Cefoxitin-based combination for ESBL-producing Enterobacteriaceae endocarditis.

BACKGROUND: Endocarditis due to extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is a rare but challenging condition. Its treatment relies on carbapenems alone or in combination, and no alternative has been described to date. The cephamycin cefoxitin has been used for treatment of mild ESBL-producing Enterobacteriaceae infections. CASE PRESENTATION: We report two patients with nosocomial endocarditis due to ESBL-producing Escherichia coli and Klebsiella pneumoniae who underwent clinical failure or adverse event, respectively, during treatment with imipenem-cilastatin. The first patient was subsequently treated with cefoxitin combined with ciprofloxacin with a favorable outcome. In the second patient, the endocarditis relapsed following a 6-week treatment with cefoxitin and fosfomycin. In time-kill assays, the cefoxitin/ciprofloxacin and cefoxitin/fosfomycin combinations showed synergistic effect. CONCLUSION: These cases illustrate that cefoxitin is an interesting alternative to carbapenems, even in severe infections such as endocarditis. Pharmacokinetic optimization and combination with another synergistic antibiotic should be considered whenever possible.

Novel homozygous mutation (c.175delG) in platelet glycoprotein ITGA2B gene as cause of Glanzmann's thrombasthenia type I.

BACKGROUND: Glanzmann's thrombasthenia (GT), is a rare autosomal recessive bleeding disorder. Platelets from patients with GT show quantitative or qualitative defects of the platelet membrane glycoprotein (GP) IIb/IIIa complex. A variety of genetic defects in ITGA2B and ITGB3 (genes for GPIIb and GPIIIa) has been described causing the clinical entity of GT. PATIENTS: A newborn with bleeding symptoms (petechiae) platelet analyses revealed an inherited primary hemostasis disorder. METHODS/RESULTS: Analyses of patient's platelets using flow cytometry and immunoblotting showed absence of GPIIb protein and reduced amount of GPIIIa. Using restriction fragment length polymorphism heterozygosity for the deletion could be identified in the parents and in two siblings. Expression studies in mammalian cells revealed that the mutant GPIIb is missing and additionally affects the expression of wildtype GPIIIa. This deletion leads to a truncation at the very N-terminal region of the GPIIb protein. CONCLUSION: The present study describes a patient with GT associated with a novel homozygous deletion (c.175delG) in exon 1 of ITGA2B. This deletion led to a reading frameshift and caused a severely truncated form of GPIIb.

Dynamics of glycine receptor insertion in the neuronal plasma membrane.

The exocytosis site of newly synthesized glycine receptor was defined by means of a morphological assay to characterize its export from the trans-Golgi Network to the plasma membrane. This was achieved by expressing in transfected neurons an alpha1 subunit bearing an N-terminal tag selectively cleavable from outside the cell by thrombin. This was combined with a transient temperature-induced block of exocytic transport that creates a synchronized exocytic wave. Immunofluorescence microscopy analysis of the cell surface appearance of newly synthesized receptor revealed that exocytosis mainly occurred at nonsynaptic sites in the cell body and the initial portion of dendrites. At the time of cell surface insertion, the receptors existed as discrete clusters. Quantitative analysis showed that glycine receptor clusters are stable in size and subsequently appeared in more distal dendritic regions. This localization resulted from diffusion in the plasma membrane and not from exocytosis of transport vesicles directed to dendrites. Kinetic analysis established a direct substrate-product relationship between pools of somatic and dendritic receptors. This indicated that clusters represent intermediates between newly synthesized and synaptic receptors. These results support a diffusion-retention model for the formation of receptor-enriched postsynaptic domains and not that of a vectorial intracellular targeting to synapses.

Fast and reversible trapping of surface glycine receptors by gephyrin.

Variations in receptor number at a given synapse are known to contribute to synaptic plasticity, but methods used to establish this idea usually do not allow for the determination of the dynamics of these phenomena. We used single-particle tracking to follow in real time, on the cell surface, movements of the glycine receptor (GlyR) with or without the GlyR stabilizing protein gephyrin. GlyR alternated within seconds between diffusive and confined states. In the absence of gephyrin, GlyR were mostly freely diffusing. Gephyrin induced long confinement periods spatially associated with submembranous clusters of gephyrin. However, even when most receptors were stabilized, they still frequently made transitions through the diffusive state. These data show that receptor number in a cluster results from a dynamic equilibrium between the pools of stabilized and freely mobile receptors. Modification of this equilibrium could be involved in regulation of the number of receptors at synapses.

