Contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta

ABSTRACT: BACKGROUND: Electrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments. RESULTS: Intracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644). CONCLUSION: The isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.     

Transcript and Protein Analysis Reveals Better Survival Skills of Monocyte-Derived Dendritic Cells Compared to Monocytes during Oxidative Stress

Background

Dendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.

Methods

Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H₂DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.

Results

Tert-BHP increased CM-H₂DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.

Conclusions

Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.     

Imbalanced Lignin Biosynthesis Promotes the Sexual Reproduction of Homothallic Oomycete Pathogens

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-l-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-l-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment.     

Down-regulation of peroxisome proliferator-activated receptor-gamma gene expression by sphingomyelins

We recently demonstrated that the sphingomyelin (SM) content of adipocyte membranes was negatively correlated with the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) in the subcutaneous adipose tissue of obese women with variable degrees of insulin resistance. We have now investigated whether SM really does have an impact on the expression of PPARgamma in 3T3-F442A adipocytes. Adding SM to the culture medium for 24 h caused a significant increase in SM content of adipocyte membranes and an acyl chain length-dependent decrease in the levels of PPARgamma mRNA and protein. The longer the acyl chain of the fatty acid of SM, the greater was the decrease in PPARgamma. These data suggest that the nature of the fatty acid is important in the regulation of PPARgamma by the SM pathway.     

Modification of the protein glycosylation pathway in the methylotrophic yeast Pichia pastoris

-1,2-Mannosidase from Trichoderma reesei was used to modify the N-linked glycosylation pathway of the methylotrophic yeast Pichia pastoris. Expression of foreign influenza glycoproteins with more extensively processed N-linked oligosaccharides was observed when -1,2-mannosidase was secreted in the culture medium. However, intracellular removal of mannose residues may stimulate mannosyltransferase activity and leads to hyperglycosylation. [3H]Mannose suicide selection or high concentrations of orthovanadate, commonly used to isolate glycosylation mutants of Saccharomyces cerevisiae, had no profound effect on Pichia pastoris. © Rapid Science Ltd. 1998     

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.     

Patterns of plasma progesterone, androgen and oestrogen concentrations and in-vitro ovarian steroidogenesis during embryonic diapause and implantation in the mink (Mustela vison)

Peripheral plasma progesterone concentrations exhibited an increase 10 days before implantation, coinciding with the resumption of blastocyst growth and with a decrease in plasma androgen values (DHA, androstenedione, testosterone). No definite pattern of oestrone was observed and oestradiol concentrations remained undetectable. The production of steroids by dispersed luteal cells showed that the growth of the corpora lutea paralleled that of blastocysts and resulted in hypertrophy followed by hyperplasia of the luteal cell. The production of progesterone in the medium increased with blastocyst size up to implantation; it was enhanced by mink charcoal-treated serum, but prolactin, LH, FSH or a combination of these hormones did not affect the progesterone production, whatever the stage of diapause. DHA and androstenedione secretion increased in the two last stages of blastocyst growth and was enhanced by LH. The conversion of androstenedione and testosterone into oestrone and oestradiol was observed at all stages of embryonic diapause, indicating that corpora lutea contain aromatase activity even at an early stage. The secretion of oestrone was higher than that of oestradiol. The non-luteal tissue contributed up to 50% of the steroid production; while progesterone and androgen production remained constant, that of oestradiol decreased at the end of the delay period. These results indicated a change in the size and the secretory capacity of the luteal cell related to blastocyst development and implantation. Although progesterone was the main product of the corpora lutea, androgens and oestrogens were also secreted.