Streptozocin model of diabetes mellitus in the mollusc Anodonta cygnea: functional state of the adenylyl cyclase mechanisms of action of peptides of the insulin superfamily and their effect on carbohydrate metabolism enzymes

In terms of development of evolutionary biomedicine using invertebrate animals as models for study of molecular grounds of various human diseases, for the first time the streptozocin (ST) model of insulin-dependent diabetes in the mollusc Anodonta cygnea has been developed. This model is based on the following authors' data: (1) redetection of insulin-related peptides (IRP) in mollusk tissues: (2) discovery of the adenylyl cyclase signal mechanism (ACSM) of action of insulin and other peptides of the insulin superfamily in tissues of mammals, human, and mollusc. A. cygnea; (3) concept of molecular defects in hormonal signal systems as causes of endocrine diseases. Studies on the ST model have revealed in mollusc smooth muscle on the background of hyperglycemia at the 2nd, 4th, and 8th day after the ST administration a decrease of the ACSM response to activating action of insulin, IGF-1, and relaxin. These functional disturbances were the most pronounced at the 2nd day of development and rather less marked at the 4th and 8th day. Analysis of data on effect of hormonal and non-hormonal (NaF, GIDP, and forskolin) ACSM activators has shown that the causes of impair of signal-transducing function of this mechanism are (1) a hyperglycemia-induced increase of the basal AC activity and as a consequence--a decrease of the enzyme catalytic potentials in response to hormone; (2) a decrease of functions of Gs-protein and of its coupling with AC. Besides, administration of ST produced in the mollusc muscles an attenuation of regulation by insulin of carbohydrate metabolism enzyme (glucose-6-phosphate dehydrogenase, glycogensynthase). The pattern of disturbances in the studied parameters in the mollusc is very similar to that revealed by the authors in rat and human muscle tissues in type 1 diabetes.     

Study of structural-functional arrangement of the adenylyl cyclase signaling mechanism of action of insulin-like growth factor 1 revealed in muscle tissue of representatives of vertebrates and invertebrates

Based on the earlier discovered by the authors adenylyl cyclase signaling mechanisms (ACSM) of action of insulin and relaxin, a study was performed of the existence of a similar action mechanism of another representative of the insulin superfamily-the insulin—like growth factor 1 (IGF-1) in the muscle tissue of vertebrates (rat) and invertebrates (mollusc). For the first time there was detected participation of ACSM in the IGF-1 action, including the six-component signaling cascade: receptor tyrosine kinase → Gi-protein (βγ-dimer) → phosphatidylinositol-3-kinase (PI-3K) → protein kinase Cζ (PKCζ) → Gs-protein → adenylyl cyclase. By structural-functional organization at postreceptor stages, it coincides completely with that of insulin and relaxin, which we revealed in rat skeletal muscle. In smooth muscle of the mollusc Anodonta cygnea this ACSM of action of IGF-1 has only one difference-the protein kinase C included in this mechanism is represented not by the PKCζ isoform, but by another isoform close to PKCε of the vertebrate brain. Earlier we revealed the same differences in muscles of this mollusc in the ACSM of action of insulin and relaxin.     

Adenylyl Cyclase Signaling Mechanism of Relaxin Action

In connection with our discovery of the adenylyl cyclase signaling mechanism (ACSM) of action of some peptides belonging to the insulin superfamily, a possibility of its involvement in action of another insulin superfamily peptide, relaxin, was studied. It was shown for the first time that human relaxin-2 (10^–12–10^–8 M) activated adenylyl cyclase (AC) in a dose-dependent manner. The maximal peptide effect was revealed at a concentration of 10^–8 M. Under condition of the hormonal action the basal enzyme activity increased by +310% in human myometrium, by +117%, in rat skeletal muscles, and by +49%, in foot smooth muscles of the bivalve mollusc Anodonta cygnea. Insulin and mammalian insulin-like growth factor-I (IGF-I) also produced the AC activating effect in these muscles. The order of efficiency of these peptides, based on their ability to induce the maximal AC stimulating effect, was as follows: relaxin > IGF-I > insulin (human myometrium); IGF-I > relaxin > insulin (rat skeletal muscle); insulin-like peptide of Anodonta (ILPA) > IGF-I > insulin > relaxin (molluscan muscle). The relaxin activating effect on AC was potentiated by a guanine nucleotide, the non-hydrolyzed analog of GTP, guanylylimidodiphosphate (Gpp[NH]p), which indicates participation of G_s-protein in realization of this effect. This effect was inhibited by a tyrosine kinase selective blocker, tyrphostin 47, and a phosphatidylinositol-3-kinase (PI-3-K) selective blocker, wortmannin. Thus, for the first time, participation of ACSM in the relaxin action has been established. This mechanism, as suggested at the present time state of its study, includes the following signal pathway: receptor-tyrosine kinase PI-3-K G_s-protein AC.     