Functional heterogeneity of gephyrins.

Postsynaptic clustering of the glycine receptor requires the cytoplasmic protein gephyrin, which interacts with the receptor beta subunit. Several variants of gephyrin are generated by alternative splicing and differ by the presence of short amino acid sequences (cassettes) in the N-terminal half of the molecule. In this work, seven isoforms of gephyrin were cloned from adult rat spinal cord, some of then containing new cassettes. The relationships between gephyrin structure and recognition of glycine receptor beta subunit were analyzed. This was carried out by GST-pulldown assays using the beta subunit cytoplasmic loop and cotransfection experiments of GFP-tagged gephyrins with an alpha1 subunit bearing the gephyrin-binding site of the beta subunit. Data demonstrated that not all gephyrin molecules can bind to the beta subunit. Identified cassettes modulate this interaction. It is thus concluded that the function of gephyrin in synapse formation can rely on a structure acquired through cassette combinations.

Formation of glycine receptor clusters and their accumulation at synapses.

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substantial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investigate the formation of postsynaptic glycine receptor domains, we have examined the surface insertion of epitope-tagged receptor alpha subunits in cultured spinal cord neurons after gene transfer by polyethylenimine-adenofection. Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoforms and an alpha mutant subunit bearing the gephyrin-binding motif of the beta subunit. In COS-7 cells, transfected glycine receptor alpha subunits had a diffuse surface distribution. Following cotransfection with gephyrin, only the mutant subunit formed cell surface clusters. In contrast, in neurons all subunits were able to form cell surface clusters after transfection. These clusters were not colocalized with detectable endogenous gephyrin, and the GlyR beta subunit could not be detected in transfected cells. Therefore, exogenous receptors were not assembled as heteromeric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, since association of both proteins increased as a function of time after the onset of receptor synthesis. This phenomenon was accelerated when glycine receptor contained the gephyrin-binding domain. Together with previous results, these data support a two-step model for glycinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation of clusters.

The preclinical assessment of the risk for QT interval prolongation.

Some drugs have been reported to induce severe ventricular arrhythmias, including torsades de pointes, and have been responsible in some cases for sudden death of patients. Although the mechanisms of these arrhythmias are not well understood, they are often, but not always, associated with QT interval prolongation. Regulatory authorities (CPMP in Europe) have recently pointed out the necessity to assess most carefully the potential, especially of non-cardiovascular drugs, for QT interval prolongation. Different methodological approaches are presented in this paper and experimental protocols are suggested; limitations and advantages of the presently available in vitro and in vivo models are discussed. It appears that both in vitro and in vivo approaches are complementary. In particular it is pointed out that only the in vitro models using isolated cardiac tissues (Purkinje fibres or papillary muscles) enable assessment of the drug properties under low cardiac rhythm conditions. This model allows us to mimic pathological situations of long QT interval (such as acquired or congenital long QT syndrome) in which most of the major clinical problems are encountered. Finally, a strategy for the preclinical assessment of the potential of a molecule for QT interval prolongation is presented.

Determinants of core temperature at the time of admission to intensive care following cardiac surgery.

OBJECTIVE: To determine the predictors of core temperature on arrival in the intensive care unit (ICU) after cardiac surgery. DESIGN: Prospective, randomized trial. SETTING: Tertiary care medical center, operating rooms (ORs), and ICU. PATIENTS: 72 patients presenting for coronary artery bypass surgery. INTERVENTIONS: Randomized assignment for ambient OR temperature (16-18 degrees C vs. 21-23 degrees C) and rewarming endpoint on cardiopulmonary bypass (CPB; nasopharyngeal and urinary bladder temperatures >/=36.5 degrees C and 34.0 degrees C, respectively, vs. nasopharyngeal and urinary bladder temperatures >/=37.5 degrees C and 36.0 degrees C, respectively) at the time of separation from bypass. MEASUREMENTS AND MAIN RESULTS: The best (and only significant) predictor of core temperature on arrival in the ICU was rewarming endpoint at the time of separation from CPB (p = 0.004). Patient weight, height, body habitus, and nitroprusside administration did not significantly predict core temperature. Ambient temperature affected only body temperature when the duration of time in the OR after separation from bypass was prolonged (>90 min). A weighted average body temperature was a better predictor of complete rewarming than was any single monitoring site. CONCLUSIONS: To reduce the incidence of hypothermia after cardiac surgery, the most important variable is rewarming endpoint achieved before separation from bypass. A warm ambient temperature (>21 degrees C) may be beneficial if the duration of time in the OR after bypass is prolonged (>90 min).

