Biomedical subjects

D Atlas

Publications and source records attributed to D Atlas.

At least 55 records · Page 3Linked to original sources

PubMed1989

Synergy between membrane depolarization and muscarinic receptor activation leads to potentiation of neurotransmitter release (II)

Amplification of muscarinic agonist-induced [3H]noradrenaline ([3H]NE)-release by depolarizing agents, was studied in rat brain cortical slices. [3H]NE basal outflow was enhanced by either K+ (25 mM) or veratridine (2 microM) in a Ca2+-dependent manner and was potentiated beyond additivity in the presence of muscarinic agonists. Facilitation of the [3H]NE-induced release by the simultaneous presence of muscarinic agonists and depolarizing agents is calcium-dependent with a maximal effective concentration of 0.6-0.8 mM. The efficacy of muscarinic agonist to induce basal outflow of [3H]NE is as follows: CCh greater than arecoline greater than oxotremorine M greater than bethanechol greater than pilocarpine, which is similar to their potentiatory effects observed in the presence of depolarizing agents. Potentiation of muscarinic agonist-induced release of [3H]NE by elevated K+ is more pronounced (up to 7-fold) in comparison to potentiation by veratridine (up to 4-fold), irrespective of the various muscarinic agonists. The sequential presence of muscarinic agonists followed by depolarizing agents is not sufficient for eliciting a synergy of [3H]NE outflow, whether receptor activation was initiated prior to depolarization or depolarization was initiated prior to receptor activation. Receptors which do not mediate phosphatidyl inositol (PI) turnover such as nicotine induced [3H]NE-release which is not affected by the presence of depolarizing agents and yielded in their presence additive fractional release only. In this report we establish synergy of [3H]NE release by muscarinic agonists under depolarizing conditions, similar to synergism of inositol phosphate (IP) production which was observed by muscarinic agonists and depolarization agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Characterization of an imidazoline/guanidinium receptive site distinct from the alpha 2-adrenergic receptor.

alpha 2-Adrenergic receptors recognize a number of molecules with diverse chemical structures, including the yohimban diastereoisomers yohimbine and rauwolscine, catecholamines, guanidinium analogs, and imidazolines, such as clonidine. The affinity of the receptor protein for some of these ligands can vary by 10-100-fold among various tissues and species, suggesting a heterogeneous class of binding sites. Certain cellular effects elicited by the compounds possessing an imidazoline or guanidinium moiety may actually be mediated by a membrane receptor distinct from the alpha 2-adrenergic receptor. To determine whether this imidazoline/guanidinium receptive site (IGRS) and the alpha 2-adrenergic receptor represent distinct proteins, we solubilized and partially characterized the two binding sites in rabbit kidney. This tissue expresses both alpha 2-adrenergic receptors and high affinity imidazoline/guanidinium binding sites, the latter which are rauwolscine-insensitive but can be identified with the benzodioxan [3H]idazoxan. The IGRS and alpha 2-adrenergic receptor in rabbit kidney exhibit distinct ligand recognition properties, which are maintained after solubilization and partial purification. In addition, the two receptors can be physically separated by heparin-agarose or lectin affinity chromatography indicating that the two binding sites are distinct entities. [3H]Idazoxan binding is trypsin-sensitive, indicating that the IGRS is a protein rather than a lipid component of the plasma membrane. [3H]Idazoxan binding is not inhibited by endogenous agonists for known neurotransmitter receptors. However, the IGRS does recognize clonidine-displacing substance, a small non-catechol compound isolated from calf brain, suggesting the existence of a previously uncharacterized hormonal/neurotransmitter receptor system.

Animals↗

PubMed1989

Cholinergic-induced [3H] noradrenaline release in rat brain cortical slices is mediated via a pertussis toxin sensitive GTP binding protein and involves activation of protein kinase C.

