Alpha-adrenergic receptors on human platelets.

[³H] dihydroergocyrptine, an α-adrenergic antagonist, binds specifically to sites on human platelet membranes. Prostaglandin E₁ (PGE₁) stimulates the production of cyclic AMP (cAMP) in human platelets. Alpha-adrenergic agonists, 1-epinephrine and 1-norepinephrine, and antagonists, phentolamine, phenoxybenzamine, and dihydroergocyrptine inhibit the binding of [³H] dihydroergocryptine. The α-adrenergic agonists inhibit PGE₁-stimulated cAMP production and the α-adrenergic antagonists phentolamine and dihydroergocryptine reverse this inhibition. The β-adrenergic agonist 1-isoproterenol and the β-adrenergic antagonists d1-propranolol and 1-alprenolol do not significantly alter binding or PGE₁-stimulated cAMP production. Clonidine, dopamine, and serotonin inhibit binding, but clonidine and dopamine are weak inhibitors of PGE₁-stimulated cAMP production, and serotonin is without effect. Tyramine, an amine without direct adrenergic activity fails to inhibit binding. Alpha-adrenergic agonists decrease the apparent affinity of a PGE₁-receptor activating cAMP production. The inhibition of PGE₁-stimulated cAMP production is a physiological measure of α-adrenergic agonist binding to the α-receptor.     

Discovery of highly potent and selective biphenylacylsulfonamide-based β3-adrenergic receptor agonists and molecular modeling based on the solved X-ray structure of the β2-adrenergic receptor: Part 6

As an extension of research, we have investigated modification of left-hand side (LHS) of biphenyl analogues containing an acylsulfonamide moiety in the development of potent and selective human β₃-adrenergic receptor (AR) agonists. Result of structure–activity relationships (SAR) and cassette-dosing evaluation in dogs showed that the hydroxynorephedrine analogue 16 had an excellent balance of in vitro and in vivo potency with pharmacokinetic profiles. In addition, to facilitate structure-based drug design (SBDD), we also have performed a docking study of biphenyl analogues based on the X-ray structure of the β₂-adrenergic receptor.     

The nature of receptors regulating the formation of cyclic AMP in brain tissue.

Extensive studies of the past seven years have provided fundamental information on the pharmacological properties of receptors controlling formation of cyclic AMP in brain slices and homogenates. Receptors for adenosine, α- and β-adrenergic agonists, dopamine, serotonin, H₁- and H₂-histaminergic agonists, and prostaglandins of the E series have been defined and evidence for a glutamate receptor has been presented. Extrapolation of such pharmacological data to studies with whole animals should provide important information as to the physiological significance of specific cyclic AMP-generating systems to the function of the intact brain.     

Carbonic anhydrases in plants and algae

Carbonic anhydrases catalyse the reversible hydration of CO₂, increasing the interconversion between CO₂ and HCO₃⁻ + H⁺ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C₃ dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.     

The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

Biochemical evidence for pharmacological similarities between alpha-adrenoreceptors and voltage-dependent Na+ and Ca++ channels

The α-adrenergic antagonists yohimbine, prazozin and phentolamine, but not the α-adrenergic agonists, block voltage-dependent Na⁺ channels of rat brain synaptosomes. The lipid-soluble neurotoxins (veratridine, aconitine, grayanotoxins and ceveratrum alkaloids), which cause a permanent activation of the Na⁺ channels by acting at the same receptor site as yohimbine, compete with [³H]yohimbine for its binding to rat brain α₂-adrenoreceptors. The calcium channel inhibitors verapamil and D₆₀₀ also block the Na⁺ channel and recognize α₂-adrenoreceptors.     