Structural and functional organization of the adenylyl cyclase signaling mechanism of insulin-like growth factor 1 revealed in the muscle tissue in vertabrates and invertebrates

Based on the earlier discovered by the authors adenylyl cyclase signaling mechanisms (ACSM) of action of insulin and relaxin, the study was performed of the presence a similar action mechanism of another representative of the insulin superfamily--the insulin-like growth factor 1 (IGF-1) in the muscle tissues of vertebrates (rat) and invertebrates (mollusc). For the first time there was detected participation of ACSM in the IGF-1 action, including the six component signaling cascade: receptor tyrosine kinase --> G(i)-protein (betagamma-dimer) --> phosphatidylinositol-3-kinase (PI-3-K) --> protein kinase Czeta (PKCzeta) --> G(-)protein --> adenylyl cyclase. By this mechanism structural-functional organization at postreceptor stages, in coincides completely with the mechanism of insulin and relaxin, which we revealed in rat skeletal muscle. In smooth muscle of the mollusc Anodonta cygnea this ACSM of action of IGF-1 has only one difference--the protein kinase C included in this mechanism is represented not by PKCzeta isoform, but by another isoform close to PKCepsilon of the vertabrate brain. Earlier we revealed the same differences in muscle of this mollusc in the ACSM of action of insulin and relaxin.     

On the mechanism of relaxin action: the involvement of adenylyl cyclase signalling system

The molecular mechanism of relaxin action was studied taking into account the evolutionary relationship of the peptides belonging to the insulin superfamily and using the authors' previous data on the involvement of the adenylyl cyclase (AC) signalling system in the action of insulin and related peptides. Human relaxin 2 (10(-12)-10(-8) M) has been shown to cause a dose-dependent activating effect on AC in the human myometrium (+370%), in rat skeletal muscles (+117%) and the smooth foot muscles of the bivalve mollusc Anodonta cygnea (+73%). In these tissues mammalian insulin and insulin-like growth factor-1 (IGF-1) also had the AC activating effect. The order of efficiency of the above peptides based upon their ability to induce the maximal AC activating effect was as follows: relaxin > IGF-1 > insulin (human myometrium); IGF-1 > relaxin > insulin (rat skeletal muscle); molluscan insulin-like peptide > IGF-I > insulin > relaxin (molluscan muscle). The relaxin AC activating effect was inhibited with a selective tyrosine kinase blocker tyrphostin 47 and potentiated with Gpp[NH]p providing evidence for the participation of the receptor-tyrosine kinase and G-protein of the stimulatory type (Gs) in the regulatory action of relaxin. The conclusion is that the signalling chain: receptor tyrosine kinase ==> Gs protein ==> AC is involved in the mechanism of relaxin action.     

Comparative study of biological activity of insulins of lower vertebrates in the novel adenylyl cyclase test-system

