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The use of dual-mobility cups has gained popularity in recent years. Thus, surgeons can expect an increase in known and new causes of failure. We report a previously undescribed form of a late intraprosthetic dislocation consisting of a complete breakage of a polyethylene mobile bearing that suffered a dislocation 3 months after its implantation. Two years later, he began feeling anterior groin pain and suffered gait changes. Computed tomography scan revealed an eccentric alignment of the mobile polyethylene bearing suggestive of poly wear. During the revision surgery, the polyethylene was found to be split in 2. Possible causes of this complication are proposed. Our case shows a previously unreported implant-specific complication, so surgeons can identify it.

Recurrent posterior glenohumeral instability is an entity that demands a high clinical suspicion and a detailed study for a correct approach and treatment. Its classification must consider its biomechanics, whether it is due to functional muscular imbalance or to structural changes, volition, and intentionality. Due to its varied clinical presentations and different structural alterations, ranging from capsule-labral lesions and bone defects to glenoid dysplasia and retroversion, the different treatment alternatives available have historically had a high incidence of failure. A detailed radiographic assessment, with both CT and MRI, with a precise assessment of glenoid and humeral bone defects and of glenoid morphology, is mandatory. Physiotherapy focused on periscapular muscle reeducation and external rotator strengthening is always the first line of treatment. When conservative treatment fails, surgical treatment must be guided by the structural lesions present, ranging from soft tissue repair to posterior bone block techniques to restore or increase the articular surface. Bone block procedures are indicated in cases of recurrent posterior instability after the failure of conservative treatment or soft tissue techniques, as well as symptomatic demonstrable nonintentional instability, presence of a posterior glenoid defect >10%, increased glenoid retroversion between 10 and 25°, and posterior rim dysplasia. Bone block fixation techniques that avoid screws and metal allow for satisfactory initial clinical results in a safe and reproducible way. An algorithm for the approach and treatment of recurrent posterior glenohumeral instability is presented, as well as the author’s preferred surgical technique for arthroscopic posterior bone block.

Molecular dynamics (MD) simulations have become a standard tool to correlate the structure and function of biomolecules and significant advances have been made in the study of proteins and their complexes. A major drawback of conventional MD simulations is the difficulty and cost of obtaining converged results, especially when exploring potential energy surfaces containing considerable energy barriers. This limits the wide use of MD calculations to determine the thermodynamic properties of biomolecular processes. Indeed, this is true when considering the conformational entropy of such processes, which is ultimately critical in assessing the simulations' convergence. Alternatively, a wide range of structure-based models (SBMs) has been used in the literature to unravel the basic mechanisms of biomolecular dynamics. These models introduce simplifications that focus on the relevant aspects of the physical process under study. Because of this, SBMs incorporate the need to modify the force field definition and parameters to target specific biophysical simulations. Here we introduce SBMOpenMM, a Python library to build force fields for SBMs, that uses the OpenMM framework to create and run SBM simulations. The code is flexible and user-friendly and profits from the high customizability and performance provided by the OpenMM platform.

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an "in silico" approach. Homology models of the catalytic domain of sGC in "inactive" or "active" conformations were constructed using the structure of previously described crystals of the catalytic domains of "inactive" sGCs (2WZ1, 3ET6) and of "active" adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 μs. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of "inactive" and "active" conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the "active" structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop β2 -β3 , does not overlap with the forskolin binding site on adenylate cyclases. Proteins 2016; 84:1534-1548. © 2016 Wiley Periodicals, Inc.

Aims It has been shown that sarcoplasmic reticulum calcium ATPase (SERCA) plays a critical role in reperfusion injury. Moreover, ischaemic post-conditioning (PoCo) results in protein kinase G (PKG) activation which has been proposed to modulate phospholamban (PLB) and SERCA. We assessed whether PLB phosphorylation contributes to the cardioprotective effects of PoCo. Methods and results Isolated Sprague–Dawley rat hearts were submitted to 40 min of ischaemia and reperfusion with and without a PoCo protocol that reduced infarct size by 48%. Reperfusion caused a rapid phosphorylation in PLB at Ser16 and Thr17 that was delayed by PoCo. NO-independent activation of soluble guanylate cyclase (sGC) (ataciguat) and cAMP-dependent protein kinase (PKA) inhibition (KT5720) mimicked the reduction in Ser16 phosphorylation in reperfused control hearts, while in PoCo hearts the inhibitors of PKG (KT5823) and phosphodiesterase 2 (BAY-60-7550) reverted it. CaMKII activity measured by Thr287 phosphorylation was reduced in PoCo. In reperfused control hearts, inhibition of PLB phosphorylation or SERCA (thapsigargin) simulated the cardioprotective effects of PoCo. Ataciguat reduced cytosolic Ca2+ oscillations and improved Ca2+ recovery in cardiomyocytes subjected to anoxia–reoxygenation and infarct size by 32% in rats with 30 min of the left anterior descending coronary artery occlusion and 2 h of reperfusion. Blockade of Na+/Ca2+-exchanger (NCX; KB-R7943) impaired Ca2+ control in cardiomyocytes and abolished cardioprotection in PoCo hearts. Conclusions PoCo reduces SERCA activity at the onset of reperfusion by delaying PLB phosphorylation through activation of PKG and inhibition of PKA and CaMKII. This effect contributes to PoCo protection by favouring cytosolic Ca2+ extrusion through NCX, and it may be mimicked by pharmacological stimulation of sGC.