Structural basis of the selectivity of the β2-adrenergic receptor for fluorinated catecholamines

The important and diverse biological functions of adrenergic receptors, a subclass of G protein-coupled receptors (GPCRs), have made the search for compounds that selectively stimulate or inhibit the activity of different adrenergic receptor subtypes an important area of medicinal chemistry. We previously synthesized 2-, 5-, and 6-fluoronorepinehprine (FNE) and 2-, 5-, and 6-fluoroepinephrine (FEPI) and found that 2FNE and 2FEPI were selective β-adrenergic agonists and that 6FNE and 6FEPI were selective α-adrenergic agonists, while 5FNE and 5FEPI were unselective. Agonist potencies correlated well with receptor binding affinities. Here, through a combination of molecular modeling and site-directed mutagenesis, we have identified N293 in the β₂-adrenergic receptor as a crucial residue for the selectivity of the receptor for catecholamines fluorinated at different positions.     

Substituted 1,2,3,4-tetrahydroquinolin-6-yloxypropanes as β3-adrenergic receptor agonists: Design,synthesis, biological evaluation and pharmacophore modeling

In search of potent β₃-adrenergic receptor agonists, a series of novel substituted 1,2,3,4-tetrahydroquinolin-6-yloxypropanes has been synthesized and evaluated for their β₃-adrenergic receptor agonistic activity (ranging from ?17.73% to 90.64% inhibition at 10 μM) using well established Human SK-N-MC neuroblastoma cells model. Four molecules viz. 11, 15, 22 and 23 showed β₃-AR agonistic IC₅₀ value of 0.55, 0.59, 1.18 and 1.76 μM, respectively. These four candidates have been identified as possible leads for further development of β₃-adrenergic receptor agonists for obesity and Type-II diabetes pharmacotherapy. The free OH and NH functions are found to be essential for β₃-adrenergic receptor agonistic activity. Among the synthesized β₃-adrenergic receptor agonists having 1,2,3,4-tetrahydroquinoline scaffold, the N-benzyl group is found to be superior over N-arylsulfonyl group. A putative pharmacophore model has been modeled considering the above four active molecules which distinguishes well between the active and inactive molecules.     

II. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung

β-Adrenergic receptors were identified in membrane fractions of fetal and postnatal rat lung with the β-adrenergic antagonist (?)?[³H] dihydroalprenolol, (?)?[³H] DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to day 28 of postnatal life, 46±7 to 491±69 femtomole·mg^?1 protein. Neither the K_D, approximately 0.8nM for [³H]DHA, nor the β-adrenergic subtype changed with age. Classical agonists competed for the β-receptor with properties characteristic of β₂-adrenergic binding. Analysis of the inhibition of receptor binding by selective β-adrenergic agents demonstrated approximately 75% β₂ and 25% β₁ β-adrenergic subtypes in fetal rat lung membranes. The increase in β-adrenergic receptor during development was associated with adenylate cyclase activity which was sensitive to catecholamines at all ages studied, supporting the possible role of the β-adrenergic receptor system in the postnatal regulation of pulmonary function.     

Synthesis and characterization of novel substituted thiophene derivatives and discovery of their carbonic anhydrase and acetylcholinesterase inhibition effects

Novel substituted thiophene derivatives ( 1, 2a‐e, 3, and 4 ) were synthesized and their structures were characterized by infrared radiation, nuclear magnetic resonance, and mass analysis. These novel substituted thiophene derivatives were effective inhibitor compounds of the carbonic anhydrase I and II isozymes (hCA I and II), and acetylcholinesterase (AChE) enzyme with K _i values in the range of 447.28 to 1004.65 nM for hCA I, 309.44 to 935.93 nM for hCA II, and 0.28 to 4.01 nM for AChE, respectively. Novel substituted thiophene derivatives can be good candidate drugs for the treatment of some diseases like neurological disorders, epilepsy, glaucoma, gastric and duodenal ulcers, mountain sickness, or osteoporosis as carbonic anhydrase isozymes inhibitors, and for the treatment of Alzheimer’s and Parkinson’s diseases as acetylcholinesterase inhibitors.     