Forced-air warming decreases vasodilator requirement after coronary artery bypass surgery.

Postoperative hypothermia is common and associated with adverse hemodynamic consequences, including adrenergically mediated systemic vasoconstriction and hypertension. Hypothermia is also a known predictor of dysrhythmias and myocardial ischemia in high-risk patients. We describe a prospective, randomized trial designed to test the hypothesis that forced-air warming (FAW) provides improved hemodynamic variables after coronary artery bypass graft. After institutional review board approval and written informed consent, 149 patients undergoing coronary artery bypass graft were randomized to receive postoperative warming with either FAW (n = 81) or a circulating water mattress (n = 68). Core temperature was measured at the tympanic membrane. A weighted mean skin temperature was calculated. Heart rate, mean arterial blood pressure, central venous pressure, cardiac output, and systemic vascular resistance were monitored for 22 h postoperatively. Mean arterial blood pressure was maintained by protocol between 70 and 80 mm Hg by titration of nitroglycerin and sodium nitroprusside. The two groups had similar demographic characteristics. Tympanic and mean skin temperatures were similar between groups on intensive care unit admission. During postoperative rewarming, tympanic temperature was similar between groups, but mean skin temperature was significantly greater in the FAW group (P < 0.05). Heart rate, mean arterial pressure, central venous pressure, cardiac output, and systemic vascular resistance were similar for the two groups. The percent of patients requiring nitroprusside to achieve the hemodynamic goals was less (P < 0.05) in the FAW group. In conclusion, aggressive cutaneous warming with FAW results in a higher mean skin temperature and a decreased requirement for vasodilator therapy in hypothermic patients after cardiac surgery. This most likely reflects attenuation of the adrenergic response or opening of cutaneous vascular beds as a result of surface warming. IMPLICATIONS Forced-air warming after cardiac surgery decreases the requirement for vasodilator drugs and may be beneficial in maintaining hemodynamic variables within predefined limits.

Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2.

Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [35S]sulfate transfer from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS); and (ii) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. & Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896-2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an approximately 13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.

Strychnine-sensitive stabilization of postsynaptic glycine receptor clusters.

The cellular and molecular mechanisms underlying the postsynaptic aggregation of ionotropic receptors in the central nervous system are not understood. The glycine receptor (GlyR) and its cytoplasmic domain-associated protein, gephyrin, are clustered at the postsynaptic membrane and constitute a good model for addressing these questions. The glycine receptor is inhibited by strychnine. The effects of chronic strychnine treatment on the expression and cellular distribution of gephyrin and glycine receptor were therefore tested using primary cultures of spinal cord neurons. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the glycine receptor alpha1, alpha2, beta subunits and gephyrin mRNAs were expressed at comparable levels in strychnine-treated and untreated cultures. The number of immunoreactive cells and the subcellular distribution of gephyrin and GlyR subunits was determined with standard and confocal immunofluorescence. The proportion of gephyrin and glycine receptor-immunoreactive (IR) cells was unaffected by strychnine treatment. Confocal microscopy revealed that the glycine receptor was mainly localized intracellularly near the nucleus. This cytoplasmic glycine receptor was not associated with the Golgi apparatus nor with the rough endoplasmic reticulum and therefore is not likely to correspond to neosynthesized proteins. The number of GlyR clusters on the somato-dendritic membrane was dramatically reduced on neurons displaying intracellular staining. In contrast, the subcellular distribution and the number of gephyrin clusters was not modified by the treatment. The fact that gephyrin postsynaptic localization was not modified by strychnine suggests that the aggregation of glycine receptor and gephyrin is governed by different mechanisms. The distribution of other cell surface molecules such as NCAM or GABAA receptor beta2/3 subunits was not modified by strychnine treatment. Chronic exposure of the cultures to tetrodotoxin did not affect gephyrin or glycine receptor cluster formation. Taken together, these results indicate that functional glycine receptor, but not electrical synaptic activity, is required for the formation of glycine receptor clusters.

Tension response of the cardiotonic agent (+)-EMD-57033 at the single cell level.