Resumen Introduccion y objetivos La distension de la region isquemica se ha relacionado con una mayor incidencia de arritmias ventriculares espontaneas tras la oclusion coronaria. Analizamos si la distension isquemica regional predice una mayor inducibilidad de fibrilacion ventricular tras la oclusion coronaria en cerdos. Metodos En 18 cerdos anestesiados con torax abierto, se ocluyo la descendente anterior durante 60 min. Se monitorizo la longitud segmentaria en la region isquemica mediante cristales ultrasonicos. Se realizo estimulacion programada basal, y despues continuamente entre 10 y 60 min tras la oclusion. Resultados La oclusion coronaria indujo un rapido aumento en la longitud telediastolica en la region isquemica, que alcanzo el 109,4 ± 0,9% de los valores basales a los 10 min (p Conclusiones La distension regional isquemica predice una mayor inducibilidad de fibrilacion ventricular tras la oclusion coronaria. Estos resultados subrayan la influencia potencial de los factores mecanicos, que actuan no solo sobre los desencadenantes, sino tambien sobre el sustrato, en la genesis de las arritmias ventriculares malignas durante la isquemia aguda.

Distension of the ischemic region has been related to an increased incidence of spontaneous ventricular arrhythmias following coronary occlusion. This study analyzed whether regional ischemic distension predicts increased ventricular fibrillation inducibility after coronary occlusion in swine.In 18 anesthetized, open-chest pigs, the left anterior descending coronary artery was ligated for 60 min. Myocardial segment length in the ischemic region was monitored by means of ultrasonic crystals. Programmed stimulation was applied at baseline and then continuously between 10 and 60 min after coronary occlusion.Coronary occlusion induced a rapid increase in end-diastolic length in the ischemic region, which reached 109.4% (0.9%) of baseline values 10 min after occlusion (P.001). On average, 6.6 (0.5) stimulation protocols were completed and 5.4 (0.6) ventricular fibrillation episodes induced between 10 and 60 min of coronary occlusion. Neither baseline serum potassium levels nor the size of the ischemic region were significantly related to ventricular fibrillation inducibility. In contrast, the increase in end-diastolic length 10 min after coronary occlusion was associated directly (r=0.67; P=.002) with the number of induced ventricular fibrillation episodes and inversely (r=-0.55; P=.018) with the number of extrastimuli needed for ventricular fibrillation induction.Regional ischemic expansion predicts increased ventricular fibrillation inducibility following coronary occlusion. These results highlight the potential influence of mechanical factors, acting not only on the triggers but also on the substrate, in the genesis of malignant ventricular arrhythmias during acute ischemia.

Altres ajuts: Programa HIVACAT, programa CERCA (Generalitat de Catalunya), Red Temática Cooperativa de Investigación en SIDA (RD12/0017/0002)), Govern Basc (IT838-13), Spanish Supercomputing Network (grant numbers BCV-2015-2-0009 and BCV-2016-2-0005) The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, holesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

Background The cause of coronary vasoconstriction in patients with angina at rest, nonsignificant coronary stenosis, and endothelial dysfunction remains unknown. Our objective was to investigate the association between enhanced coronary vasoconstriction and increased circulating levels of vasoconstrictor agents. Methods Plasma levels of big endothelin-1, serotonin, and superoxide produced by polymorphonuclear leukocytes were measured in 38 patients with stable angina at rest without significant coronary artery stenosis—23 with nonvasospastic angina and 15 with vasospastic angina—and were compared with 10 patients with stable coronary disease and 20 age-matched controls. Results Patients with angina at rest showed higher big endothelin-1 (1.28 vs 0.72 fmol/mL, P 0.001), serotonin (18.0 vs 9.1 ng/mL, P = 0.002), and superoxide produced by polymorphonuclear leukocytes (177 vs 67 nmol/10 × E8 × minutes, P = 0.001) than did controls. Serotonin and superoxide produced by polymorphonuclear leukocytes were also higher than in coronary disease patients (5.4 ng/mL, P = 0.001, and 97 nmol/10 x E8 x minutes, P = 0.005), and big endothelin-1 levels tended to be higher (0.99 fmol/mL, P = 0.073). Moreover, there were no significant differences in these 3 parameters between patients with vasospastic and nonvasospastic angina, and among the latter, between patients with a positive and those with a negative exercise stress test. Conclusion Systemic plasma levels of agents with the potential to produce coronary vasoconstriction are increased in patients with stable vasospastic or nonvasospastic angina and, hence, may contribute to their angina, increased coronary tone, and impaired vasodilatory capacity. Furthermore, they may establish a mechanistic link between the 2 conditions.

Ischemic postconditioning has been demonstrated to limit infarct size in patients, but its molecular mechanisms remain incompletely understood. Low intracellular pH (pHi) inhibits mitochondrial permeability transition, calpain activation and hypercontracture. Recently, delayed normalization of pHi during reperfusion has been shown to play an important role in postconditioning protection, but its relation with intracellular protective signaling cascades is unknown. The present study investigates the relation between the rate of pHi normalization and the cGMP/PKG pathway in postconditioned myocardium. In isolated Sprague-Dawley rat hearts submitted to transient ischemia both, postconditioning and acidic reperfusion protocols resulted in a similar delay in pHi recovery measured by (31)P-NMR spectroscopy (3.6±0.2min and 3.5±0.2min respectively vs. 1.4±0.2min in control group, P

The beneficial effects of restoration of coronary flow in patients with acute myocardial infarction may be hampered by inadequate tissue perfusion. Among other factors, it is likely that platelets contribute substantially to this phenomenon. Platelets may compromise blood flow at the microvascular level by forming a part of microemboli, by adhering to reperfused, capillary or venular endothelium or to attached leukocytes, by releasing substances producing vasoconstriction, or through toxic effects. Patients with acute coronary syndromes have an increased number of circulating activated platelets, and this systemic platelet activation has been related to the presence and extent of myocardial necrosis. The mechanisms of platelet deposition to reperfused microvessels are not fully understood, but likely involve the interaction between adhesion molecules such as selectins or glycoproteins expressed on these cells upon activation and their ligands on the surface of endothelial cells or polymorphonuclear leukocytes. While these interactions are potentially important therapeutic targets in acute myocardial infarction, reducing platelet deposition and increasing myocardial salvage by direct effects on the microvasculature is still challenging with the existing armamentarium of antiplatelet agents. This review summarizes the current knowledge on the mechanisms of platelet-mediated myocardial damage after reperfusion and the effects of pharmacological interventions aimed to reduce microvascular platelet deposition and platelet-mediated myocardial injury.