Heterogeneous origin of carbonic anhydrase activity of thylakoid membranes

Carbonic anhydrase activities of pea thylakoids as well as thylakoid fragments enriched either in Photosystem 1 (PS1-membranes) or Photosystem 2 (PS2-membranes) were studied. The activity of PS1-membranes if calculated on chlorophyll basis was much higher than the activity of PS2-membranes. Acetazolamide, a non-permeable inhibitor of carbonic anhydrases, increased carbonic anhydrase activity of PS2-membranes at concentrations lower than 10⁻⁶ M and suppressed this activity only at higher concentrations. A lipophilic inhibitor of carbonic anhydrases, ethoxyzolamide, effectively suppressed the carbonic anhydrase activity of PS2-membranes (I ₅₀ = 10⁻⁹ M). Carbonic anhydrase activity of PS1-membranes was suppressed alike by both inhibitors (I ₅₀ = 10⁻⁶ M). In the course of the electrophoresis of PS2-membranes treated with n-dodecyl-β-maltoside “high-molecular-mass” carbonic anhydrase activity was revealed in the region corresponding to core-complex of this photosystem. Besides, carbonic anhydrase activity in the region of low-molecular-mass proteins was discovered in the course of such an electrophoresis of both PS2-and PS1-membranes. These low-molecular-mass carbonic anhydrases eluted from corresponding gels differed in sensitivity to specific carbonic anhydrase inhibitors just the same as PS1-membranes versus PS2-membranes. The results are considered as evidence for the presence in the thylakoid membranes of three carriers of carbonic anhydrase activity. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 651–659.     

Furazan and furoxan sulfonamides are strong α-carbonic anhydrase inhibitors and potential antiglaucoma agents

A series of furazan and furoxan sulfonamides were prepared and studied for their ability to inhibit human carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX, and hCA XII. The simple methyl substituted products 3–5 were potent inhibitors. Differing structural modifications of these leads had differing effects on potency and selectivity. In particular, products in which the sulfonamide group is separated from the hetero ring by a phenylene bridge retained high potency only on the hCA XII isoform. The sulfonamides 3–5 exerted intraocular pressure (IOP) lowering effects in vivo in hypertensive rabbits more efficiently than dorzolamide. Some other products (39–42), although less effective in vitro hCA II/XII inhibitors, also effectively lowered IOP in two different animal models of glaucoma.     

Adenosine,adenosine receptors and glaucoma: An updated overview

Background

Glaucoma, a leading cause of blindness worldwide, is an optic neuropathy commonly associated with elevated intraocular pressure (IOP). The major goals of glaucoma treatments are to lower IOP and protect retinal ganglion cells. It has been revealed recently that adenosine and adenosine receptors (ARs) have important roles in IOP modulation and neuroprotection.

Scope of review

This article reviews recent studies on the important roles of adenosine and ARs in aqueous humor formation and outflow facility, IOP and retinal neuroprotection.

Major conclusions

Adenosine and several adenosine derivatives increase and/or decrease IOP via A_2A AR. Activation of A₁ AR can reduce outflow resistance and thereby lower IOP, A₃ receptor antagonists prevent adenosine-induced activation of Cl⁻ channels of the ciliary non-pigmented epithelial cells and thereby lower IOP. A₁ and A_2A agonists can reduce vascular resistance and increase retina and optic nerve head blood flow. A₁ agonist and A_2A antagonist can enhance the recovery of retinal function after ischemia attack. Adenosine acting at A₃ receptors can attenuate the rise in calcium and retinal ganglion cells death accompanying P2X(7) receptor activation.

General significance

Evidence suggested that the adenosine system is one of the potential target systems for therapeutic approaches in glaucoma.     

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO₃^?/CO₂ as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α?carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α?carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO₃^?/CO₂. Addition of exogenous HCO₃^? or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.     

Heterocyclic acetamide and benzamide derivatives as potent and selective β3-adrenergic receptor agonists with improved rodent pharmacokinetic profiles

A series of amide derived β₃-adrenergic receptor (AR) agonists is described. The discovery and optimization of several series of compounds derived from 1, is used to lay the SAR foundation for second generation β₃-AR agonists for the treatment of overactive bladder.     