The involvement of a GTP-binding protein (G-protein) in the process of neurotransmitter release was examined using pertussis toxin and cholera toxin. Cholinergic agonists are shown to mediate [3H]noradrenaline release in rat brain slices via a pertussis toxin (1.2 micrograms/ml) sensitive, and cholera toxin (0.5 microgram/ml) insensitive G-protein. An indication for the involvement of a G-protein and phospholipase C activation in the release process was implied from the inhibitory effect of neomycin on K+-, veratridine- and carbachol-induced-norepinephrine release. Depolarizing agents mediate a neomycin-sensitive release, which is not which is not affected either by pertussis toxin or cholera toxin, suggesting a different mode of phospholipase C activation, unlike carbachol-induced release, which is both neomycin and pertussis toxin sensitive. Similarly, a hormone-sensitive carrier activated by phenylephrine not via alpha 1-adrenergic receptors, mediates a non-exocytosis efflux which is not affected by neomycin and is shown to be pertussis toxin-insensitive. The inhibitory action of protein kinase C inhibitors polymyxin B, K252a and H-7 [(1-(5-isoquinolinesulphonyl)-2-methyl-piperazine] on release, strongly suggests its participation in the process. Polymyxin B, a relatively selective protein kinase C inhibitor, inhibited carbachol-induced release (IC50 = 0.53 microM) as well as the K+ and the veratridine induced [3H] noradrenaline release, K252a, an inhibitor of various protein kinases at the ATP site, and H-7, another protein kinase C inhibitor, inhibited carbachol-induced noradrenaline released with IC50 = 35 nM and 3 microM respectively. Consistent with its inability to activate phospholipase C, phenylephrine-induced noradrenaline efflux was unaffected by polymyxin B (greater than 70 microM). These results offer more supportive evidence for a major role played by the dual messengers inositol trisphosphate and diacylglycerol (IP3/DG) in the mechanisms of neuronal release.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1988

Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation.

The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.

Animals↗

PubMed1988

Muscarinic agonists evoke neurotransmitter release: possible roles for phosphatidyl inositol bisphosphate breakdown products in neuromodulation.

Carbachol (CCh), a muscarinic agonist that elicits the formation of inositol trisphosphate (IP3) and diacylglycerol (DG), induces a calcium-dependent [3H]norepinephrine ([3H]NE) release [IC50 = (2.7 +/- 0.5) X 10(-4) M] in rat brain slices. Similarly, other muscarinic agonists evoke [3H]NE release which is specifically inhibited by muscarinic antagonists such as 3-quinuclidinyl benzilate, atropine, and N-methyl-4-piperidyl benzilate. The atropine-sensitive evoked release is effectively inhibited by neomycin (IC50 = 50 microM), a phospholipase C inhibitor that interferes with IP3-dependent cellular processes. In addition, polymyxin B, a rather selective inhibitor of protein kinase C (PK-C), abolishes the agonist-mediated release with a half-maximal effective concentration of 0.53 microM (750 ng/ml). These results have a significant implication for the mechanism by which agonists generating IP3 and DG act as inducers of neurotransmitter release in the CNS. However, since both neomycin and polymyxin B act also as N-calcium-channel blockers, other possible mechanisms are discussed. The CCh-induced release suggests that in the CNS an agonist-receptor interaction leads to a calcium-dependent neurotransmitter release, most likely via promoting the IP3/DG as second messengers followed by activation of PK-C.

Animals↗

PubMed1987

A low molecular weight brain substance interacts, similarly to clonidine, with alpha 2-adrenoceptors of human platelets.

In the present study, we explored the effects of a clonidine-displacing substance (CDS) which was isolated and partially purified from bovine brain. The low molecular weight brain substance competes with clonidine and rauwolscine in rat brain membranes, and mimics clonidine's inhibitory action in rat vas deferens. We find that CDS competes with [3H]rauwolscine-labeled alpha 2-adrenoceptors in human platelets. Further characterization of CDS in human platelets reveals that, like clonidine, it inhibits the epinephrine-induced aggregation, potentiates the ADP- and the collagen-induced aggregation however, by itself, CDS is unable to induce aggregation. Unlike clonidine, CDS does not affect the prostacyclin (PGI2)-stimulated cAMP accumulation in intact platelets. The presence of CDS in human plasma, as we have recently shown, implies a possible role of CDS in the regulation of platelet action.

Animals↗

PubMed1987

Neomycin inhibits K+- and veratridine-stimulated noradrenaline release in rat brain slices and rat brain synaptosomes.