SummaryReperfusion injury may cause myocardial cell death and limit the benefit achieved by restoration of coronary artery patency in patients with acute myocardial infarction. The mechanism includes altered Ca2+ handling with cytosolic and mitochondrial Ca2+ overload, Ca2+- and ATP-dependent hypercontraction, cytoskeletal fragility, mitochondrial permeability transition and gap junction-mediated propagation of cell death, as well as alterations in non-cardiomyocyte cells, in particular platelets and endothelial cells. cGMP modulates favorably all these mechanism, mainly through PKG-mediated actions, but cGMP synthesis is altered in reperfused cardiomyocytes and endothelial cells by mechanisms that are only partially understood. Stimulation of cGMP synthesis during initial reperfusion by means of natriuretic peptides has been found protective in different animal models and in patients. Moreover, increasing evidence indicates that cGMP is an important step in signal transduction of endogenous cardioprotection. Thus, the cGMP pathway appears as a key element in the pathophysiology of myocardial ischaemiareperfusion and as a promising therapeutic target in patients with acute myocardial infarction.

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37°C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an "in silico" approach. Homology models of the catalytic domain of sGC in "inactive" or "active" conformations were constructed using the structure of previously described crystals of the catalytic domains of "inactive" sGCs (2WZ1, 3ET6) and of "active" adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 μs. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of "inactive" and "active" conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the "active" structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop β2 -β3 , does not overlap with the forskolin binding site on adenylate cyclases. Proteins 2016; 84:1534-1548. © 2016 Wiley Periodicals, Inc.

The tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) gene encodes the TNF-R1, one of the main TNF receptors that mediates its inflammatory actions. In a recent study, serum levels of the soluble TNF-R1 and mRNA levels of the full-length receptor were found to be significantly increased in multiple sclerosis (MS) patients carrying the R92Q mutation. Interestingly, R92Q-mutated patients were younger at disease onset and progressed slower as compared to non-carriers. Building on these previous findings, here we aimed to investigate by means of both in silico and in vitro approaches the mechanisms relating the R92Q substitution with functional changes of the receptor and their potential effects modulating MS disease course. Models of the extracellular domains of the human TNF-R1 and human TNF-R1 carrying the R92Q mutation, alone or bound to TNF, were constructed and submitted to molecular dynamics. TRAF2 and CASP3 mRNA expression levels were determined by real-time PCR in peripheral blood mononuclear cells (PBMC) from 61 MS patients, 9 R92Q carriers and 52 non-carriers (CT and CC genotypes for SNP rs4149584, respectively). Molecular dynamic studies revealed that the R92Q mutation increased the contact area between receptor and TNF (1070 and 1388A(2) for native and mutated receptor) and decreased the distance between them (28.7 to 27.9A), while Van der Waals and electrostatic interaction energies were increased. In PBMC from MS patients carrying the R92Q mutation, CASP3 mRNA expression levels were significantly increased compared to non-carriers, whereas a trend was observed for TRAF2. These data suggest that the R92Q mutation gives rise to a stronger interaction between the receptor and its ligand, which results in the potentiation of TNF-mediated pathways. Although further studies are needed, these functional changes may be related with the modulation in disease course reported in MS patients carrying the R92Q mutation.

Objectives: It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect. Methods: Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia-5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed. Results: IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P

Portal hypertension induces neuroendocrine activation and a hyperkinetic circulation state. This study investigated the consequences of portal hypertension on heart structure and function. Intrahepatic portal hypertension was induced in male Sprague-Dawley rats by chronic bile duct ligation (CBDL). Six weeks later, CBDL rats showed higher plasma angiotensin-II and endothelin-1 (P

Objective: Inhibition of Na+–H+ exchange (NHE) delays the onset of myocardial rigor contracture during ischemia. The aim of this study was to analyse the effects of NHE inhibition on cell-to-cell electrical uncoupling during myocardial ischemia/reperfusion. Methods: Twenty-six isolated rat hearts and 23 in situ porcine hearts were submitted to no-flow ischemia followed by reperfusion, with or without pre-treatment with cariporide (7 μM in rats and 3 mg/kg in pigs). Ischemic rigor and hypercontracture, conduction velocity and myocardial electrical impedance were monitored. Results: Pre-treatment with cariporide delayed ATP depletion (luminescence assay in rat myocardium) and onset of rigor contracture (tension recordings or ultrasonic crystals) during ischemia both in rat and pig hearts ( P

Objective: There is recent evidence that Ca2+ influx via reverse mode Na+/Ca2+ exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca2+]i during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability. Methods: The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca2+]i and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion. Results: In myocytes, KB-R7943 at concentrations up to 15 μM reduced [Ca2+]i rise and the probability of hypercontracture during re-energization ( P

