Nitric oxide directly activates large conductance Ca2+-activated K+ channels (rSlo).

We investigated whether nitric oxide (NO) directly activates the cloned alpha-subunit of large conductance Ca2+-activated K+ (Maxi-K) channels from rat brain (rSlo), expressed either in HEK293 cells or Xenopus oocytes. In inside-out patches, the application of S-nitroso-N-acetylpenicillamine (SNAP), a NO-releasing compound, reversibly activated the channel shifting the voltage dependent activation curve of the macroscopic Maxi-K current to the left by about 15 mV. Pretreatment of the patches with N-ethylmaleimide to alkylate free sulfhydryl groups did not prevent the effect of SNAP, suggesting that NO may directly interact with the channels. These results suggest that Maxi-K channels might be one of the physiological targets of NO in the brain.

Functional characteristics of two BKCa channel variants differentially expressed in rat brain tissues.

cDNAs encoding large-conductance Ca2+-activated K+ channel alpha-subunit (rSlo) were obtained from rat brain. From the DNA sequence of multiple rslo clones, we identified a specific sequence variation of 81 nucleotides, which is either absent from or present at the N-terminal region of a putative Ca2+-sensing domain of the channel. Transcripts containing such variations were detected in different ratios from several brain regions, and their functional significance was further examined. When heterologously expressed in Xenopus oocytes, both rSlo variants, named rSlo0 and rSlo27, generated Ca2+-activated and voltage-activated K+ currents characteristic of neuronal large-conductance Ca2+-activated K+ (BKCa) channels. Single-channel recordings of the two channels showed almost identical permeation characteristics and steady-state gating behavior. Noticeable differences between rSlo0 and rSlo27 were revealed when the macroscopic currents were measured at various voltages and intracellular Ca2+ concentrations. rSlo27 activated was more rapidly than rSlo0 in the presence of the same voltage stimulus, and the differences in these activation kinetics were dependent on the concentration of intracellular Ca2+. Despite their similar apparent affinities for Ca2+, rSlo0 and rSlo27 showed significant differences in their co-operative gating behavior. The Hill coefficient for intracellular Ca2+ was estimated to be about 3.7 for rSlo27 regardless of the membrane voltage, and that for rSlo0 was reduced from about 5 to 2 as the membrane voltage changed from 40 to 140 mV. As activation of BKCa channels is involved in rapid hyperpolarization of action potentials, the differential processing of rslo transcripts, and the generation of channels with different activation kinetics and Ca2+ cooperativity may be a mechanism for tuning the excitability of neurons in different brain regions.

Regulation of renal laminin in mice with type II diabetes.

This study examines the regulation of renal laminin in the db/db mouse, a model of type II diabetes characterized by extensive remodeling of extracellular matrix. Immunohistochemistry demonstrated an increase in the contents of laminin chains including beta1 chain in the mesangium and tubular basement membranes at 1, 2, 3, and 4 mo of diabetes. Immunofluorescence with an antibody against the recently discovered laminin alpha5 chain showed that in the normal mouse, the protein had a restricted distribution to the glomerular and tubular basement membranes with scant expression in the mesangium of older mice. In the diabetic mouse, the laminin alpha5 chain content of the glomerular and tubular basement membranes was increased, with marked expression in the mesangium. Northern analysis revealed a significant decrease in the renal cortical contents of alpha5, beta1, and gamma1 chain mRNA in the diabetic mice compared to control, at each of the time points. In situ hybridization showed decreased abundance of alpha5 transcripts in the glomeruli of diabetic mice compared to nondiabetic controls. Analysis of mRNA changes by Northern and in situ hybridization studies demonstrated that the reduction in laminin transcripts involved both glomerular and tubular elements. These observations demonstrate that laminin accumulation in the db/db mice with type II diabetes is due to nontranscriptional mechanisms. Because previous investigations in rodents with type I diabetes have shown that the increase in renal laminin content was associated with a corresponding increment in laminin chain transcript levels, it appears that the mechanisms underlying augmentation in renal matrix laminin content may be distinct in the two types of diabetes.

The regulation of prodynorphin gene expression in cultured spinal cord cells: involvement of second messengers.