The biological activity of insulins of lower vertebrates (teleosts-Oncorhynchus gorbuscha, Scorpaena porcus, chondrosteans-Acipenser guldenstaedti and cyclostomates-Lamperta fluviatilis) was studied and compared with that of standard pig insulin. The determination of biological activity was made using the novel adenylyl cyclase (AC) test-system based on the adenylyl cyclase signaling mechanism (ACSM) of insulin action discovered earlier by the authors. The biological activity of insulins was estimated as EC(50), i.e. concentration leading to half-maximal activating effect of the hormone (10(-11)-10(-7) M), in vitro, on adenylyl cyclase in two types of the target tissues: in membrane fractions of the muscles of rat and mollusc Anodonta cygnea. In rat, the efficiency of insulins was found to decrease in the following order: pig insulin>scorpaena insulin>gorbuscha insulin>sturgeon insulin>lamprey insulin. In the mollusc, the order was different: sturgeon insulin>scorpaena insulin>pig insulin>gorbuscha insulin. Lamprey insulin at the same concentrations did not apparently reach the maximal adenylyl cyclase activating effect. The suggestion was made that differences in the biological activity of insulins depend on the hormone structure and a number of indexes characteristic of the adenylyl cyclase test-system in the vertebrate and invertebrate tissues. The proposed adenylyl cyclase test-system is highly sensitive to insulin at physiological concentrations, has good reproduction and is easy to apply.     

Functional defects in adenylyl cyclase signaling mechanisms of insulin and relaxin in skeletal muscles of rat with streptozotocin type 1 diabetes

Functional disturbance in the novel adenylyl cyclase signaling mechanism (ACSM) of insulin and relaxin action in rat streptozotocin (STZ) type I diabetes was studied on the basis of the authors’ conception of molecular defects in hormonal signaling systems as the main causes of endocrine diseases. Studying the functional state of molecular components of the ACSM and the mechanism as a whole, the following changes were found in the skeletal muscles of diabetic rats compared with control animals: 1) increase of insulin receptor binding due to an increase in the number of insulin binding sites with high and low affinity; 2) increase of the basal adenylyl cyclase (AC) activity and the reduction of AC-activating effect of non-hormonal agents (guanine nucleotides, sodium fluoride, forskolin); 3) reduction of ACSM response to stimulatory action of insulin and relaxin; 4) decrease of the insulin-activating effect on the key enzymes of carbohydrate metabolism, glycogen synthase and glucose-6-phosphate dehydrogenase. Hence, the functional activity of GTP-binding protein of stimulatory type, AC and their functional coupling are decreased during experimental type 1 diabetes that leads to the impairment of the transduction of insulin and relaxin signals via ACSM.     

Structural-functional characteristics of the adenylyl cyclase signaling system regulated by biogenic amines and peptide hormones in muscles of the earthworm Lumbricus terrestris

It has been shown for the first time that biogenic amines (catecholamines and tryptophane derivatives) stimulate dose-dependently activity of adenylyl cyclase (AC) and GTP-binding of G-proteins in muscle of the skin-muscle sac of the earthworm Lumbricus terrestris. By efficiency of their stimulating action on the AC activity, biogenic amines can be arranged in the following sequence: octopamine > tyramine > tryptamine ≈ serotonin > dopamine > isoproterenol ≈ adrenalin. The sequence of efficiency of their action on GTP-binding is somewhat different: serotonin > tryptamine > octopamine > dopamine ≈ tyramine > adrenaline > isoproterenol. Sensitivity of AC and G-proteins in the worm muscle to biogenic amines is similar with that in smooth muscle of the mollusc Anodonta cygnea (invertebrates), but differs markedly by this parameter from the rat myocardium (vertebrates). It has also been revealed that AC in the worm muscle is regulated by peptide hormones, relaxin and somatostatin, whose action is comparable with that in the mollusc muscle, but much weaker that the action of these hormones on the rat myocardium AC activity. Use of Cterminal peptides of α-subunits of G-proteins of the stimulatory (385–394 Gαs) and inhibitory (346–355 Gαi2) types that disrupt selectively the hormonal signal transduction realized via Gsand Giproteins, respectively, allowed establishing that the AC-stimulating effects of relaxin, octopamine, tyramine, and dopamine in the worm muscle are realized via the receptors coupled functionally with Gs-protein; the AC-inhibiting effect of somatostatin is realized via the receptor coupled with Gi-protein, whereas serotonin and tryptamine activate both types of G-proteins.     