Objectives: To investigate the roles of 2 polymorphisms of the tumor necrosis factor (TNF) receptor superfamily member 1A ( TNFRSF1A ) gene, rs1800693 (a common variant) and rs4149584 (a coding polymorphism that results in an amino acid substitution—R92Q), as genetic modifiers of multiple sclerosis (MS), and to evaluate their potential functional implications in the disease. Methods: The effects of rs1800693 and rs4149584 on 2 measures of disease severity, age at disease onset and Multiple Sclerosis Severity Score, were analyzed in 2,032 patients with MS. In a subgroup of patients, serum levels of the soluble form of TNF-R1 (sTNF-R1) were measured by ELISA; mRNA expression levels of the full-length TNF-R1 and ∆6-TNF-R1 isoform were investigated in peripheral blood mononuclear cells (PBMC) by real-time PCR; cell surface expression of the TNF-R1 was determined in T cells by flow cytometry. Results: For rs4149584, R92Q carriers were younger at disease onset and progressed slower compared to noncarriers. However, no association with disease severity was observed for rs1800693. Serum levels of sTNF-R1 and mRNA expression levels of the full-length receptor were significantly increased in patients with MS carrying the R92Q mutation ( p = 0.003 and p = 0.011, respectively), but similarly distributed among rs1800693 genotypes; cell surface TNF-R1 expression in T cells did not differ between rs4149584 and rs1800693 genotypes. The truncated soluble ∆6-TNF-R1 isoform was identified in PBMC from patients carrying the risk allele for rs1800693. Conclusions: These findings suggest that both rs1800693 and rs4149584 TNFRSF1A polymorphisms have functional consequences in the TNF-R1.

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.

Attention has focused on particular neurons as the source of nitric oxide (NO) within the parenchyma of the CNS. In contrast, glial cells have been viewed mainly as potential reservoirs of l-arginine, the substrate for nitric oxide synthase (NOS), and as likely targets for neuronally derived NO because of their proximity and their expression of soluble guanylyl cyclase (sGC). However, it is becoming evident that astrocytes display both constitutive and inducible NOS activity under various conditions, and that activated microglia express an inducible NOS. The NO-producing capacity of oligodendrocytes is not yet known. Glial-derived NO has significant implications for CNS pathophysiology, given the anatomical location and abundance of these cells, and the wide variety of potential interactions that NO can have with cellular biochemistry. Our intention here is to evaluate the evidence for NO production from non-neuronal CNS sources and thus prompt discussion about potential ‘nitrinergic' roles for glial cells.

Cyclic GMP accumulation induced by noradrenaline in astrocyte-enriched primary cultures from rat cerebrum involves synthesis of NO, as evidenced by the competitive inhibition exerted by the NO synthase inhibitor NG-monomethyl-L-arginine (IC50 = 3 microM). Furthermore, the noradrenaline effect was potently inhibited by haemoglobin (IC50 = 25 nM) and potentiated by superoxide dismutase, indicating that NO synthesis and cyclic GMP formation may occur in different subsets of astrocytes. Investigation of the receptors implicated by using selective adrenoceptor agonists and antagonists indicates that about 75% of the NO-dependent noradrenaline response is mediated by alpha 1-adrenoceptors and the rest by beta-adrenoceptors, with no evidence for potentiating effects between the two receptor types. This noradrenaline effect appears to require Ca2+ entry, since it is strongly dependent on extracellular Ca2+ but is not affected by conditions that will abolish intracellular Ca2+ mobilization (incubation with neomycin or pretreatment with carbachol). Inhibition by pretreatment with pertussis toxin is in agreement with involvement of the alpha 1A-adrenoceptor subtype in this Ca(2+)-dependent effect. However, implication of an unknown alpha 1-adrenoceptor subtype cannot be disregarded, because a similar inhibition is exerted by the presumably selective alpha 1B- and alpha 1C-adrenoceptor blocking agent chloroethylclonidine. Treatment of the cultures with the protein kinase C activator phorbol 12-myristate 13-acetate inhibits to a great extent the noradrenaline-induced cyclic GMP formation.

The ability of various compounds to stimulate cyclic GMP accumulation was studied in neuronal and astrocyte-enriched primary cultures from rat cerebrum. Glutamate was the only agonist eliciting a response in neurons whereas several agonists had an effect in astrocytes but only those due to norepinephrine and glutamate were of considerable magnitude. The responses were markedly inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The effect of glutamate appears to be mediated predominantly by NMDA receptors in neurons and by quisqualate AMPA-insensitive receptors in astrocytes.

Background Activated platelets might contribute to endothelial dysfunction in non-ischaemic territories during acute myocardial infarction. We assessed platelet deposition, coronary flow reserve and contractile function in remote cardiac regions after transient coronary occlusion and their association with systemic platelet activation. Materials and methods In 10 pigs (series A) subjected to 48-min occlusion of the left anterior descending coronary artery (LAD), 99mTc-platelet content in the right coronary artery (RCA) and its dependent myocardium was counted after reflow. In 10 pigs (series B) receiving the same occlusion of the RCA, the hyperaemic response at the LAD and systolic shortening in LAD-dependent myocardium were monitored after reperfusion. P-selectin expression on circulating platelets was assessed in both series by flow cytometry. Results In series A, platelet counts in the RCA and non-ischaemic myocardium were correlated with platelet content, polymorphonuclear leukocyte infiltration and infarct size in the reperfused zone, as well as with the percentage of P-selectin-positive platelets after reflow. In series B, a transient reduction in peak hyperaemic response in the LAD and sustained contractile dysfunction in non-ischemic myocardium were observed after releasing the RCA occlusion, these changes being also correlated with platelet activation status. Conclusions Ischaemic injury triggers macro- and microvascular platelet deposition and causes an impairment in coronary flow reserve and contractile function in distant regions of the heart, which are related to activation of circulating platelets.