The effects of the Ca sensitizer (+)-EMD-57033 were tested on single chemically skinned cells isolated from rat ventricle. The present study demonstrates that (+)-EMD-57033 (10 microM) increased maximal force by 20% (at pCa 4.5) and myofilament Ca sensitivity by 0.2 pCa unit. However, the force-length dependency was not affected by the addition of (+)-EMD-57033, since similar Ca-sensitizing effects occurred at different sarcomere lengths. Consequently, the Ca-sensitizing effect of the drug and of the sarcomere length might be additive. Cross-bridge kinetics were also investigated in the presence of the thiadiazinone derivative. (+)-EMD-57033 induced marked increases in the rate of tension redevelopment (ktr) after brief slack release/restretch, particularly at low Ca concentrations. These results suggest that the Ca-sensitizing effects of (+)-EMD-57033 are due, at least in part, to an increased number of attached cross bridges during one cyclo. This observation, together with the increase in peak force, is discussed in relation to the reduction in energy cost induced by such Ca-sensitizing agents.

Biology of the postsynaptic glycine receptor.

Glycine is one of the major inhibitory neurotransmitters, and upon binding to its receptor it activates chloride conductances. Receptors are accumulated immediately opposite release sites, at the postsynaptic differentiations, where they form functional microdomains. This review describes recent advances in our understanding of the structure-function relationships of the glycine receptor, a member of the ligand-gated ion channel superfamily. Following purification of the receptor complex and identification of its integral and peripheral membrane protein components, molecular cloning has revealed the existence of several subtypes of the ligand-binding subunit. This heterogeneity is responsible for the distinct pharmacological and functional properties displayed by the various receptor configurations that are differentially expressed and assembled during development. This review also focuses on the molecular aspects of glycinergic synaptogenesis, highlighting gephyrin, the peripheral component of the receptor. The role of this cytoplasmic protein in anchoring and maintaining the channel complex in postsynaptic clusters is discussed. The glycine receptor recently moved into the spotlight as a paradigm in the approach to cell biology of the formation of the postsynaptic membrane.

Ca-dependence of isometric force kinetics in single skinned ventricular cardiomyocytes from rats.

OBJECTIVES: The effects of Ca2+ on the rate of tension redevelopment following a brief release/restretch were investigated in single chemically-skinned ventricular myocytes from the rat. METHODS: The myocytes were enzymatically isolated and skinned using Triton-X100. They were then attached with an optical adhesive glue to glass micropipettes fixed to a piezoelectric translator and a force transducer. Tension redevelopment was measured at various levels of Ca activation after disrupting force-generating crossbridges by a brief (20 ms) step release/restretch equivalent to 20% of the original 2.1 microns sarcomere length. Most of tension redevelopment was well fitted by a monoexponential function. RESULTS: At maximal Ca concentrations, pCa 4.5 maximal force was obtained at 2.1 microns sarcomere length and averaged 11.8 +/- 0.7 microN. The rate of tension redevelopment (ktr) increased with increasing Ca concentrations up to 5.19 +/- 0.37.s-1 at maximal Ca activation. The relation between the rate of tension redevelopment and Ca concentration was sigmoidal and could be fitted by the Hill equation with coefficients similar to those describing the tension-pCa relation. The relation between relative rate of tension redevelopment and relative steady activated tension was curvilinear increasing with increasing Ca concentration. CONCLUSIONS: In cardiac muscle, Ca2+ modulates both the number and the kinetics of force-generating crossbridges in a manner similar to that previously reported in skeletal muscle.

Oxidative effects of selenite on rat ventricular contractility and Ca movements.