The possible involvement of phosphoinositides' turnover in the process of neurotransmitter release in the central nervous system (CNS) was studied using rat brain slices and synaptosomes. A depolarizing concentration of potassium chloride (25 mM) induces an 8.6 +/- 0.4% increase of [3H]noradrenaline [( 3H]NA) fractional release in cerebral cortical slices above spontaneous release, and 15 mM KCl induces a 3-fold increase of [3H]NA release in rat brain synaptosomes. Neomycin, an aminoglycoside which binds phosphoinositides, inhibits the potassium-induced release in cortical slices with an IC50 = 0.5 +/- 0.07 mM and with IC50 = 0.2 +/- 0.03 mM in synaptosomes. Veratridine, a veratrum alkaloid which increases membrane permeability to sodium ions and causes depolarization of neuronal cells, induces a net 13.4 +/- 0.3% increase of [3H]NA fractional release above spontaneous release in cortical slices. In analogy to K+ stimulation, neomycin inhibits the veratridine-stimulated release in cortical slices with an IC50 = 0.65 +/- 0.1 mM. It appears that the recycling of phosphoinositides, which is necessary for Ca2+ mobilization, participates in the Ca2+-dependent induced neurotransmitter release in the central nervous system.

Animals↗

PubMed1987

The brain's own clonidine: purification and characterization of endogenous clonidine displacing substance from brain.

An endogenous clonidine-like substance was isolated from bovine brain and characterized by its chemical and pharmacological properties. The clonidine displacing substance (CDS) is a noncatecholamine, nonpeptide compound. It is devoid of NH2-group, is thermostable, and has a molecular weight of 587.8 +/- 2 daltons. CDS binds selectively to alpha 2-adrenergic receptors in rat brain membranes as measured by displacement of specifically bound [3H]clonidine, an alpha 2-adrenergic agonist, and [3H]rauwolscine, an alpha 2-antagonist, with no affinity for alpha 1-adrenergic receptors. In physiological studies CDS mimics clonidine's action as an inhibitor of the electrically induced twitch response and as a partial agonist of the epinephrine-induced platelet aggregation. At the central nervous system CDS antagonizes clonidine action, increases mean arterial pressure when injected into the nucleus reticularis lateralis of rats, and reduces the hypotensive effect upon intracisternal injection. CDS, which interacts at alpha 2-adrenergic receptors and increases mean arterial pressure upon intracerebral injection, may represent a neuroregulator that plays an important role in cardiovascular events.

Animals↗

PubMed1987

Central cardiovascular effects of a noncatecholamine endogenous ligand for clonidine receptors.

Cats and rats anaesthetized with pentobarbital were used to study the central cardiovascular effects of the clonidine displacing substance (CDS). The potential influence of centrally applied CDS on the cardiovascular effects of clonidine was also investigated in both species. CDS is a brain substance which is not a catecholamine (CA) and which displaces completely the binding of tritiated clonidine to brain membranes. It was observed that direct application of CDS unilaterally to the nucleus reticularis lateralis (NRL), a privileged site of action for clonidine in the cat, increases markedly the mean arterial pressure (MAP) and does not affect the heart rate (HR). CDS also causes hypertension when infused into the left vertebral artery in the cat. When MAP reverted to baseline, the hypotensive effect of clonidine given by the same route is significantly prevented. In rats, the intracisternal administration of CDS increased MAP without affecting HR. Here also, the CDS pretreatment antagonized the hypotensive effect of i.v. clonidine as compared with control animals. It is concluded that CDS is an endogenous non-catecholamine ligand for receptors involved in the hypotensive effect of clonidine. This substance interferes with clonidine on these receptors. It is a candidate as a neuromodulator or a neurotransmitter involved in the blood pressure regulation at least in the NRL region.

Animals↗

PubMed1987

Raised levels of an endogenous nonadrenergic substance in the serum of pregnancy-induced hypertension patients.