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction and myocardial infarction. Several drugs that increase the activity of this enzyme are now in clinical phase of development: some of them are heme-dependent and might interact with the catalytic domain and others are heme-independent and supposedly bind to the sensory domain. The absence of reliable structural information is one of the factors that have precluded knowledge of the precise site of interaction of these molecules and of the mechanism of activation of the enzyme.

Increased mechanical tension in the ischemic region during acute coronary occlusion might favor the occurrence of phase Ib ventricular arrhythmias. We aimed to investigate whether intracoronary administration of Gd3+, a stretch-activated channel blocker, into the ischemic zone reduces the incidence of these arrhythmias. In thiopental-anesthetized, open-chest pigs, the left anterior descending coronary artery (LAD) was ligated for 45 or 48 min. Phosphate-free, HEPES-buffered saline bubbled with 100% N2 was infused into the ischemic region for 4 min, starting 5 min ( series A; n = 16) or 20 min ( series B; n = 16) after coronary occlusion, at a rate doubling the baseline blood flow. Animals were blindly allocated to receive 40 μM Gd3+ or only the buffer during the final 2 min of the infusion. There were no differences between groups with respect to hemodynamic variables, plasma K+ levels, or size of the ischemic region. In neither series was the number of phase Ib premature ventricular beats reduced by Gd3+ (46 ± 20 in untreated vs. 91 ± 37 in Gd3+-treated animals in series A and 19 ± 7 vs. 22 ± 13, respectively, in series B; both P = not significant). The occurrence of ventricular tachycardia or fibrillation was significantly associated with the magnitude of early ischemic expansion of the LAD region, as measured by ultrasonic crystals, but was also not prevented by Gd3+. These results argue against a major role of stretch-activated channels inside the area at risk in the genesis of phase Ib ischemic ventricular arrhythmias.

Our understanding of the end-effectors involved in preconditioning protection is still very limited. This is partially due to an incomplete knowledge of the mechanisms responsible for acute sarcolemmal rupture and cell death during the first minutes of reperfusion, including the relative roles of hypercontracture-mediated sarcolemmal rupture and mitochondrial permeability transition pore (MPTP) opening-mediated cell death. In the present article, the role of proposed end-effectors of preconditioning protection, defined as molecules directly involved in cell death that are modified by ischemic preconditioning (IP), is examined. IP attenuates hypercontracture-mediated cell death, probably through several mechanisms, including attenuated calpain activation during reperfusion leading to preserved cytoskeletal integrity and accelerated recovery of Na+/K+-ATPase function, but probably also protein kinase G (PKG)-mediated improved calcium handling. The potential role of gap junctions in preconditioning protection is controversial, but the recently discovered mitochondrial localisation of connexin43 seems to play an important role in protection that has not yet been completely defined. Several recent studies suggest that IP can reduce MPTP opening during reperfusion and limit infarct size through this mechanism, although the contribution of this widely accepted mechanism to the infarct size reduction induced by IP in the intact heart needs to be established.

Objective: Although the importance of the cyclic GMP (cGMP) signaling pathway in cardiac myocytes is well established, little is known about its regulation. Ca2+-dependent translocation of nitric oxide (NO) sensitive guanylyl cyclase (GCNO) to the cell membrane has been recently proposed to play a role. The aim of this study was to determine the possible functional relevance of GCNO bound to the cardiomyocyte membrane. Methods: Cytosolic and particulate fractions of adult rat cardiomyocytes were isolated and blotted, and their GCNO activity was assayed in parallel experiments. Results: In untreated cardiomyocytes, approximately 30% of β1-and α1-subunits of GCNO and a similar proportion of GCNO activity were found in the particulate fraction. The dependence of GCNO activity on pH, Ca2+, GTP and NO donor concentrations was similar in particulate and cytosolic fractions. Treatment of cardiomyocytes with the ionophore A23187 caused GCNO to translocate to the sarcolemma, increased GCNO activity in this fraction, and potentiated NO-mediated cGMP synthesis. These effects appeared to be mediated by Ca2+-dependent changes on the phosphorylation status of GCNO, since they were enhanced by the non-selective inhibitor staurosporine and by the selective inhibitor of Ca2+/calmodulin-dependent protein kinase KN-93. The effect of drugs increasing intracellular Ca2+ on cGMP synthesis was clearly correlated with their effects on membrane-associated GCNO activity but not with their effects on cytosol-associated GCNO. Conclusion: These results are the first evidence that 1) GCNO is associated with the cell membrane in cardiomyocytes, 2) the regulation of membrane-associated GCNO differs from that of cytosolic GCNO, and 3) membrane association may have a crucial role in determining the response of cells to NO.

Objectives The goal of this study was to assess whether selectin blockade reduces myocardial platelet deposition and platelet-mediated injury after transient ischemia. Background Selectins participate in platelet adhesion to reperfused endothelium. Methods Thiopental-anesthetized, open-chest pigs were subjected to mechanical injury of the left anterior descending coronary artery followed by a 48-min occlusion and 2 (n = 20) or 4 (n = 16) h of reperfusion. Fifteen minutes before occlusion, animals were blindly allocated to receive a continuous intravenous infusion of the selectin blocker fucoidan (30 μg/kg/min, plus a 1-mg/kg bolus in the latter group) or saline. In isolated rat hearts infused with thrombin-activated platelets, the effects of fucoidan (30 μg/ml) administered during reperfusion after 40 min of global ischemia were also analyzed. Results Fucoidan did not prevent the development of cyclic reductions in coronary flow, but reduced the content of 99mTc-labeled platelets in reperfused myocardium after 2 h of reperfusion (23.4 ± 3.3 vs. 42.1 ± 8.3 × 106platelets/g in treated and untreated animals, p = 0.03) and attenuated the impairment in the coronary flow reserve and reduced infarct size after 4 h (53 ± 2% vs. 73 ± 5% of the ischemic region, respectively, p = 0.003). Treated animals showed a trend toward less neutrophil infiltration early after reperfusion, but not after 4 h. In isolated hearts, fucoidan improved functional recovery and reduced coronary resistance and lactate dehydrogenase release, lacking any beneficial effects if given in the absence of platelets. Conclusions The results suggest that selectin-dependent adhesion is a prominent mechanism of platelet deposition in reperfused cardiac microvessels and highlight its potential as a therapeutic target in patients with acute myocardial infarction.