OBJECTIVE: The study aimed at characterizing the effects of selenite, known for its reactivity with thiols, on cardiac contractility and excitation-contraction coupling. METHODS: The inotropic effects of selenite were studied on rat papillary muscles. Freshly isolated rat ventricular myocytes were used to determine the selenite-induced alterations in thiol contents, free Ca2+ levels (in fura-2 loaded cells), Ca2+ currents and contractile properties of skinned cells. RESULTS: Selenite, at concentrations > or = 0.1 mM, affected muscle contractions by inducing a transient positive inotropic effect (up to 120 +/- 3% of control in 1 mM selenite) followed by a gradual decline of developed tension together with an increase in resting tension (respectively to 37 +/- 3 and 166 +/- 5% of their control values after 20 min exposure). These changes, irreversible on washout, could be reversed by the disulfonic reducing agent dithiothreitol (DTT, 1 mM). Lowering temperature from 35 degrees to 22 degrees C or preincubating the muscles with the disulfonic stilbene SITS (0.2 mM) completely prevented the selenite-induced transient positive inotropy and rise in resting tension. In isolated myocytes, 10 min exposure to 1 mM selenite induced a 40 +/- 9% decrease of total sulfhydryl content. At this concentration, selenite rapidly caused a rise of basal [Ca2+]i together with a diminution of the Ca2+ spike amplitude (respectively to 165 +/- 15 and 45 +/- 9% of their control values after 5 min exposure). In addition, selenite significantly enhanced at each Ca2+ concentration the force generated by skinned myocytes. Ca2+ currents, measured at 22 degrees C, decreased by 28 +/- 8% in the presence of 1 mM selenite. These effects were reversed by DTT. CONCLUSIONS: The results demonstrate that selenite, through alterations of cellular thiol redox status, induced a dual action on muscle contraction that can be imputed to a combined action on Ca2+ channels, Ca2+ transporters and contractile proteins. Extracellular negative effects of selenite are due to a partial reduction of Ca2+ current magnitude. Intracellular effects are mediated both by a diminution of Ca2+ handing by intracellular organelles and by a sensitization of the contractile to Ca2+ ions. The results further indicate that selenite uptake into the cardiac cells occurs mainly through the temperature-sensitive anion exchanger.

Expression of tyrosine-sulfated secretory proteins in Xenopus laevis oocytes. Differential export of constitutive and regulated proteins.

Xenopus laevis oocytes were used to study the tyrosine sulfation and secretion of exogenous proteins. Secretogranin II (SgII), a tyrosine-sulfated protein found in secretory granules of a wide variety of endocrine cells and neurons, became tyrosine-sulfated by the oocytes when expressed by injection of poly(A)-rich RNA isolated from the neuroendocrine cell line PC12. The same result was observed when SgII was expressed from cloned SgII cRNA, showing that its tyrosine sulfation did not require the coexpression of exogenous tyrosylprotein sulfotransferase (TPST) but occurred by means of the endogenous oocyte TPST. Sulfophilin, an artificial protein consisting of 12 repeats of a heptapeptide tyrosine-sulfation site, was highly sulfated upon injection of its RNA, indicating the presence of TPST levels sufficient for stoichiometric sulfation of appropriate reporter proteins. Comparison of the secretion of [35S]sulfate-labelled SgII with that of sulfophilin and an exogenous heparan sulfate proteoglycan (HSPG), two proteins delivered to the cell surface by the constitutive pathway of secretion, revealed striking differences. The majority of sulfophilin and the HSPG was found in the medium, whereas that of SgII was found intracellularly. Prolactin, another secretory granule protein, showed the same secretion behaviour as SgII. These results show that oocytes express TPST and that these cells secrete constitutive and regulated secretory proteins in a differential manner.

Fatty acids regulate the expression of lipoprotein lipase gene and activity in preadipose and adipose cells.

During fasting, a reduction in lipoprotein lipase (LPL) activity has been observed in rat fat pad with no change in enzyme mass, whereas LPL mRNA and synthesis are increased, suggesting that insulin and/or fatty acids (FA) regulate LPL activity post-translationaly [Doolittle, Ben-Zeev, Elovson, Martin and Kirchgessner (1990) J. Biol. Chem. 265, 4570-4577]. To examine the role of FA, either preadipose Ob1771 cells or Ob1771 and 3T3-F442A adipose cells were exposed to long-chain FA and to 2-bromopalmitate, a non-metabolized FA. A rapid (2-8 h) and dose-dependent increase (up to 6-fold) in LPL mRNA occurred, primarily due to increased transcription, which is accompanied by a decrease (down to 4-fold) in LPL cellular activity. Under these conditions, secretion of active LPL was nearly abolished. Removal of FA led to full recovery of LPL activity. LPL gene expression in 3T3-C2 fibroblasts was not affected by FA treatment. However fatty acid-activated receptor transfected-3T3-C2 cells, which show FA responsiveness, had increased LPL gene expression upon FA addition. LPL synthesis and cellular content appeared unaffected by FA treatment, whereas secretion of LPL was inhibited. These results indicate that FA regulate the post-translational processing of LPL. It is proposed that the regulation of LPL activity by FA is important with regard to the fine-tuning of FA entry into adipocytes during fasting/feeding periods.

Thermoregulation and heart rate variability.

