Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase

Earlier, it has been shown that some amino acids and their derivatives are able to regulate activities of adenylyl cyclase (AC) and guanylyl cyclase (GC) in free-living infusoria Dileptus anser and Tetrahymena pyriformis. The goal of this work consisted in studying the molecular mechanisms of action of methionine, tyrosine, alanine, and neurohormone serotonin on the activity of enzyme-cyclases and in identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both infusoria; the effect of serotonin on AC in T. pyriformis took place with participation of the Ca²⁺-dependent form of AC and of the heterotrimeric G-proteins. The AC-stimulating effect of tyrosine and alanine was expressed weakly and was revealed only in D. anser. Serotonin in both infusoria and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine did not affect GC. Methionine and serotonin were bound with a high affinity to the surface receptors of infusoria. The K_D for [methyl-³H]methionine binding to D. anser and T. pyriformis were equal to 7.5 and 35.6 nM, and for [³H]serotonin binding, they were 2.7 and 4.7 nM, respectively. Alanine and tyrosine were bound to infusoria with low affinity. Thus, in the infusoria D. anser and T. pyriformis, there are chemosignal systems regulated by amino acids and their derivatives, including enzymes with cyclase activity. These systems are suggested to be similar to the hormonal signal systems of the higher eukaryotes and to be their predecessors.     

Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system in the infusorian <Emphasis Type="Italic">Dileptus anser</Emphasis>

In some unicellular eukaryotes, cAMP performs functions not only of the secondary messenger, but also of hormone, the primary messenger. We have found that cAMP is bound to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and the enzyme, adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K _D = 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N⁶,O^2′-dibutyryl-cAMP, a non-hydrolyzed cAMP analogue, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated by the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.     

Effects of natural amino acids and sugars on activity of infusiorian cyclases

Natural amino acids and sugars in unicellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, tyramine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis rose in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of the infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large specter of comparatively simple natural molecules has a hormone-like action.     

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian <Emphasis Type="Italic">Dileptis anser</Emphasis>

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca²⁺ was studied and it was found that calcium cations at concentrations of 0.5–10 μM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca²⁺ concentrations to 100 μM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca²⁺. Calmodulin antagonists—chlorpromazine, W-7, and W-5—caused a dose-dependent decrease of the enzyme activity stimulated by 5 μM Ca²⁺ with IC₅₀ values of 35, 137, and 174 M, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 μM Ca²⁺, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 μM calcium ions, but were markedly inhibited by 100 μM Ca²⁺. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca²⁺, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity depends on calcium cations and possibly is regulated by Ca²⁺/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

Effect of EGF on transmission of the proliferative signal in infusoria Tetrahymena pyriformis

It has been established that in infusoria Tetrahymena pyriformis, transmission of a proliferative signal induced by epidermal growth factor (EGF) is not associated with autophosphorylation of receptor tyrosine kinases. In these microorganisms, EGF triggers the mitogenic pathway that involves membrane proteins of the tyrosine kinase type (without phosphorylation at tyrosine), adenylyl cyclase, and tyrosine-and Ca²⁺-dependent ERK-like kinases.     

Structure-functional organization of adenylyl cyclases of unicellular eukaryotes and molecular mechanisms of their regulation

Adenylyl cyclases, the enzymes which catalyze the formation of the second messenger cAMP, are presently known to exist in yeast and related fungi, the amoeba Dictyostelium discoideum, flagellates, plasmodium, and infusoria. However, their structure-functional organization and molecular mechanisms of regulation differ considerably. Thus, in flagellates, tens of structurally similar adenylyl cyclase one-pass transmembrane proteins performing receptor functions have been discovered. In the amoeba D. discoideum, three types of adenylyl cyclases were detected, which differ by their topology, domain organization, and sensitivity to regulatory molecules and physical factors, one of which, adenylyl cyclase-A (AC-A), is similar to mammalian membrane-bound adenylyl cyclases and regulated by extracellular cAMP. Yeasts, in turn, have been shown to possess adenylyl cyclases that do not have transmembrane domains, but are able to form intermolecular complexes stabilized by interactions between repeated regions enriched in leucine residues. The data presented in this review indicate that the main molecular mechanisms underlying the actions of vertebrate adenylyl cyclases evolved as early as in the unicellular organisms and fungi. The structures and functions of adenylyl cyclases of the lower eukaryotes are much more diverse, which might be due both to the peculiarities of their life cycles and to the development at the initial stages of evolution of different models for the functioning and regulation of cAMP-dependent signaling cascades.     