It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect.Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia-5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed.IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P0.001). At the end off reperfusion, calpain activity was reduced by 66% (P0.001) in IPC, and calpain-dependent degradation of sarcolemmal proteins was attenuated. Addition of the calpain inhibitor MDL-28170 mimicked the effects of IPC on protein degradation and reduced LDH release by 48% (P0.001). The effects of IPC on calpain, alpha-fodrin, and LDH release were blunted by the application of the PKA inhibitor H89 or alprenolol during IPC, while transient stimulation of PKA with CPT-cAMP or isoproterenol before ischemia attenuated calpain activation, alpha-fodrin degradation, and markedly reduced LDH release (P0.001). In hearts exposed to Na(+)-free perfusion, IPC attenuated calpain activation by 67% (P0.001) and reduced by 56% (P0.001) LDH release associated to massive edema occurring during Na(+) readmission without modifying its magnitude.These results are consistent with PKA-dependent attenuation of calpain-mediated degradation of structural proteins being an end-effector mechanism of the protection afforded by IPC.

The mechanism by which inhibition of Na+/H+ exchanger (NHE) reduces cell death in ischemic-reperfused myocardium remains controversial. This study investigated whether cariporide could inhibit mitochondrial NHE during ischemia, delaying H+ gradient dissipation and ATP exhaustion. Mouse cardiac myocytes (HL-1) were submitted to 1 h of simulated ischemia (SI) with NaCN/deoxyglucose (pH 6.4), with or without 7 μM cariporide, and mitochondrial concentration of Ca2+ (Rhod-2), 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and the charge difference across the mitochondrial membrane potential (Δψm, JC-1) were assessed. ATP content was measured by bioluminescence and mitochondrial swelling by spectrophotometry in isolated mitochondria. Cariporide significantly attenuated the acidification of the mitochondrial matrix induced by SI without modifying Δψm decay, and this effect was associated to a delayed ATP exhaustion and increased mitochondrial Ca2+ load. These effects were reproduced in sarcolemma-permeabilized cells exposed to SI. In these cells, cariporide markedly attenuated the fall in mitochondrial pH induced by removal of Na+ from the medium. In isolated mitochondria, cariporide significantly reduced the rate and magnitude of passive matrix swelling induced by Na+ acetate. In isolated rat hearts submitted to 40-min ischemia at different temperatures (35.5°, 37°, or 38.5°C) pretreatment with cariporide limited ATP depletion during the first 10 min of ischemia and cell death (lactate dehydrogenase release) during reperfusion. These effects were mimicked when a similar ATP preservation was achieved by hypothermia and were abolished when the sparing effect of cariporide on ATP was suppressed by hyperthermia. We conclude that cariporide acts at the mitochondrial level, delaying mitochondrial matrix acidification and delaying ATP exhaustion during ischemia. These effects can contribute to reduce cell death secondary to ischemia-reperfusion.

The effect of simulated ischemia [hypoxia, no glucose, extracellular pH (pHo) 6.4] on cGMP synthesis induced by stimulation of soluble (sGC) or particulate guanylyl cyclase (pGC) was investigated in adult rat cardiomyocytes. Intracellular cGMP content was measured after stimulation of sGC by S-nitroso- N-penicillamine (SNAP) or stimulation of pGC by natriuretic peptides [urodilatin (Uro), atrial natriuretic peptide (ANP), or C-type natriuretic peptide (CNP)] for 1 min in the presence of phosphodiesterase inhibitors. After 2 h of simulated ischemia, a decrease of >50% was observed in pGC-dependent cGMP synthesis, but no significant change was observed in sGC-dependent cGMP synthesis. The reduction in cGMP synthesis caused by simulated ischemia was mimicked by extracellular acidosis (pHo6.4), which decreased pGC-mediated cGMP synthesis without altering sGC-mediated cGMP synthesis. An extreme sensitivity of pGC activity to low pH was also observed in membrane cell fractions. Hypoxia without acidosis (pHo7.4) profoundly depressed cellular ATP content but did not change the response to SNAP, Uro, or ANP (selective agonists of pGC type A receptor). Only cGMP synthesis in response to CNP (a selective agonist of pGC type B receptor) was significantly reduced by ATP depletion. These data support the relevance of intracellular pH as a modulator of cGMP and suggest that, in ischemic cardiomyocytes, synthesis of cGMP would be mainly nitric oxide dependent.

Myocardial stretch induces several electrophysiological changes and arrhythmias, but little is known on its possible role in triggering ventricular fibrillation (VF) during acute coronary occlusion. In thiopental-anesthetized, open-chest pigs submitted to a 40-min ligation of the left anterior descending coronary artery, the association between the early increase in end-diastolic length (measured by means of ultrasonic crystals) in the ischemic region and subsequent VF was analyzed. Animals received no treatment (n = 35) or intravenous nitroglycerin (2.5 microg/kg/min for 20 min, starting 10 min after coronary occlusion, n = 8) or Gd(3+) (80 microM/kg for 35 min, starting 5 min before occlusion, n = 15). Twenty-four animals (41 %) had VF, 16 to 39 min after coronary occlusion. The magnitude of ischemic dilation and the incidence of VF were similar among groups. End-diastolic length in the ischemic region 15 min after coronary occlusion was 115.7 +/- 1.2 % of baseline in animals with VF and 111.4 +/- 0.9 % in those without (P = 0.007), and was the strongest predictor of this arrhythmia (P = 0.003) after adjusting for treatment and other possible confounding variables. Thus, the dilation of the ischemic region is closely and independently associated with VF following coronary occlusion. Although the interventions tested in the present study failed to protect against this arrhythmia, the results strongly suggest an influence of ischemic dilation on VF.

