Isometric contraction induces the Ca2+-independent activation of the endothelial nitric oxide synthase

Shear stress and tyrosine phosphatase inhibitors have been shown to activate the endothelial NO synthase (eNOS) in a Ca2+/calmodulin-independent manner. We report here that isometric contraction of rabbit aorta activates eNOS by a pharmacologically identical pathway. Endothelium-intact aortic rings were precontracted under isometric conditions up to 60% of the maximal phenylephrine-induced tone. The NO synthase inhibitor NGnitro-L-arginine (L-NA) and the soluble guanylyl cyclase inhibitor NS 2028 induced an additional contraction, the amplitude of which depended on the level of precontraction. The maximal production of NO by isometrically contracted aortic rings (as estimated by the increase in cGMP in detector smooth muscle cells in a superfusion bioassay) was observed during the initial phase of isometric contraction and was greater than that detected following the application of acetylcholine. The supplementary L-NA-induced increase in vascular tone was inhibited by the nonselective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A. Another tyrosine kinase inhibitor, genistein, the calmodulin antagonist calmidazolium, and the selective protein kinase C inhibitor, Ro 31-8220, had no effect. Coincident with the enhanced NO formation during isometric contraction was an increase in the tyrosine phosphorylation of endothelial proteins, which also correlated with the level of precontraction. Thus, isometric contraction activates eNOS via a Ca2+-independent, tyrosine kinase inhibitor-sensitive pathway and, like shear stress, seems to be an independent determinant of mechanically induced NO formation.     

Inhibition of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase by nitric oxide

The effects of nitric oxide on the activities of thapsigargin-sensitive sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Ca2+ uptake by sarcoplasmic reticulum (SR) membranes prepared from white skeletal muscle of rabbit femoral muscle were studied. Pretreatment of the SR preparations with nitric oxide at concentrations of up to 250 microM for 1 min decreased the SERCA activity concentration dependently, and also decreased their Ca2+ uptake. Both these effects of nitric oxide were reversible. Inhibitors of guanylyl cyclase and protein kinase G (PKG) had no significant effect on the nitric oxide-induced inhibitions of SERCA and Ca2+ uptake. Moreover, dithiothreitol did not reverse the inhibitory effects of nitric oxide on SERCA and Ca2+ uptake. These findings suggest that nitric oxide inhibits SERCA, mainly SERCA 1, of rabbit femoral skeletal muscle by an action independent of the cyclic GMP-PKG system or oxidation of thiols, and probably by a direct action on SERCA protein.     

Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway

With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba2+ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 microM) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTPgammaS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). In conclusion, the M2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.     

Ca2+ differentially regulates conventional protein kinase Cs' membrane interaction and activation

The regulation of conventional protein kinase Cs by Ca2+ was examined by determining how this cation affects the enzyme's 1) membrane binding and catalytic function and 2) conformation. In the first part, we show that significantly lower concentrations of Ca2+ are required to effect half-maximal membrane binding than to half-maximally activate the enzyme. The disparity between binding and activation kinetics is most striking for protein kinase C betaII, where the concentration of Ca2+ promoting half-maximal membrane binding is approximately 40-fold higher than the apparent Km for Ca2+ for activation. In addition, the Ca2+ requirement for activation of protein kinase C betaII is an order of magnitude greater than that for the alternatively spliced protein kinase C betaI; these isozymes differ only in 50 amino acids at the carboxyl terminus, revealing that residues in the carboxyl terminus influence the enzyme's Ca2+ regulation. In the second part, we use proteases as conformational probes to show that Ca2+dependent membrane binding and Ca2+-dependent activation involve two distinct sets of structural changes in protein kinase C betaII. Three separate domains spanning the entire protein participate in these conformational changes, suggesting significant interdomain interactions. A highly localized hinge motion between the regulatory and catalytic halves of the protein accompanies membrane binding; release of the carboxyl terminus accompanies the low affinity membrane binding mediated by concentrations of Ca2+ too low to promote catalysis; and exposure of the amino-terminal pseudosubstrate and masking of the carboxyl terminus accompany catalysis. In summary, these data reveal that structural determinants unique to each isozyme of protein kinase C dictate the enzyme's Ca2+-dependent affinity for acidic membranes and show that, surprisingly, some of these determinants are in the carboxyl terminus of the enzyme, distal from the Ca2+-binding site in the amino-terminal regulatory domain.     