Tissue specificity of metabolism in the bivalve mollusc <Emphasis Type="Italic">Anadara inaequivalvis</Emphasis> Br. under conditions of experimental anoxia

Peculiarities of the course of metabolic processes in tissues of the bivalve mollusc Anadara inaequivalvis Br. were studied under conditions of experimental anoxia. In the absence of oxygen, in gill and foot the protein catabolism processes were found to be enhanced; this led to a decrease of the protein content and to an increase of the free amino acid and urea levels. Predominantly hydrolyzed were low molecular peptides, which was indicated by a decrease of the cathepsin D activity on the background of a rise of the γ-glutamyltranspeptidase activity. Anoxia was accompanied by enhancement of the succinate thiokinase and fumarate reductase reactions controlled by alanine and aspartate aminotransferases. This prevented accumulation of toxic lactate in tissues and allowed obtaining an additional macroerg resource. Metabolic processes in the mollusc hepatopancreas were oriented to production of amino acids.     

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.     

Insulin-Regulated Adenylyl Cyclase Signaling System in Rat Skeletal Muscles under Conditions of in vivo Insulin Administration and of Insulin Insufficiency Produced by Streptozotocin Diabetes

Possibility of the appearance of functional defects in the adenylyl cyclase (AC) signaling mechanism (ACSM) of insulin action, which was discovered by the authors earlier [1–3], is studied in skeletal muscles of rats with acute insulin insufficiency produced by streptozotocin diabetes (24 h). This ACSM includes the signaling chain: receptor-tyrosine kinase Gi-protein phosphatidylinositol 3-kinase protein kinase C-zeta Gs-protein adenylyl cyclase protein kinase A. At comparative evaluation of the functional state of individual molecular blocks of ACSM and the entire mechanism as a whole in skeletal muscles of diabetic rats in comparison with control animals, the following facts have been revealed: (1) an increase of the AC basal activity and a decrease of effects of non-hormonal activators of AC (guanine nucleotides, NaF, forskolin) ; (2) reduction of reactivity of the whole ACSM to insulin (10_–8 M, in vitro) and to combined action of the hormone and GIDP (10_–6 M) ; (3) a decrease of the activating action of insulin on key enzymes of carbohydrate metabolism—glycogen synthase and glucose-6-phosphate dehydrogenase (G6PDG). It is concluded that insulin insufficiency leads to several disturbances in the insulin ACSM: at the level of its catalytic component—AC, Gs protein and its coupling with AC, as well as to a decrease of regulatory metabolic effects of the hormone. These data indicate a decrease of sensitivity of skeletal muscles of diabetic rats to insulin and an involvement of this hormone in maintenance of functionally active status of the ACSM of insulin signal transduction.     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.     

Comparative study of molecular mechanisms of natural and synthetic polycationic peptides action on the activity of the adenylyl cyclase signaling system

The molecular mechanisms of action of natural and synthetic polycationic peptides, forming amphiphilic helices, on the heterotrimeric G-proteins and enzyme adenylyl cyclase (AC), components of hormone-sensitive AC system, were studied. It is shown that synthetic peptides C-epsilonAhx-WKK(C10)-KKK(C10)-KKKK(C10)-YKK(C10)-KK (peptide I) and (GRGDSGRKKRRQRRRPPQ)2-K-epsilonAhx-C(Acm)(peptide II) in dose-dependent manner stimulate the basal AC activity, inhibit forskolin-stimulated AC activity and decrease both stimulating and inhibiting AC effects of the hormones in the tissues (brain striatum, heart muscle) of rat and in smooth muscles of the mollusc Anodonta cygnea. AC effects of these peptides are decreased after membrane treatment by cholera and pertussis toxins and are inhibited in the presence of the peptides, corresponding to C-terminal regions 385-394 alphas- and 346-355 alphai2-subunits of G-proteins. These data give evidence that the peptides I and II act on the signaling pathways which are realized through Gs- and Gi-proteins. At the same time, natural polycationic peptide mastoparan acts on AC system through Gi-proteins and blocks hormonal signals mediated via Gi-proteins only. Consequently, the action of mastoparan on G-proteins is selective and differs from the action of the synthetic peptides. It is also shown that peptide II, with branched structure, directly interacts not only with G-proteins (less effective in comparison with peptide I with hydrophobic radicals and mastoparan), but also with enzyme AC, the catalytic component of AC system. On the basis of data obtained the following conclusions were made: 1) the formation of amphiphilic helices is not enough for selective activation of G-protein by polycationic peptides, and 2) the primary structure of the peptides, the distribution of positive charged amino acids and hydrophobic radicals in them are very important for selective interaction between polycationic peptides and G-proteins.     