A clonidine-displacing substance (CDS) was isolated from bovine brain and shown to mimic clonidine in peripheral tissues. CDS acts--contrary to clonidine--by increasing the mean arterial pressure in the CNS. Methanolic extract from sera of patients with pregnancy-induced hypertension (PIH) showed a significant increase in CDS levels as compared with sera of either normal or chronic hypertensive pregnancy. Although small amounts of CDS were found in the placenta, no positive correlation between placental levels of CDS and serum CDS levels was observed. The pharmacological properties of CDS and its higher levels in sera of PIH patients suggest that CDS is a sympathetic agent involved in the pathophysiology of PIH.

Adult↗

PubMed1986

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.

Animals↗

PubMed1986

An endogenous brain substance, CDS (clonidine-displacing-substance), inhibits the twitch response of rat vas deferens.

The effect of CDS, an endogenous brain substance that specifically displaces bound [3H]clonidine and [3H]rauwolscine in rat brain membranes and human platelets, has been tested in isolated, field-stimulated rat vas deferens. CDS, obtained after an extensive purification procedure as a single peak from an HPLC sizing column, inhibited the electrically stimulated rat vas deferens similarly to the inhibitory action of clonidine, an alpha 2-agonist. The effective dose of CDS as an inhibitor of the vas deferens is equivalent to its effective dose in displacing specifically bound [3H]-clonidine in rat brain membranes. Furthermore, the CDS inhibition of the twitch response is reversed by two alpha 2-adrenergic antagonists, yohimbine and phentolamine. From these results, it is suggested that CDS extracted from brain, with affinity for clonidine sites, may be involved in the nonadrenergic fast response of the sympathetic transmission of the vas deferens.

Animals↗

PubMed1986

Respiratory monitoring with a new impedance plethysmograph.

A new impedance plethymograph respiratory monitor, (AR-8800), was tested on anaesthetised dogs. Decreases in tidal volume and total apnoea were brought about by partial and complete airway obstruction and by partial and total muscle paralysis. The changes in respiration were reliably detected by the monitor and its alarms activated within 15-20 seconds. The misinterpretation of gross body movement as normal breathing did not occur. Our findings suggest that the AR-8800 is a safe and dependable monitor which has distinct advantages and may be of particular use in the monitoring of patients at risk of developing respiratory depression or obstruction, whether in the intensive care unit, the general ward or at home.

Animals↗

PubMed1985

Beta-adrenergic activity and conformation of the antihypertensive specific alpha 2-agonist drug, guanabenz.

In recent research a new series of specific drugs, one of which is guanabenz (GBZ, 2,6(dichlorobenzyliden)-aminoguanidine) has been introduced into the clinical treatment of centrally mediated hypertension. Guanabenz (GBZ) is considered to be among the most specific alpha 2-adrenergic agonists, acting similarly to clonidine by decreasing the sympathetic outflow from the brain to the peripheral circulatory system. In the present report we show that GBZ displays a significant affinity for beta-adrenoceptors. In displacement studies of the iodinated beta-antagonist [125I]cyanopindolol (CYP) from turkey erythrocyte membranes, the dissociation constant of GBZ was 3.8 microM. Inhibition of the (-) epinephrine induced adenylate cyclase activity by GBZ is competitive, with an apparent dissociation constant of 30 microM. A similar value was obtained by studies of GBZ's effect on the (-) epinephrine-induced [3H]cAMP accumulation in intact turkey erythrocytes. In view of its unexpected affinity for beta-adrenoceptors, we examined the three-dimensional structure of crystalline GBZ. In these studies substantial differences between clonidine and GBZ were observed, despite their strong structural resemblance. These dissimilarities (angle of rotation phi = 39.7 degrees as compared to 76 degrees in clonidine, and the rotational restriction of clonidine as compared to the greater mobility in rotation of GBZ) could explain the difference of specificity between these two compounds.

Adenylyl Cyclases↗

PubMed1984

Isolation and partial purification of a clonidine-displacing endogenous brain substance.