Tubulin and microtubules from bovine kidney: purification, properties, and characterization of ligand binding.

Tubulin was purified from bovine renal medulla by in vitro assembly of microtubules in the presence of dimethyl sulfoxide and glycerol. Light scattering measurements of the polymerization process demonstrate that dimethyl sulfoxide and glycerol decrease the critical concentration of tubulin required for polymerization. The minimum concentration of tubulin from bovine renal medulla is about 1% of the total soluble protein. Assembly occurs in the absence of detectable amounts of high-molecular weight proteins or τ-protein. Microtubules polymerized in the absence and presence of 10% dimethyl sulfoxide and 4 m glycerol are similar morphologically as detected by electron microscopy. Molecular weights of α- and β-tubulin from bovine renal medulla are 54,000 ± 700 and 52,000 ± 800, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Colchicine-binding activity of renal medullary tubulin decays in an apparent first-order process which is temperature dependent. The half-time of decay in buffer is 5.1 h and addition of 5 μm vinblastine sulfate increases the half-time of decay to 10.9 h at 37 °C. Calculations based on measurements of the rate of decay of colchicine-binding activity at different temperatures indicates that vinblastine sulfate stabilizes the binding activity by decreasing the entropy of activation of the decay process. Colchicine decreases the rate of decay about 3.5-fold both in the absence and presence of vinblastine sulfate at 37 °C. Values of the apparent colchicine-binding constant, K_A, of bovine renal medullary tubulin are 5.9 × 10⁶ and 7.8 × 10⁶m^?1 at 37 °C in the absence and presence of vinblastine sulfate. Vinblastine sulfate decreases the rate of decay and increases the apparent binding constant of colchicine binding. Lumicolchicine does not affect the binding of colchicine. Podophyllotoxin apparently competitively inhibits the binding of colchicine; the apparent K_i for podophyllotoxin is 4.0 × 10^?7m at 37 °C. Thus, tubulin from bovine renal medulla has ligand-binding characteristics which exhibit differences and similarities to the corresponding characteristics of the brain tubulin. These biochemical properties of the colchicine-binding activity of bovine renal medullary tubulin support previous physiologic studies which demonstrate that microtubules are required for the function of vasopressin in mammalian kidneys.     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

P-Protein in sieve elements

Summary The ultrastructure of P1 and P2 proteins in the sieve elements of Heracleum mantegazzianum is described. P1-protein tubules are closely associated with stacks of membranes, are often linked by short cross-bridges, and occasionally display a hexagonal packing. Incubation with the alkaloids vinblastine and colchicine had no discernible effects on the ultrastructure of the sieve elements at various stages during differentiation. Evidence for and against any similarities between P1-protein tubules and cytoplasmic microtubules is discussed.     

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT_1BR) and 6 (5-HT₆R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT_1BR-peptide ARERKATKTL^307–316K-amide mimicking agonist-activated 5-HT_1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT_1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT_1BR-antagonists. The 5-HT₆R-peptides mimicking 5-HT₆R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT₆R-agonists on AC and G_s proteins, but in the presence of 5-HT₆R-antagonists their action was blocked. Of all the 5-HT₆R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL^258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT₆R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT_1BR and 5-HT₆R selectively activate G_i and G_s proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.     

