Daphne Atlas

FUNCTIONAL INTERACTION OF SYNTAXIN AND SNAP-25 WITH VOLTAGE-SENSITIVE L- AND N-TYPE CA2+ CHANNELS

We have used an electrophysiological assay to investigate the functional interaction of syntaxin 1A and SNAP‐25 with the class C, L‐type, and the class B, N‐type, voltage‐sensitive calcium channels. Co‐expression of syntaxin 1A with the pore‐forming subunits of the L‐ and N‐type channels in Xenopus oocytes generates a dramatic inhibition of inward currents (>60%) and modifies the rate of inactivation (tau) and steady‐state voltage dependence of inactivation. Syntaxin 1–267, which lacks the transmembrane region (TMR), and syntaxin 2 do not modify channel properties, suggesting that the syntaxin 1A interaction site resides predominantly in the TMR. Co‐expression of SNAP‐25 significantly modifies the gating properties of L‐ and N‐type channels and displays modest inhibition of current amplitude. Syntaxin 1A and SNAP‐25 combined restore the syntaxin‐inhibited N‐type inward current but not the reduced rate of inactivation. Hence, a distinct interaction of a putative syntaxin 1A‐SNAP‐25 complex with the channel is apparent, consistent with the formation of a synaptosomal SNAP receptors (SNAREs) complex. The in vivo functional reconstitution: (i) establishes the proximity of the SNAREs to calcium channels; (ii) provides new insight into a potential regulatory role for the two SNAREs in controlling calcium influx through N‐ and L‐type channels; and (iii) may suggest a pivotal role for calcium channels in the secretion process.

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis

Increasing evidence suggests that enhanced production of reactive oxygen species (ROS) activates the MAP kinases, c‐Jun N‐terminal protein kinase (JNK) and mitogen‐activated protein kinase MAPK (p38). These phosphorylated intermediates at the stress‐activated pathway induce expression of matrix metalloproteinases (MMPs), leading to inflammatory responses and pathological damages involved in the etiology of multiple sclerosis (MS). Here we report that N‐acetylcysteine amide (AD4) crosses the blood–brain barrier (BBB), chelates Cu2+, which catalyzes free radical formation, and prevents ROS‐induced activation of JNK, p38 and MMP‐9. In the myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, oral administration of AD4 drastically reduced the clinical signs, inflammation, MMP‐9 activity, and protected axons from demylination damages. In agreement with the in vitro studies, we propose that ROS scavenging by AD4 in MOG‐treated animals prevented MMPs induction and subsequent damages through inhibition of MAPK pathway. The low toxicity of AD4 coupled with BBB penetration makes this compound an excellent potential candidate for the therapy of MS and other neurodegenerative disorders.

Imidazoline receptors in rat liver cells : a novel receptor or a subtype of α2-adrenoceptors ?

An imidazoline/guanidine receptor has been characterized in rat liver cells. Binding of [3H]idazoxan, a selective benzodioxan antagonist, to imidazoline receptor on intact fresh hepatocytes (Bmax = 801 +/- 23 fmol/mg protein, Kd = 11 +/- 0.8 nM) and to liver membranes (Bmax = 400 +/- 38 fmol/mg protein, Kd = 10 +/- 2 nM) was saturable at 4 degrees C within 3.5 h and at 30 degrees C within 30 min, respectively. Rat lung membranes had more imidazoline sites (Bmax = 578 +/- 30 fmol/mg protein, Kd = 14 +/- 1.4 nM) than alpha 2-adrenoceptors (Bmax = 175.0 +/- 20.0 fmol/mg protein, Kd = 4.8 +/- 2.0 nM). We also screened other tissues for imidazoline sites; the ratio of adrenoceptors to total sites labeled with [3H]idazoxan displaced by cirazoline was lower in rat lung compared to rat brain and human platelets. The imidazoline receptor has common pharmacological properties with alpha 2-adrenoceptors, although it is not a subtype of the adrenoceptor, since it bound neither the endogenous agonists norepinephrine and epinephrine, nor the selective alpha 2-antagonists yohimbine and phentolamine. All guanidine type alpha 2-adrenoceptor drugs (e.g. guanbenz, guanoxan) and imidazolines (e.g., UK-14,304, naphazoline) competed with high affinity for the liver imidazoline receptor. The lack of effect by Gpp(NH)p, a non-hydrolysable GTP analogue, on the affinity of guanidine- and imidazoline-type ligands for liver imidazoline receptors suggests that the mode of action of these drugs at imidazoline receptors is different than at conventional alpha 2-adrenoceptors. Ionic changes were considered as a possible mechanism underlying the alpha 2-adrenoceptor effects in various cells. Opening of K+ channels by alpha 2-adrenoceptors agonists is a pathway which might be shared by imidazoline-type agonists at imidazoline sites. Indeed, 4-aminopyridine, a K+ channel blocker, inhibited the specific binding of [3H]idazoxan to liver cells with an IC50 of 0.34 +/- 0.07 mM a concentration which is effective in blocking K+ channels in neuronal cells. Similarly, Cs+ and NH4+ effectively interfered with [3H]idazoxan binding, suggesting a possible coupling of imidazoline sites to K+ gating. The endogenous ligand clonidine-displacing substance (CDS), which was isolated from bovine brain and which binds to alpha 2-adrenoceptors in brain membranes and human platelets competed with idazoxan at rat liver imidazoline receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Imidazoline binding sites in human placenta: evidence for heterogeneity and a search for physiological function.

