± 7 vs. 100 ± 15 pA) while the number of APs at 2× rheobase was higher (4.4 ± 0.8 vs. 2 ± 0.5) than placebo (n = 13-20). The potential of half-maximum activation (V(1/2)) of TTX-R Na(+) currents was shifted to more hyperpolarized potential in the chronic morphine group (-37 ± 1 mV) vs. placebo (-28 ± 1 mV) without altering the V(1/2) of inactivation (-41 ± 1 vs. -33 ± 1 mV) (n = 8-11). Recovery rate from inactivation of TTX-R Na(+) channels or the mRNA level of any Na(+) channel subtypes did not change after chronic morphine. Also, chronic morphine significantly (P < 0.05) enhanced the magnitude of TRPV1 currents (-64 ± 11 pA/pF) vs. placebo (-18 ± 6 pA/pF). The increased excitability of sensory neurons by chronic morphine may be due to the shift in the voltage threshold of activation of TTX-R Na(+) currents. Enhanced TRPV1 currents may have a complementary effect, with TTX-R Na(+) currents on opiate-induced hyperexcitability of sensory neurons causing hypernociception. In conclusion, chronic morphine-induced hypernociception is associated with hyperexcitability and functional remodeling of TTX-R Na(+) and TRPV1 channels of sensory neurons.

The role of ß-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract.

ß-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of ß-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 µM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from ß-arrestin2 knockout mice. ß-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of ß-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 µM) exposure. Naloxone prevented the decrease in ß-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in ß-arrestin2 occurred. We conclude that a decrease in ß-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Blockade of endocannabinoid hydrolytic enzymes attenuates precipitated opioid withdrawal symptoms in mice.

Δ(9)-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB(1) and CB(2) cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB(1) receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-carboxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB(1) receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.

Adrenomedullin relaxes rat uterine artery: mechanisms and influence of pregnancy and estradiol.

Uterine arteries play a major role in regulating uteroplacental blood flow. Failure to maintain blood flow to the uteroplacental compartment during pregnancy often results in intrauterine growth retardation. Immunohistochemical staining of adrenomedullin (AM), an endogenous vasoactive peptide, in uterine artery was intense in pregnant compared to nonpregnant rats, but it is not known whether AM directly relaxes uterine artery or not. In this study, we elucidated the mechanisms of uterine artery relaxation by AM and its regulation by pregnancy and female sex steroids. AM was able to relax uterine artery, and this relaxation was influenced positively by pregnancy and estradiol as evidenced by the increased pD(2) and E(max) values of AM. Both pregnancy and estradiol treatment to ovariectomized rats amplified RAMP(3) expression in uterine arteries while progesterone had no effect. AM-induced uterine artery relaxation is predominantly endothelium-dependent. The AM receptor antagonist CGRP(8-37) is more potent than AM(22-52) in inhibiting the AM relaxation, indicating the involvement of AM(2) receptor subtype. Moreover, AM uses the classical nitric oxide-cyclic guanosine monophosphate pathway along with K(Ca) channels to mediate the vasodilatory effect in uterine artery. In conclusion, sensitivity of uterine artery to AM-induced relaxation is increased with pregnancy or estradiol treatment by increasing RAMP(3) expression, suggesting an important role for AM in regulating the uterine hemodynamics, probably maintaining uterine blood flow during pregnancy and in pre- and postmenopausal cardiovascular adaptation differences.

Colonic inflammation alters Src kinase-dependent gating properties of single Ca2+ channels via tyrosine nitration.

Nitration of L-type calcium channels during colonic inflammation impairs phosphorylation by the tyrosine kinase, Src kinase. This results in decreased calcium currents. The purpose of this study was to determine the mechanism of the downregulation of Ca2+ currents in colonic inflammation. In whole cell voltage clamp of mouse single smooth muscle cells, long-duration depolarization produced noninactivating calcium currents that were significantly reduced by the Src kinase inhibitor, protein phosphatase 2 (PP2). Unitary Ba2+ currents were recorded upon repolarization from positive potentials in cell-attached patches of smooth muscle and hCa(v)1.2b-transfected cells to assess the properties of the single channels attributed to the noninactivating open state. Repolarization to -40 mV from 0 mV resulted in single-channel events with conductance of approximately 23 pS. The ensemble average of the tail currents from 1,000 sweeps was 337 +/- 27 fA in control and 218 +/- 49 fA (P < 0.05) in inflamed cells. Neither open-probability nor open-time constants were significantly different between control and inflamed cells. However, the transition to the open state measured as channel availability was significantly reduced from 19 +/- 3% to 6.4 +/- 1%. Similarly, peak ensemble average current and channel availability were significantly reduced by PP2 and treatment with peroxynitrite in control cells. Mutation of COOH-terminal tyrosine residues in hCa(v)1.2b Chinese hamster ovarian cells also decreased peak ensemble average tail currents and availability. The present findings suggest that the transition of Ca2+ channels to the noninactivating open state is Src kinase dependent. Tyrosine nitration prevents Src-mediated transitions, leading to decreased calcium currents.