Effects of thiols and blockers of sulfhydryl groups on negative regulation of the adenylyl cyclase signaling system by biogenic amines

Effect of a thiol-containing compound, β-mercaptoethanol, and of blockers of cysteine sulfhydryl groups (para-chlormercury benzoate andN-ethylmaleimide) on hormonal negative regulation of functional activity of the adenylyl cyclase system was studied in smooth muscles of the freshwater bivalve molluscAnodonta cygnea (agonist of ß-adrenoreceptors—isoproterenol) and in the rat skeletal muscles (serotonin). It was found that in the presence of ß-mercaptoethanol the inhibitory effects of isoproterenol (mollusc) and serotonin (rat) were preserved. At the same time, the blockers of SH-groups disturb essentially both the hormonal effects and their potentiation in the presence of Gpp[NH]p, this process being practically irreversible in the presence ofN-ethylmaleimide. Thus, it has been shown that the negative hormonal regulation in the muscle membranes of the mollusc and rat is dependent on the SH-group state in the protein components of the hormonesensitive adenylyl cyclase system.     

Effect of SH-Specific Reagent 5,5′-Dithobis(2-Nitrobenzoic Acid) on Functional Activity of Components of Adenylyl Cyclase Signal System

Sensitivity of adenylyl cyclase signal system to 5,5′-dithobis(2-nitrobenzoic acid) (DTNB) oxidizing SH-groups of cystein residues to disulfide bonds was studied. It was shown that treatment of plasma membranes fractions of smooth muscles of the mollusc Anodonta cygnea and of rat skeletal muscles as well as of homogenate of mouse fibroblasts culture of L strain with micromole concentrations of DTNB led to a decrease of activity of adenylyl cyclase (AC) stimulated by GIDP, sodium fluoride, and, to a lesser degree, forskolin. Dithiothreitol (DTT) partly restored the stimulating effects of GIDP, NaF, and forskolin, the effect of this dithiol being dose-dependent. AC stimulated by biogenic amines—serotonin in mollusc muscles, isoproterenol in rat muscles, and both hormones in mouse fibroblasts—is more sensitive to DTNB than the enzyme stimulated by non-hormonal agents. Thus, the stimulatory effects of hormones decreased dose-dependently in the presence of 10–100 μM DTNB and were almost completely blocked by 250 μM reagent. These effects were partly restored in the presence of 5 mM DTT. The obtained data indicate a high sensitivity of the hormone-stimulated AC to action of the reagents interacting specifically with SH-groups of the proteins components of the AC system. In the rat muscle membranes treated with 25 μM DTNB, no significant rightward shift was observed of the curve of competitive replacement of the β-adrenergic receptor antagonist [³H]-dihydroalprenolol by the β-agonist isoproterenol in the presence of GTP and the affinity of the agonist to the receptor somewhat decreased, which indicates a disturbance of functional coupling of the β-adrenergic receptor with G-protein after treatment with DTNB.     

Receptor of Insulin-Like Growth Factor 1 in Tissues of the Mollusc Anodonta cygnea

To identify insulin-like receptors in the mollusc Anodonta cygnea, specific binding of ¹²⁵I-insulin and ¹²⁵I-IGF-1 by WGA-purified glycoprotein fractions of foot muscles and neural ganglia is studied. The binding sites for IGF-1 are detected for the first time in invertebrates, both in the muscles, and in the neural tissue of the mollusc. The level of ¹²⁵I-IGF-1 binding in the muscle tissue was equal to 2.8 ± 0.1, in the neural tissue, to 4.0 ± 0.2% per 5 µg of protein. The equilibrium dissociation constant (K _d) was equal to 4.8 ± 0.3 and 4.3 ± 0.2 nM, respectively. The relative affinity of the binding sites to insulin did not exceed 1% of their affinity to IGF-1. Binding of ¹²⁵I-insulin in the muscle tissue was not detected; the level of labeled insulin binding in the neural tissue was equal to 0.5% per 5 µg of protein. In the sarcolemmal fraction of the mollusc foot, IGF-1 and, to a lesser degree, insulin at a dose of 100 nM initiated phosphorylation of tyrosine in a protein with mol. mass of 70 kDa. The minor band of the phosphorylation was also detected in the zone of protein of 80 kDa. The conclusion is made about the existence in molluscan tissues of high-conserved receptors-tyrosine kinases identical by functional parameters to the mammalian receptor of IGF-1. From this, it is suggested that the peptides close by structure to vertebrate IGF-1 may be involved in physiological processes in A. cygnea. The problem of the nature of the insulin-binding sites in the molluscan neural tissue is discussed.     