1 An α2‐adrenoceptor antagonist, idazoxan, that binds to both α2‐adrenoceptors and to imidazoline sites (IR), has been used to characterize human placental IR. Human placenta is shown to be the richest source of IR (1800 ± 100 fmol mg−1 protein; Kd 38.9 ± 3.4 nm). 2 Primary cells derived from human placenta and grown in monolayers, also displayed a high density of receptors (3209 ± 136 fmol mg−1 in cytotrophoblasts and 3642 ± 144 fmol mg−1 protein in syncytiotrophoblast enriched cell culture). 3 [3H]‐idazoxan did not show binding characteristics of α2‐adrenoceptors in human placental membranes or human trophoblastic cells, thus making it a ligand of choice to study the imidazoline site. The tissue appeared to be lacking α2‐adrenoceptors in that other α2‐adrenoceptor ligands, [3H]‐rauwolscine and [3H]‐clonidine, do not bind to α2‐adrenoceptors in human placenta. 4 IRs are localized on the cell surface, as determined by the release of bound [3H]‐idazoxan from cells, when washed with high ionic/acidic medium. 5 Imidazoline receptors of human placenta display high affinity for amiloride (72 ± 27 nm). The high affinity was used as a criterion to classify IR to IRa subtype (placenta, rabbit kidney, rabbit liver and rabbit adipose cells) as opposed to the IRb subtype which display low affinity for amiloride (> 2 μm, in all the other tissues). 6 Several novel ligands comprising a guanido functional group attached to an aromatic residue (e.g. benziliden‐amino‐guanidine (BAG), guanido pyrole) display pronounced selectivity for IR over the α2‐adrenoceptors as the affinity of BAG is about 40 fold higher (Kd = 18.9 ± 13.8 nm in human placenta), than the affinity for α2‐adrenoceptors (Kd = 768 ± 299 nm in human platelets). Imidazoline sites bind selectively BAG and other guanido ligands thus indicating a distinct structural requirement at its site of binding. 7 K+ channel blockers and monovalent ions (e.g. Cs+ and NH4+) interfere with idazoxan binding to IR, indicating a possible involvement of IR in K+ transport.

An endogenous, non-catecholamine clonidine antagonist increases mean arterial blood pressure

We report here that topical application of clonidine displacing substance (CDS), an endogenous brain extract, directly in the nucleus reticularis lateralis (NRL) region of anaesthetized cats regularly produced hypertension. CDS (5 units) increased the mean blood pressure by 40 +/- 8%. Pretreatment of anaesthetized rabbits with intracisternal CDS (500 units) shifted to the right the dose-response curve obtained with clonidine alone injected the same way. This brain extract might be considered as an endogenous antagonist for the hypotensive effects of clonidine at least in the NRL region.

Synaptotagmin restores kinetic properties of a syntaxin‐associated N‐type voltage sensitive calcium channel

© 1997 Federation of European Biochemical Societies.

Isolation of an endogenous clonidine-displacing substance from rat brain

An endogenous substance which specifically displaces clonidine, yohimbine and rauwolscine from rat brain α2‐adrenergic receptors, has been isolated. The new compound, designed clonidine‐displacing‐substance (CDS), has been partially purified by ion exchange chromatography, zone electrophoresis and high performance liquid chromatography (HPLC). CDS binds specifically to α2‐adrenergic receptors by competing with either α2‐adrenergic agonists or α2‐antagonists, but has no effect on the specific binding of [3H]prazosin to α1‐adrenergic receptors in rat brain membranes. In the course of isolation, CDS was shown to be neither the endogenous neurotransmitter (−)norepinephrine (NE) nor the guanyl nucleotide GTP which lowers the specific binding of α2‐agonists to the α2‐adrenergic receptors.

Ligand specificity and characteristics of the β-adrenergic receptor in turkey erythrocyte plasma membranes

Detailed kinetic studies of adenylate cyclase activation by catecholamines in turkey erythrocyte plasma membranes were carried out in order to characterize the ligand specificity of the β -adrenergic receptor. A general kinetic method was used to evaluate the affinity of the β -receptor towards full-agonists, partial agonists and antagonists. This method allowed us to study ligands that bind with high affinity as well as ligands that bind with low affinity. Direct binding studies of [ 3 H]propranolol to the β -receptor were used to study the ligand specificity and the modulation of that specificity by regulatory ligands known to activate or inhibit adenylate cyclase stimulation by l -catecholamines. A very close correspondence between the binding constants obtained by the kinetic method and the binding method was found. It was found that the allosteric activator of adenylate cyclase, guanylylimidodiphosphate, and the allosteric inhibitor, Ca 2+ , do not affect propranolol binding.

A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha.

Reactive oxygen species (ROS) play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. Recent studies have demonstrated that antioxidants are able to reduce airway inflammation and hyperreactivity in animal models of allergic airway disease. A newly developed antioxidant, small molecular weight thiol compound, N-acetylcysteine amide (AD4) has been shown to increase cellular levels of glutathione and to attenuate oxidative stress related disorders such as Alzheimers disease, Parkinsons disease, and multiple sclerosis. However, the effects of AD4 on allergic airway disease such as asthma are unknown. We used ovalbumin (OVA)-inhaled mice to evaluate the role of AD4 in allergic airway disease. In this study with OVA-inhaled mice, the increased ROS generation, the increased levels of Th2 cytokines and VEGF, the increased vascular permeability, the increased mucus production, and the increased airway resistance in the lungs were significantly reduced by the administration of AD4. We also found that the administration of AD4 decreased the increases of the NF-κB and hypoxia-inducible factor-1α (HIF-1α) levels in nuclear protein extracts of lung tissues after OVA inhalation. These results suggest that AD4 attenuates airway inflammation and hyperresponsiveness by regulating activation of NF-κB and HIF-1α as well as reducing ROS generation in allergic airway disease.