Mast cell activation causes delayed neurodegeneration in mixed hippocampal cultures via the nitric oxide pathway

Mast cells are pleiotropic bone marrow-derived cells found in mucosal and connective tissues and in close apposition to neurons, where they play important roles in tissue inflammation and in neuroimmune interactions. Connective tissue mast cells, with which intracranial mast cells share many characteristics, contain cytokines that can cause inflammation. Here, we report that myelin basic protein, a major suspected immunogen in multiple sclerosis, as well as an antigenic stimulus, provokes mast cells to trigger a delayed cytotoxicity for neurons in mixed neuron-gila cultures from hippocampus. Neurotoxicity required a prolonged period (12 h) of mast cell incubation, and appeared to depend largely on elaboration of the free radical nitric oxide by astrocytes. Activation of astrocytes was mediated, in part, by mast cell-secreted tumor necrosis factor-alpha. Myelin basic protein and 17 beta-estradiol had a synergistic action on the induction of mast cell-associated neuronal injury. The cognate mast cell line RBL-2H3, when subjected to an antigenic stimulus, released tumor necrosis factor-alpha which, together with exogenous interleukin-1 beta (or interferon-gamma), induced astroglia to produce neurotoxic quantities of nitric oxide. A small but significant proportion of mast cell-derived neurotoxicity under the above conditions occurred independently of glial nitric oxide synthase induction. Further, palmitoylethanolamide, which has been reported to reduce mast cell activation by a local autacoid mechanism, decreased neuron loss resulting from mast cell stimulation in the mixed cultures but not that caused by direct cytokine induction of astrocytic nitric oxide synthase. These results support the notion that brain mast cells could participate in the pathophysiology of chronic neurodegenerative and inflammatory diseases of the nervous system, and suggest that down-modulation of mast cell activation in such conditions could be of therapeutic benefit.     

The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+

Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.     

Ca2+-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

The nitric oxide/cyclic GMP pathway: a potential major regulator of cochlear physiology

The nitric oxide (NO)/cyclic guanosine monophosphate (GMP) pathway is now recognized as a major regulatory system in cell physiology and tissue homeostasis. This pathway may control processes as diverse as muscle relaxation, gut peristalsis, neurotransmission and hormonal secretion. It is also involved in the development and function of sensory systems such as vision and olfaction. This review will detail the NO/cyclic GMP pathway, evaluate studies in the auditory system and discuss its potential participation in cochlear blood flow, supporting cell physiology and excitotoxicity.     

Inhibition of nitric oxide synthesis increases adenosine production via an extracellular pathway through activation of protein kinase C

At the time of diagnosis, many sarcoidosis patients have no clinical indication for corticosteroid therapy, and prognostic parameters predicting deterioration are missing. In the present study, we investigated parameters derived from bronchoalveolar lavage (BAL) and serum in 77 patients with recently diagnosed sarcoidosis to test their predictive value. Patients were divided into a group with (Group A, n = 37) and a group without (Group B, n = 40) indications for therapy, and the course of the disease was evaluated after 5.7 +/- 0.4 mo. The CD4+/CD8+ lymphocyte ratio and percentage of BAL lymphocytes were of no predictive value. Release of tumor necrosis factor-alpha (TNF-alpha) from cultured alveolar macrophages (AM) was significantly increased in both groups (Group A = 1,872 +/- 428 pg/ml; Group B = 1,561 +/- 449 pg/ml) as compared with controls (220 +/- 37 pg/ml). In Group B, however, patients with a high level of TNF-alpha release had a significantly greater risk of disease progression than did those with normal TNF-alpha release (43.8% versus 8.3%, respectively). From the serologic parameters investigated, consisting of neopterin, angiotensin converting enzyme (ACE), and soluble interleukin-2 receptor (sIL-2R), only the last was of significant predictive value; 42.1% of sarcoidosis patients in Group B with a high level of sIL-2R experienced disease progression, whereas none of those with a normal level did. We conclude that TNF-alpha release and sIL-2R are suitable parameters for predicting disease progression in sarcoid patients who have no indication for therapy at the time of disease diagnosis.     

Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms

Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor alpha-naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.     

Modulation of Ca2+ channels by cyclic nucleotide cross activation of opposing protein kinases in rabbit portal vein

Cyclic nucleotides are known to modify voltage-gated (L-type) Ca2+ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca2+ channel function by using both conventional and perforated-patch-clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 micromol/L), significantly increased (14% to 16%) peak Ba2+ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba2+ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba2+ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca2+ channel currents were significantly inhibited by the PKA blocker 8-Bromo-2'-O-monobutyryladenosine-3',5'-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 micromol/L) and enhanced by the PKG inhibitor beta-Phenyl-1,N2-etheno-8-bromoguanosine-3',5'-monophosphorothioate, Rp-isomer (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cGMPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGMP (0.1 mmol/L) enhanced Ba2+ currents (13% to 39%). The excitatory effect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo cAMP (0.05 mmol/L) and forskolin (10 micromol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba2+ currents. Ba2+ currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca2+ channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase.     

Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway

Nitric oxide (NO) has been invoked as an important pathogenic factor in a wide range of immunologically mediated diseases. The present study demonstrates that macrophage-derived NO may conversely function to fine tune T cell-mediated inflammation via reversible dephosphorylation of intracellular signaling molecules, which are involved in the control of T cell proliferation. Thus, T cells activated in the presence of alveolar macrophages are unable to proliferate despite expression of IL-2R and secretion of IL-2. This process is reproduced by the NO generator S-nitroso-N-acetylpenicillamine and is inhibitable by the NO synthase inhibitor N(G)-methyl-L-arginine. Analysis of T cell lysates by immunoprecipitation with specific Abs and subsequent immunoblotting indicated marked reduction of tyrosine phosphorylation of Jak3 and STAT5 mediated by NO. Further studies indicated that NO-mediated T cell suppression was reversible by the guanylate cyclase inhibitors methylene blue and LY-83583 and was reproduced by a cell-permeable analogue of cyclic GMP, implicating guanylate cyclase activation as a key step in the inhibition of T cell activation by NO.     

Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.     

Ether-à-go-go encodes a voltage-gated channel permeable to K+ and Ca2+ and modulated by cAMP

The Drosophila ether-à-go-go (eag) mutant is responsible for altered potassium currents in excitable tissue. These mutants exhibit spontaneous, repetitive firing of action potentials in the motor axons of larval neuromuscular junctions. The eag gene encodes a polypeptide that shares sequence similarities with several different ionic channel proteins, including voltage-gated potassium channels, an inward rectifier as well as cyclic-nucleotide-gated channels. These formal similarities in the derived primary sequences indicate that eag polypeptides might express a new type of ion channel. Here we report the expression by eag RNA in Xenopus oocytes of such a channel which incorporates properties of both voltage- and ligand-gated channels. The permeability of these eag channels to potassium and calcium is dependent on voltage and cyclic AMP. The ability to mediate potassium-outward and calcium-inward currents endows this channel with properties likely to be important in the modulation of synaptic efficiency in both central and peripheral nervous systems.     

Does a decrease in subplasmalemmal Ca2+ explain how store-operated Ca2+ channels are opened?

The activity of each of 99 intraparietal neurons was studied in three awake-behaving rhesus monkeys (Macaca mulatta) while subjects performed 100-900 delayed saccade trials. On each trial, a saccadic target was presented at one location selected randomly from a grid of 441 locations spanning 40 degrees of horizontal and vertical visual space. Individual neurons in our population were sensitive to both the direction and amplitude of saccades. Response fields, which plotted firing rate as a function of the horizontal and vertical amplitude of movements for each neuron, were characterized by a Cartesian two-dimensional gaussian model. The goodness-of-fit of these gaussian models was tested by: (1) comparing observed responses with predicted responses for each movement; and (2) by computing the percentage of variance explained by each model. Cartesian Gaussian models provided a good fit to the response fields of most neurons. Across our population, the Gaussian fit to the response field of each neuron accounted for more of the variance in neuronal activity when the data were plotted with regard to the horizontal and vertical amplitude of the saccade than when the same data were plotted with regard to the position of the saccadic target. The Gaussian functions were used to estimate the eccentricity and spatial tuning breadth of each neuronal response field. Modal response field radius was less than 5 degrees, whereas mean response field radius was about 10 degrees. Linear regression analysis demonstrated that response field eccentricity accounted for less than 30% of the variance in response field radius. Analysis of the horizontal distribution of response field centers showed an approximately normal distribution around central fixation. Most histologically recovered neurons were located on the lateral bank of the intraparietal sulcus, although a small number of saccade-related neurons were recorded from Brodmann's area 5 on the medial bank of the intraparietal sulcus.     

Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P < 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.     

Altered activation of the L-arginine nitric oxide pathway during and after cardiopulmonary bypass

This paper describes the incidence of coronal caries in a sample of older adults. A 3-year follow-up study was conducted of 493 community-dwelling adults aged 50 years and over in Ontario, Canada. The incidence of coronal caries was 57.0%, and the mean net DFS increment was 1.9 surfaces. In bivariate analysis, several variables were significantly associated with incidence and/or mean DFS increment. These included: age, marital status, baseline coronal DFS, number of teeth at baseline, mean periodontal attachment loss of 4 mm or more, and wearing partial dentures. In logistic regression analysis only four factors had significant independent effects. These were level of education, marital status, mean periodontal attachment loss and number of teeth at baseline. The predictive ability of this model was fair: accuracy 65.7%, sensitivity 80.2%, and specificity 46.2%. When logistic analysis was repeated separately for two age groups, different predictors had significant independent effects, and sensitivity and specificity values differed substantially. These findings indicate predictive models for caries incidence should include both clinical and non-clinical variables because both types of variables may help to explain different aspects of coronal caries experience. Further research is required to identify other factors associated with coronal caries in older adults.