Catecholamine binding to CNS adrenergic receptors

The properties of ³H-catecholamine binding to α- and β-adrenergic receptors in CNS are reviewed. ³H-epinephrine and ³H-norepinephrine label one class of α-receptors throughout the brain, with high affinities for agonists and some antagonists. Agonist affinities at this site are increased in low temperature conditions but are reduced by guanine nucleotides and monovalent cations. Divalent cations reverse both effects. This α-receptor may be coupled to adenylate cyclase by GTP and/or sodium, and uncoupled by divalent cations. ³H-epinephrine labels β₂, but not β₁, receptors in CNS, especially in bovine cerebellum. The same β-receptor does not show agonist-specific GTP-sensitivity, but does exhibit Na⁺-sensitivity. This receptor appears to be linked to adenylate cyclase, and sodium rather than GTP may be the coupling agent.     

Endogenous and Exogenous Regulation of Human α- and β-Adrenergic Receptors

Abstract

Regulation of human β2-adrenergic receptors in lymphocytes (determined by (±)-125 iodocyanopindolol (ICYP) binding) and α₂-adrenergic receptors in platelets (determined by ³H-yohimbine binding) was studied. While α₂-adrenergic receptor number did not change with age, a significant negative correlation between the number of α₂-adrenergic receptors and age was found; plasma catecholamines, on the contrary, were elevated in the elderly.In healthy women during normal menstrual cycle the number of α₂-adrenergic receptors decreased with increasing plasma estradiol levels.Incubation of lymphocyte membranes with isoprenaline (100 μM) and of platelet membranes with clonidine (1-100 μM) led to a reduction of the number of β₂- and α₂-receptors, respectively, without changes in the K_D-values. Treatment of hypertensive patients with clonidine (3x150 μg/die) for 7 days reduced the number of α₂-adrenergic receptors in platelets. In platelet membranes from such treated patients inhibition of ³H-yohimbine binding by clonidine and adrenaline was not affected by 10-⁴MGTP. It is concluded, that human α- and β-adrenergic receptors undergo regulatory mechanisms similar to those recently described for adrenergic receptors in a variety of animal models.     

The effects of carbonic anhydrase inhibitors formate,bicarbonate, acetazolamide,and imidazole on photosystem II in maize chloroplasts

Inhibitors of carbonic anhydrase were tested for their effects on Photosystem II (PS II) activity in chloroplasts. We find that formate inhibition of PS II turnover rates increases as the pH of the reaction medium is lowered. Bicarbonate ions can inhibit PS II turnover rates. The relative potency of the anionic inhibitors N₃^?, I^?, OA_c^?, and Cl^? is the same for both carbonic anhydrase and PS II. The inhibitory effect of acetazolamide on PS II increases as light intensity decreases, indicating a lowering of quantum yields in the presence of the inhibitor. Imidazole inhibition of PS II increases with pH in a manner suggesting that the unprotonated form of the compound is inhibitory. Formate, bicarbonate, acetazolamide, and imidazole all inhibit DCMU-insensitive, silicomolybdate-supported oxygen evolution, indicating that the site(s) of action of the inhibitors is at, or before, the primary stable PS II electron acceptor Q. This inhibitory effect of low levels of HCO₃^? along with the known enhancement by HCO₃^? of quinone-mediated electron flow suggests an antagonistic control effect on PS II photochemistry. We conclude that the responses of PS II to anions (formate, bicarbonate), acetazolamide, and imidazole are analogous to the responses shown by carbonic anhydrase. These findings suggest that the enzyme carbonic anhydrase may provide a model system to gain insight into the “bicarbonate-effect” associated with PS II in chloroplasts.     

The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves

Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive feature of the effect of Triton X-100 on this activity also suggested that the source of the activity is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic anhydrase β-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the thylakoid lumen of higher plants.