Platelets (Plt) accumulate in reperfused myocardium but their effect on myocardial necrosis has not been established. We tested the hypothesis that the effect of Plt depends on their activation status. Pig Plt were obtained before 48 min of coronary occlusion (pre-CO-Plt), 10 min after reperfusion (R-Plt), or after a 60-min sham operation (sham-Plt). Plt were infused into isolated rat hearts ( n = 124) and subsequently submitted to 60 min of ischemia and 60 min of reperfusion. P-selectin expression was higher ( P = 0.02) in R-Plt than in pre-CO-Plt or sham-Plt. Lactate dehydrogenase (LDH) release during reperfusion was similar in hearts receiving pre-CO-Plt, sham-Plt, or no Plt, but R-Plt increased LDH release by 60% ( P = 0.004). Activation of pre-CO-Plt with thrombin increased P-selectin expression and LDH release ( P < 0.001), and these results were unaffected by tirofiban. There was a close correlation between P-selectin expression and LDH release ( r = 0.84; P < 0.001), and myocardial Plt accumulation ( r = 0.85; P < 0.001). We conclude that the deleterious effect of Plt on reperfused myocardium depends on their activation status as represented by P-selectin expression, which is enhanced by ischemia-reperfusion.

To characterize the effects of ischemia on cGMP synthesis in microvascular endothelium, cultured endothelial cells from adult rat hearts were exposed to hypoxia or normoxia at pH 6.4 or 7.4. Cellular cGMP and soluble (sGC) and membrane guanylyl cyclase (mGC) activities were measured after stimulation of sGC (S-nitroso-N-acetyl-penicillamine) or mGC (urodilatin) or after no stimulation. Cell death (lactate dehydrogenase release) was negligible in all experiments. Hypoxia at pH 6.4 induced a rapid approximately 90% decrease in cellular cGMP after sGC and mGC stimulation. This effect was reproduced by acidosis. Hypoxia at pH 7.4 elicited a less pronounced (approximately 50%) and slower reduction in cGMP synthesis. Reoxygenation after 2 h of hypoxia at either pH 6.4 or 7.4 normalized the response to mGC stimulation but further deteriorated the sGC response; normalization of pH rapidly reversed the effects of acidosis. At pH 7.4, the response to GC stimulation correlated well with cellular ATP. We conclude that simulated ischemia severely depresses cGMP synthesis in microvascular coronary endothelial cells through ATP depletion and acidosis without intrinsic protein alteration.

Objective: Stimulation of cGMP synthesis protects cardiomyocytes against reoxygenation-induced hypercontracture. The purpose of this study was to determine whether l-arginine supplementation has a protective effect against reperfusion-induced hypercontracture and necrosis in the intact animal. Methods: Twenty-four Large-White pigs were randomized to receive either 100 mg/kg of l-arginine IV or vehicle 10 min before 48 min of coronary occlusion and 2 h of reperfusion. Hemodynamic variables, coronary blood flow and myocardial segment length changes (piezoelectric crystals) were monitored. Postmortem studies included quantification of myocardium at risk (in vivo fluorescein), infarct size (triphenyltetrazolium reaction), myocardial myeloperoxidase activity and histological analysis. Systemic, coronary vein, and myocardial cGMP concentration were measured in additional animals. Results: Administration of l-arginine had no significant effect in hemodynamics or coronary blood flow. During reperfusion, myocardial cGMP content was reduced in the LAD as compared to control myocardium ( P =0.02). l-Arginine increased myocardial cGMP content and caused a transient increase in plasma cGMP concentration during the initial minutes of reperfusion ( P =0.02). The reduction in end-diastolic segment length induced by reperfusion, reflecting hypercontracture, was less pronounced in the l-arginine group ( P =0.02). Infarct size was smaller in pigs receiving l-arginine (47.9±7.2% of the area at risk) than in controls (62.9±4.9%, P =0.047). There were no differences between groups in leukocyte accumulation in reperfused myocardium ( P =0.80). Conclusion: l-Arginine supplementation reduces myocardial necrosis secondary to in situ ischemia–reperfusion by a direct protective effect against myocyte hypercontracture.

Objectives: Hypercontracture is an important mechanism of myocyte death during reperfusion. cGMP modulates the sensitivity of contractile myofilaments to Ca2+, and increasing cGMP concentration during the last minutes of anoxia prevents reoxygenation-induced hypercontracture in isolated cardiomyocytes. The purpose of this study was to determine whether stimulation of particulate guanylyl cyclase with the natriuretic peptide urodilatin, given at the time of reperfusion, reduces myocardial necrosis in the rat heart submitted to transient ischemia. Methods: Isolated rat hearts ( n =38) were submitted to either 40 or 60 min of no-flow ischemia and 2 h of reperfusion, and were allocated to receive or not receive 0.05 μM urodilatin during the first 15 min of reperfusion or non-reperfusion treatment. Results: A marked reduction in myocardial cGMP concentration was observed in control hearts during reperfusion after 40 or 60 min of ischemia. Urodilatin significantly attenuated cGMP depletion during initial reperfusion, markedly improved contractile recovery after 40 min of ischemia ( P