1. Heart rate variability is modulated by multiple control systems, including autonomic and hormonal systems. Long-term variability, i.e. the very low-frequency band of the power spectra, has been postulated to reflect thermoregulatory vasomotor control, based upon thermal entrainment experiments. However, the relationship between thermoregulatory responses (vasoconstriction and shivering) and heart rate variability has not been studied. 2. We performed two distinct protocols in a series of human subjects. In the first protocol, core temperature was reduced by intravenous infusion of cold saline, while skin temperature was unchanged. The second protocol involved skin-surface warming and cooling until shivering developed. Power spectral analysis was performed using a fast Fourier transformation, and the area in three distinct band-widths was determined. 3. Very low-frequency power (0.0039-0.04 Hz) increased significantly in response to core cooling, peripheral vasoconstriction and shivering, while both very low- and low- (0.04-0.15 Hz) frequency power increased in response to skin-surface cooling. Heart rate decreased during core cooling-induced vasoconstriction, suggesting a direct thermal response, and increased in relation to the metabolic demands associated with shivering. 4. Our results suggest that very low-frequency power is modulated by thermal stimuli which result in core hypothermia and thermoregulatory activity, while skin-surface cooling without core hypothermia does not selectively modulate this frequency band.

Functional tissue and developmental specificities of myofibrils and mitochondria in cardiac muscle.

To understand the factors underlying the functional differences between atrial and ventricular tissues, intrinsic properties of myofibrils and mitochondria of atrial skinned fibers were compared with those of fibers from adult or immature (1 and 2 weeks old) ventricular muscle. Isometric mechanical parameters were determined at various calcium concentrations in fibers treated with Triton X-100 to solubilize all cellular membranes. Maximal active tension and stiffness measured at pCa 4.5, as well as calcium sensitivity, were not different in adult atria and ventricles. Both force and stiffness increased in adult ventricles, while calcium sensitivity diminished in adult ventricles, compared with immature muscles. Myofibrillar contractile kinetics, assessed by the rate constant of tension fall following quick stretches, were similar in adult atria (79.7 +/- 6.9 s-1) and ventricles (72.4 +/- 6.8 s-1) and higher in adult atria and ventricles than in immature ventricles (24.1 +/- 2.3 s-1 in 1-week-old rats and 49.3 +/- 4.2 s-1 in 2-week-old rats). Sensitivity of rigor tension development to MgATP in the presence and in the absence of phosphocreatine was not markedly different in the different tissues. Mitochondrial function was assessed in saponin-skinned fibers. Tissue oxidative capacities, expressed as nmol O2.min-1.mg-1 fiber dry weight, were lower in immature ventricles and atria than in adult ventricles. Creatine failed to stimulate respiration in ventricles of young rats and in adult atria, whereas a 74 +/- 10% increase in respiration was observed in adult ventricles. Since mitochondrial creatine kinase was present in adult atria, this suggests an absence of coupling between oxidative phosphorylation and mitochondrial creatine kinase in this tissue. Thus, adult atrial tissue differs from neonatal ventricular tissue but it exhibits contractile properties similar to adult ventricular properties and differs from adult ventricle mainly in metabolic properties.

Inhibition of dihydropyridine-sensitive calcium entry in hypoxic relaxation of airway smooth muscle.

Hypoxia dilates airways in vivo and reduces active tension of airway smooth muscle in vitro. To determine whether hypoxia impairs Ca2+ entry through voltage-dependent channels (VDC), we tested the ability of dihydropyridines to modulate hypoxia-induced relaxation of KCl- and carbamyl choline (carbachol)-contracted porcine bronchi. Carbachol- or KCl-contracted bronchial rings were exposed to progressive hypoxia in the presence or absence of 1 microM BAY K 8644 (an L-type-channel agonist). In separate experiments, rings were contracted with carbachol or KCl, treated with nifedipine (a VDC antagonist), and finally exposed to hypoxia. BAY K 8644 prevented hypoxia-induced relaxation in KCl-contracted bronchi. Nifedipine (10(-5) M) totally relaxed KCl- contracted bronchi. Carbachol-contracted bronchi were only partially relaxed by nifedipine but were completely relaxed when the O2 concentration of the gas was reduced from 95 to 0%. These data indicate that hypoxia can reduce airway smooth muscle tone by limiting entry of Ca2+ through a dihydropyridine-sensitive pathway, but that other mechanisms also contribute to hypoxia-induced relaxation of carbachol-contracted bronchi.