A new compound, designated clonidine-displacing substance (CDS), has been isolated from calf brain by ion-exchange chromatography, zone electrophoresis and high-performance liquid chromatography. CDS binds specifically to alpha 2-adrenergic receptors in rat brain and human platelet membranes, as measured in direct binding experiments using [3H]clonidine and [3H]yohimbine respectively. Unlike clonidine or other alpha 2-agonists, CDS does not affect basal levels of adenylate cyclase in human platelets at the highest concentrations obtainable. The apparent molecular mass of the compound is estimated to be 500 +/- 50 Da, as determined by gel-filtration chromatography on Sephadex G-15. The new compound is thermostable, not affected by proteolytic enzymes, such as trypsin, chymotrypsin, pronase, papain and pyroglutamase, or by boiling in 0.2 M HCl for 5 min. It does not bind to alpha 1-receptors in rat brain or to beta-adrenergic receptors in turkey erythrocytes, since it is unable to displace [3H]prazosin and [125I]cyanopindolol from alpha 1 and beta-receptors respectively.

Adenylyl Cyclase Inhibitors↗

PubMed1984

Isolation of an endogenous clonidine-displacing substance from rat brain.

An endogenous substance which specifically displaces clonidine, yohimbine and rauwolscine from rat brain alpha 2-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 alpha 2-adrenergic receptors by competing with either alpha 2-adrenergic agonists or alpha 2-antagonists, but has no effect on the specific binding of [3H]prazosin to alpha 1-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 alpha 2-agonists to the alpha 2-adrenergic receptors.

Animals↗

PubMed1984

Solubilization and reconstitution of alpha 2-adrenergic receptors from rat and calf brain.

Sodium cholate and digitonin were used to solubilize alpha2-adrenergic receptors from rat and calf brain. Sodium cholate extracted 40-50% of the membrane protein and 25-30% of the binding capacity. Digitonin extracted only 20-30% of the membrane protein and only 10-15% of the binding capacity of the native membranes. Both detergents were removed by dialysis in the presence of phospholipids, and the solubilized protein was precipitated upon addition of poly(ethyleneglycol) and magnesium. In the solubilization/reconstitution process no purification of the alpha2-adrenergic receptor was obtained, most probably due to its inactivation by the solubilization conditions. The reconstituted protein(s) tested for binding properties, using p-[3H]aminoclonidine and/or [3H]clonidine, maintained the pharmacological profile of the native alpha2-adrenergic receptor. The potency order of various alpha2-agonists and alpha2-antagonists as well as their stereoselectivity were identical to those of the native alpha2-receptor. Specific receptor binding decreases in the presence of the guanyl nucleotides GTP or guanosine 5'-[beta, gamma-imido]-triphosphate but not ATP, thus indicating a co-solubilization of GTP regulatory components (stimulatory protein Ns or inhibitory protein Ni or both). Adenylate cyclase activity of the reconstituted preparation is stimulated threefold by sodium fluoride, suggesting the presence of both Ns-protein and the catalytic unit (C) in the reconstituted protein(s).

Adenylyl Cyclases↗

PubMed1983

Characterization of benextramine as an irreversible alpha-adrenergic blocker and as a blocker of potassium-activated calcium channels.

An irreversible alpha-adrenergic blocker, benextramine [N,N'-bis(o-methoxybenzylamine-n-hexyl)-cysteamine] was used as a probe to study the possible interrelationship between alpha-adrenoceptors and the K+-activated Ca2+-channels. Benextramine, a tetraamine disulfide, acts irreversibly both on the alpha 1-adrenoceptor (t 1/2 = 3 min) and the alpha 2-adrenoceptors. These studies were carried out on rat brain synaptosomes, [3H]prazosin and [3H]clonidine binding. Benextramine blocked Ca2+ influx in rat brain synaptosomes under both depolarizing (75 mM KCl) and normal conditions (5 mM KCl). Its action at the channel is reversible with IC50 = 10 +/- 5 microM of the net Ca2+ influx. This makes benextramine a most potent Ca2+ blocker compared to verapamil or nicardipine (IC50 = 200 microM and 170 microM, respectively). Pretreatment of rat brain slices with benextramine gave a synaptosomal preparation which was devoid of either alpha 1-adrenergic or alpha 2-adrenergic binding capacity due to the irreversible binding of benextramine, but with an undisturbed Ca2+ influx. Thus, these results suggest that the alpha-adrenoceptors and the Ca2+-channels are independent of each other, and that full occupancy of the alpha-receptors does not affect the net calcium flux.

Adrenergic alpha-Antagonists↗