The objective of this study was to investigate the effect ofl-arginine supplementation on myocardial cell death secondary to hypoxia-reoxygenation. Isolated rat hearts ( n = 51) subjected to 40 min of hypoxia and 90 min of reoxygenation received 3 mMl-arginine and/or 1 μM 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ; a selective inhibitor of soluble guanylyl cyclase) throughout the experiment or during the equilibration, hypoxia, or reoxygenation periods. The incorporation ofl-[3H]arginine into myocytes during energy deprivation was investigated in isolated adult rat myocytes. The addition ofl-arginine to the perfusate throughout the experiment resulted in higher cGMP release ( P < 0.05), reduced lactate dehydrogenase release ( P < 0.05), and increased pressure-rate product ( P < 0.05) during reoxygenation. These effects were reproduced whenl-arginine was added only during equilibration, but addition ofl-arginine during hypoxia or reoxygenation had no effect. Addition of ODQ either throughout the experiment or only during reoxygenation reversed the beneficial effects of l-arginine.l-[3H]arginine was not significantly incorporated into isolated myocytes subjected to energy deprivation. We conclude thatl-arginine supplementation protects the myocardium against reoxygenation injury by cGMP-mediated actions. To be effective during reoxygenation,l-arginine must be added before anoxia.

Metallothioneins (MTs) are a family of low molecular weight proteins which in rodents comprise four isoforms (MT-I to -IV). MT-I+II are widely expressed isoforms which are highly inducible by factors such as heavy metals and a number of hormones. The expression of these isoforms in the brain has been regarded as basically astrocytic, and to a lower extent, neuronal. We, however, demonstrate in this report, by radioimmunoassay and immunocytochemistry, that MT-I+II are expressed in primary cultures of rat microglia and that, furthermore, they are inducible by zinc and copper. Thus all major brain cell types may express the MT-I+II isoforms and respond with increased protein levels to physiological stimuli such as increased extracellular zinc and copper content. In contrast, microglia MT-I+II levels are not affected by either the glucocorticoid dexamethasone or the cytokine IL-1 under the experimental conditions.

Ca2+ entry induced by N-methyl-D-aspartate (NMDA) in neurons and by noradrenaline (NA) in astrocytes is known to increase intracellular cyclic GMP (cGMP) levels through stimulation of the Ca2+-dependent nitric oxide synthase type I (NOS-I). The possibility that Ca2+ entry could also down-regulate intracellular cGMP by activating a Ca2+/calmodulin-dependent phosphodiesterase (CaM-PDE) has been investigated here in primary cultures enriched in granule neurons or in astroglia from rat cerebellum. We show that the same agonists that stimulate nitric oxide (NO) formation (NMDA and NA at 100 μM) and the Ca2+ ionophore A23187 (10 μM) decrease cGMP generated in response to direct stimulation of soluble guanylyl cyclase (sGC) by NO donors in both cell types. This effect requires extracellular Ca2+ and is prevented by the calmodulin inhibitor W7 (100 μM). Membrane depolarization, manipulations of the Na+ gradient, and intracellular Ca2+ mobilization also decrease NO donor-induced cGMP formation in granule cells. In astroglia Ca2+ entry additionally down-regulates cGMP generated by stimulation of the particulate GC by atrial natriuretic peptide (ANF). Decreases in cGMP produced by A23187 were more pronounced in the absence than in the presence of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), indicating that a CaM-PDE was involved. We also show that astroglial cells can accumulate similar amounts of cGMP than neurons in response to NO donors when IBMX is present but much lower levels in its absence. This may result from a lower ratio of sGC to PDE activities in astroglia. J. Neurosci. Res. 49:333–341, 1997. © 1997 Wiley-Liss, Inc.

Cyclic GMP (cGMP) formation induced by agonist stimulation of Ca2+/calmodulin-dependent nitric oxide (NO) synthase type I is known to occur in both granule cell and astrocyte cultures from rat cerebellum. Here we show that in these same cells cGMP is predominantly hydrolyzed by a Ca2+/calmodulin-dependent phosphodiesterase. At 10 microM cGMP, Ca2+ (25 microM) stimulated basal (Ca(2+)-independent) phosphodiesterase activity about 6 times in granular neurons and 15 times in astrocytes. The calmodulin antagonist calmidazolium blocked the Ca(2+)-dependent phosphodiesterase activity and exogenous calmodulin increased 3-4-fold the stimulatory potency of Ca2+ in both cell types (EC50 values 1.26 +/- 0.20 and 1.50 +/- 0.42 microM in the absence and 0.38 +/- 0.11 and 0.39 +/- 0.14 microM in the presence of 1 microM calmodulin, for neurons and astrocytes, respectively). In both cell types K(m) values for cGMP at 25 microM Ca2+ were similar (1.72 +/- 0.20 and 1.92 +/- 0.09 microM) and phosphodiesterase activities were inhibited by isozyme-selective phosphodiesterase inhibitors with potencies analogous to those described for Ca2+/calmodulin-phosphodiesterases or phosphodiesterase type 1 isoforms in other preparations. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) effectively blocked the Ca2+/calmodulin-phosphodiesterase activity in granule cell and astrocyte extracts (IC50 values at 1 microM cGMP: 31 +/- 10 microM and 46 +/- 6 microM, respectively), in contrast to the apparent inability of this compound to inhibit the Ca(2+)-dependent activity reported in whole brain extracts. These results demonstrate that comparable phosphodiesterase type 1 activities are found in the cytosols of cerebellar granule cells and astrocytes and suggest that these activities may play an important role in controlling cGMP levels in cells where the Ca(2+)-dependent NO synthase type I is stimulated.