Regulation of adenylyl cyclase signaling system by insulin, biogenic amines and glucagon at their separate and combined action in muscle membranes of mollusc Anodonta cygnea

In the smooth muscles of mollusc Anodonta cygnea the regulatory action of hormones on adenylyl cyclase signaling system (ACSS) are realized through the receptors of serpentine type (biogenic amines, isoproterenol, glucagon) and receptor tyrosine kinase (insulin) type. Intracellular mechanisms of their interaction are interconnected. Application of hormones, their antagonists and pertussis toxin in combination with insulin and biogenic amines or glucagon on adenylyl cyclase (AC) activity allows revealing the possible sites of cross-linking in the mechanisms of their action. Combined influence of insulin and serotonin or glucagon leads to decreased stimulation of adenylyl cyclase (AC) by these hormones, whereas combined application of insulin and isoproterenol suppresses AC-stimulating effect of insulin, but AC-inhibiting effect of isoproterenol is maintained in the presence and absence of non-hydrolysable analog of GTP—guanylyl imido diphosphate (GIDP). The specific blockage of AC-stimulating effect of serotonin by cyproheptadine—antagonist of serotonin receptors, did not change AC stimulation by insulin. Beta-adrenoblockers (propranolol and alprenolol) prevent inhibition of AC activity by isoproterenol, but did not change AC stimulation by insulin. Pertussis toxin blocked AC-inhibiting effect of isoproterenol and weakened AC-stimulating action of insulin. Thus, in the muscles of Anodonta cygnea negative interaction between ACS have been revealed, which are realized under combined influence of insulin and serotonin or glucagon, most probably, at the level of receptor of serpentine type (serotonin, glucagon), whereas under action of insulin and isoproterenol at the level of G_i protein and AC interaction.     

Colchicine-binding protein and the secretion of thyroid hormone

The role of microtubules in the thyrotropin- or adenosine 3'',5'' cyclic monophosphate (cyclic AMP)-stimulated accumulation of cytoplasmic colloid droplets and secretion of iodine from the mouse thyroid gland has been investigated by means of different classes of agents that affect the stability of microtubules. The onset of inhibition of secretion by colchicine, the uptake of colchicine-³H by thyroid lobes, and the binding of colchicine-³H to thyroidal soluble protein are shown to have similar time courses Colloid droplet accumulation is also inhibited and does not readily resume upon removal of colchicine from the medium. This appears to be due to the slow washout of the drug (t _½ ∼ hr). Thyroids contain a soluble colchicine-binding protein that resembles microtubule proteins of other tissues with respect to apparent K_m for colchicine, pH optimum, and stability characteristics Colchicine analogues inhibit iodine secretion and colchicine binding in a parallel manner and as a function of their antimitotic potencies. Microtubule-stabilizing agents such as hexylene glycol and D₂O also inhibit secretion. Thus, inhibition of thyroid secretion by antimitotic agents appears to be mediated by an effect on microtubules. The inhibitory locus of colchicine inhibition occurs after the generation of cyclic AMP, since stimulation of secretion by this nucleotide is blocked by colchicine, whereas thyroid-stimulating hormone—induced accumulation of cyclic AMP is not affected. Thus, the functioning microtubule appears to play a role in the induction of colloid endocytosis.     

Regulation of the Adenylyl Cyclase System of the Infusorian Tetrahymena pyriformis by Hormonal and Non-Hormonal Agents and Its Dependence on the Basal Activity Level of Adenylyl Cyclase

Effects of glucagon and biogenic amines as well as of non-hormonal agents (manganese cations, forskolin, guanine nucleotides) on activity of adenylyl cyclase (AC) was studied in the infusorian Tetrahymena pyriformis, a representative of unicellular eukaryotes. The infusorian cultures at different growth stages were chosen for the study. It has been revealed that the AC basal activity measured in the cultures at the stationary stage is much lower than in those at the exponential stage. The stimulating effects of hormones (glucagon, serotonin, isoproterenol) and of non-hormonal agents were shown to depend markedly on the tetrahymena AC basal activity. These effects are pronounced at the stationary stage of growth (the low basal activity), whereas in cultures at the active division stage (the high basal activity) they either are reduced or change their sign to opposite (inhibition of AC activity. The obtained results not only are another evidence in favor of the existence of the functionally active ACS in lower eukaryotes, but also indicate an important role of this system in regulation of sensitivity of infusoria at different growth stages to external actions.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca(2+) channels in mouse embryonic ventricular myocytes

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (I_Ca,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca²⁺ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5-E11.5) than late developmental stage (LDS, E16.5-E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, I_Ca,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca²⁺ influx via LTCCs on AC activity, leading to the inability to inhibit AC.     