Morphine tolerance in the mouse ileum and colon.

Repeated administration of morphine is associated with tolerance to its antinociceptive properties. However, constipation remains the major side effect of chronic exposure to morphine. In contrast, previous studies suggest that tolerance to opioids develops in the ileum of several species. In this study, we provide evidence that constipation may arise due to a lack of tolerance development to morphine in the colon. Mice received implants with either placebo or 75 mg of morphine pellets, and they were examined for morphine tolerance to antinociception, defecation, and intestinal and colonic transit after 72 h. Tissues were obtained from the ileum and distal colon, and contractile responses were measured from longitudinal and circular muscle preparations. In morphine-pelleted mice, a 5.5-fold tolerance developed to antinociception after 72 h, and a 53.2-fold tolerance developed in mice that received an additional daily morphine injection. In both models, intestinal transit but not defecation or colonic transit developed tolerance. In isolated longitudinal muscles, electrical field stimulation-induced cholinergic contractions were dose-dependently inhibited by morphine in both the ileum and colon of placebo pelleted with a pD(2) of 7.1 +/- 0.4 and 7.8 +/- 0.4, respectively. However, the dose response to morphine inhibition was shifted to the right for the ileum from morphine-pelleted mice (pD(2) = 5.1 +/- 0.4) but not the colon (pD(2) = 6.9 +/- 0.4). In circular muscle preparations, morphine induced atropine-insensitive contractions in both tissue segments. Tolerance to morphine developed in the ileum but not the colon upon repeated administration of morphine. These findings indicate that a lack of tolerance development in the colon is the basis for opioid bowel dysfunction.

Cyclic AMP-independent CGRP8-37-sensitive receptors mediate adrenomedullin-induced decrease of CaCl2-contraction in pregnant rat mesenteric artery.

OBJECTIVES: We tested the hypothesis that adrenomedullin reduces calcium influx independent of potassium channels in depolarized endothelium-denuded mesenteric artery from pregnant rats. RESULTS: Adrenomedullin reduced the CaCl(2)-induced contraction, while the receptor antagonist calcitonin gene-related peptide (CGRP)(8-37), but not adrenomedullin(22-52), reversed these effects. Adenylate cyclase inhibition by SQ22536 did not prevent adrenomedullin effects on CaCl(2)-induced contraction. Adrenomedullin did not inhibit depolarization-induced calcium entry to isolated vascular smooth muscle. Inhibition of myosin light-chain (MLC) phosphatase by calyculin A reversed the effects of adrenomedullin on contraction caused by submillimolar concentrations of CaCl(2), while adrenomedullin still inhibited contraction caused by higher concentrations of CaCl(2). However, the ratio of phosphorylated to total myosin phosphatase target 1, the regulatory subunit of MLC phosphatase, did not change with adrenomedullin, indicating a lack of MLC phosphatase activation. Interestingly, sodium fluoride, a nonspecific protein phosphatase inhibitor, completely blocked the effect of adrenomedullin on CaCl(2)-induced contraction. Adrenomedullin inhibited calcium mobilization from intracellular stores induced by thapsigargin. CONCLUSION: Adrenomedullin inhibits CaCl(2)-induced contraction, without affecting calcium influx, through a CGRP(8-37)-sensitive receptor, but not using the cyclic adenosine monophosphate pathway, probably through activation of protein phosphatases. Inhibition of intracellular calcium release is an additional role played by adrenomedullin in calcium homeostasis in vascular smooth muscle.

COOH-terminal association of human smooth muscle calcium channel Ca(v)1.2b with Src kinase protein binding domains: effect of nitrotyrosylation.

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca(v)) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa(v)1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa(v)1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y(2134) prevented SH2 binding and resulted in reduced phosphorylation of hCa(v)1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y(1837), Y(1861), and Y(2134) were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y(1837-2134F). These data demonstrate that the COOH terminus of hCa(v)1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.