The regulation of prodynorphin (proDYN) mRNA levels by cAMP and protein kinase C (PKC) pathways was studied in cultured rat spinal cord cells. Spinal cord cells were cultured from 14 day (E 14) embryos of Sprague-Dawley rats. After 7 days in vitro, the spinal cord cells were incubated with either forskolin (5 microM) or phorbol-13-myristate acetate (PMA; 2.5 microM) for 1, 3, 6, 9, 12, or 24 h and the total RNA was isolated for Northern blot analyses. The proDYN mRNA level began to increase 1 h, then reached and remained at a peak 3-6 h after stimulation by forskolin or PMA. proDYN mRNA levels in forskolin treated cells decreased slightly from their peak after 9 h of treatment, whereas the level of proDYN mRNA returned to the basal level in PMA-treated cells. Pretreatment of cells with cycloheximide (a protein synthesis inhibitor; 10 microM) did not affect the forskolin- or PMA-induced increase in proDYN mRNA, but pretreatment with nimodipine (a L-type Ca2+ channel blocker; 2 microM), omega-conotoxin (a N-type Ca2+ channel blocker; 1 microM), or KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor; 5 microM) inhibited induction of proDYN mRNA both by forskolin and PMA. Additionally, dexamethasone did not affect the expression of proDYN mRNA level induced by forskolin. Our results suggest that proDYN mRNA levels in spinal cord cells is regulated by both cAMP and PKC pathways. Calcium influx through both L- and N-type calcium channels and Ca2+/calmodulin-dependent protein kinase II appear to be involved in the increase of proDYN mRNA levels induced by either forskolin or PMA. Furthermore, ongoing protein synthesis is not required for forskolin- or PMA-induced responses.

Molecular mechanisms underlying the regulation of proenkephalin gene expression in cultured spinal cord cells.

The regulation of proenkephalin (proENK) mRNA levels by cAMP and protein kinase C (PKC) pathways was studied in cultured rat spinal cord cells in the present study. Spinal cord cells were cultured from 14 day (E 14) embryos of Sprague-Dawley rats. After 7 days in vitro, the spinal cord cells were incubated with either forskolin (5 microM) or phorbol-13-myristate acetate (PMA; 2.5 microM) for 1, 3, 6, 9, 12 or 24 h and total RNA and proteins were isolated for Northern and Western blot analyses. The proENK mRNA level began to increase within an hour, then reached and remained at a peak 3-12 h after stimulation by both forskolin and PMA. The increased proENK mRNA level in forskolin-treated cells was slightly decreased 24 h after the stimulation, whereas the level of proENK mRNA returned to basal levels in PMA-treated cells. A Western blot assay revealed that the intracellular level of proENK protein was not changed by treatment with either forskolin or PMA. Pretreatment of cells with cycloheximide (a protein synthesis inhibitor; 10 microM) did not affect the forskolin- or PMA-induced increase of proENK mRNA. However, pretreatment with nimodipine (an L-type Ca2+ channel blocker; 2 microM), omega-conotoxin (an N-type Ca2+ channel blocker; 1 microM), calmidazolium (a calmodulin antagonist; 1 microM) or KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor; 5 microM) attenuated the forskolin- or PMA-induced increase of proENK mRNA levels. Dexamethasone (1 microM) did not affect the forskolin-induced increase of proENK mRNA levels. Our results suggest that the elevation of proENK mRNA levels in the spinal cord is regulated by both cAMP and PKC pathways. Calcium influx through both L- and N-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II appear to be involved in the increase of proENK mRNA levels induced by either forskolin or PMA. Furthermore, ongoing protein synthesis is not required for forskolin- or PMA-induced alterations in proENK mRNA.

Cerebral vasculitis in Henoch-Schönlein purpura: a case report with sequential magnetic resonance imaging.

Neurological complications are rare during the course of Henoch-Schönlein purpura (HSP). We report a 5-year-old girl with HSP who presented with seizures. Sequential magnetic resonance imaging and electroencephalography showed bilateral multifocal cerebral lesions initially, which gradually and completely resolved with clinical improvement. These lesions were compatible with the radiological pattern of the non-hemorrhagic vasculitic involvement of cerebral parenchyma.

Antifolates in rheumatoid arthritis: a hypothetical mechanism of action.

The antifolates, methotrexate, aminopterin, 10-deazaaminopterin and sulfasalazine are clinically useful in the treatment of rheumatoid arthritis. Toxicity, rather than efficacy, appears to the the major factor limiting the usefulness of the classical antifolates (i.e., methotrexate and 10-deazaaminopterin). The fact that folate supplementation of methotrexate-treated rheumatoid arthritis patients reduces toxicity without altering efficacy also suggests that inhibition of the drug's target enzyme, dihydrofolate reductase, is not complete and not essential for efficacy. Since polyglutamates of methotrexate are direct inhibitors of thymidylate synthase and folate dependent enzymes of purine biosynthesis, the efficacy of this agent may involve blockade of these pathways. We hypothesize that blockage of aminoimidazole carboxamide ribotide transformylase, the folate dependent enzyme responsible for the insertion of carbon 2 into the purine ring, produces an immunosuppression mediated by secondary inhibition of adenosine deaminase, and S-adenosyl homocystein hydrolase by aminoimidazolecarboxamide metabolites. This mechanism of immunosuppression may explain the clinical effect of methotrexate, 10-deazaaminopterin, and possibly sulfasalazine. Since purine biosynthesis is a fundamental process, blockading this pathway may also decrease leukotriene production and interleukin-1 expression, which also could contribute to the efficacy of methotrexate.