Effect of Colchicine and Vinblastine on the Agglutination of Polymorphonuclear Leucocytes by Concanavalin A

IT is generally accepted that microtubules serve as supportive elements within cell structures such as the mitotic spindle and a role for microtubules in the mechanical stabilization of cell surfaces was suggested by the microcinematographic experiments of Vasiliev, Gelfand et al.¹. On contact, surfaces lost their ruffled movement and this immobilization could be abolished by colcemid which, like colchicine and vinblastine, binds to and dissolves microtubules. We tested the possibility that microtubular proteins might also determine or maintain the topographical organization of surface elements in several functional systems in which this is believed important.     

Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells

The mechanism by which the inhibitory effect of d-ala²-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP-dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the G_iα subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin-stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through G_i. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with G_i that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase.     

Microtubule inhibitors alter the secretion of beta-glucuronidase by human blood platelets: involvement of microtubules in release reaction II

The effects of the antimicrotubular drugs colchicine and vinblastine on the blood platelet release reaction were studied by measuring release of ¹⁴C-5-hydroxytryptamine (¹⁴C-5-HT, release I) and β-glucuronidase (release II) from gel-filtered human platelets. β-glucuronidase release induced by thrombin was significantly inhibited by colchicine (0.01-1 mM) or vinblastine (0.05–0.1 mM). Release of ¹⁴C-5-HT, however, was unaffected at low concentrations of colchicine and only slightly inhibited at higher concentrations. Inhibition of β-glucuronidase release depended on colchicine or vinblastine concentrations and decreased with longer time intervals (1′, 5′, 20′) after thrombin stimulation. Levels of the cytoplasmic enzyme, lactic acid dehydrogenase, in supernatants of colchicine treated platelets were not significantly different from controls. Colchicine also inhibited β-glucuronidse release, but not ¹⁴C-5-HT release, induced by trypsin and sodium arachidonate. Binding of ¹⁴C-colchicine by platelets was measured and it was found that platelet aggregation and release of 5-HT induced by adenosine diphosphate, epinephrine and collagen proceeded without any alteration in colchicine binding. However, significant increases in the rate and degree of colchicine binding were observed when platelets were stimulated by thrombin, trypsin and arachidonic acid which induced aggregation, release of both 5-HT and β-glucuronidase. The results suggest that an alteration in platelet microtubules is correlated with the physiologic response resulting in release II and that the cellular mechanisms effecting release I and II by platelets differ qualitatively in that the microtubules may facilitate release II.     

Chronic Treatment of C6 Glioma Cells with Antidepressant Drugs Increases Functional Coupling Between a G Protein (GS) and Adenylyl Cyclase

Abstract: It has been reported that antidepressant treatment in rats results in a significant increase of G_s-mediated stimulation of adenylyl cyclase and this effect correlates well with the clinical therapeutic response. This increased activity occurs despite a down-regulation of several receptors linked normally to the stimulation of that enzyme. To distinguish between these effects and to determine whether presynaptic components of the cell are required, C6 glioma cells were treated with antidepressants. Tricyclic (amitriptyline and desipramine) or atypical (iprindole) antidepressant exposure to C6 cells for 5 days significantly increased guanylyl-5′-imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activity in membrane preparations in a manner similar to that seen for rat brain membranes after 21-day treatment. This effect was drug dose and exposure time dependent. Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreased after antidepressant treatment. By comparison, the antidepressant-induced β-receptor desensitization occurred earlier than the enhancement of Gpp(NH)p-activated adenylyl cyclase, and extensive desensitization of β receptors by isoproterenol treatment did not enhance the Gpp(NH)p-stimulated adenylyl cyclase activity. These results indicated that the antidepressant has a direct effect on cell signaling and this enhanced Gpp(NH)p-stimulated adenylyl cyclase activity is not correlated with desensitization of β-adrenergic receptor stimulated adenylyl cyclase. These data contribute to the suggestion that G proteins (especially G_s) are the target of antidepressant actions. Immunoblotting showed that neither the number of G protein subunits (α_s, α_i, α_o, and β) nor their association with the plasma membrane was changed after antidepressant treatment. Thus, these results are consistent with the hypothesis that chronic antidepressant treatment acts directly at the postsynaptic membrane to increase the coupling between G_s and adenylyl cyclase.