Adenylyl cyclase signaling mechanisms of relaxin and insulin action: similarities and differences

The adenylyl cyclase signaling mechanism (ACSM) of relaxin H2 action was discovered and deciphered in mammalian muscles. A study of signaling blocks involved in ACSM of relaxin in comparison with that of insulin previously detected showed a close similarity throughout the post-receptor signaling chain of both hormones. The inhibitory action of tyrosine kinase blockers on the hormone AC activating effect indicates that the relaxin receptor involved in ACSM is likely to be of the tyrosine kinase type. However, a recent discovery of a relaxin receptor with serpentine architecture leaves open the question concerning the existence of receptor of the tyrosine kinase type. The structural-functional organization of the ACSM due to the action of relaxin-shown here for the first time-can be presented as the following signaling sequence: relaxin receptor ==>G(i) protein (betagamma-dimer) ==>phosphatidylinositol 3-kinase ==>protein kinase Czeta ==>G(s) protein ==>adenylyl cyclase. According to our hypothesis, the regulatory action of the insulin superfamily peptides on cell processes (proliferation, apoptosis, and metabolism) is mediated via ACSM.     

Role of cysteine sulfhydryl groups in activation of adenylyl cyclase signaling system by biogenic amines in molluscan and rat tissues

It has been shown that in smooth muscles of the freshwater bivalve molluscAnodonta cygnea as well as in skeletal muscles and brain striatum of rats a blocker of SH-groups,para-chlormercury benzoate (ChMB), and an alkylating agent,N-ethylmaleimide, inhibit both the basal adenylyl cyclase (AC) activity and the activity of the enzyme stimulated by non-hormonal agents (NaF, Gpp[NH]p) and by hormonal agents such as serotonin (mollusc muscles, rat brain) or isoproterenol (rat muscles and rat brain). The inhibitory effects of ChMB andN-ethylmaleimide on AC are partly eliminated by an SH-group containing reagent, β-mercaptoethanol (ME, 5 mM). Restoration of the basal and of the stimulated enzyme activity inhibited by ME is better in the case of the ChMB than of theN-ethylmaleimide action. It has also been found that ME stimulates both the basal and the stimulated by non-hormonal agents AC activity. In the presence of ME the hormonal stimulating effects on the enzyme are also preserved, except for the effect of isoproterenol on AC in rat skeletal muscles; this effect is inhibited by ME. Potentiation of the stimulating effect of the hormones on AC by Gpp[NH]p is only preserved in the molluscan smooth muscles (the effect of serotonin—90%). The data obtained indicate that cysteine sulfhydryl groups play a key role in hormonal regulation of the functional activity of the components of the hormone-sensitive adenylyl cyclase signaling system.     

Topical Niosome Gel Containing an Anthocyanin Complex: a Potential Oral Wound Healing in Rats

Anthocyanins from dietary sources showing potential benefits as anti-inflammatory in oral lesions were developed as an anthocyanin complex (AC), comprised of extracts of Zea mays (CC) and Clitoria ternatea (CT), and formulated into a niosome gel to prove its topical oral wound healing in vitro and in vivo investigations. The AC formed nano-sized clusters of crystalline-like aggregates, occurring through both intra- and inter-molecular interactions, resulting in delivery depots of anthocyanins, following encapsulation in niosomes and incorporation into a mucoadhesive gel. In vitro permeation of anthocyanins was improved by complexation and further enhanced by encapsulation in niosomes. Collagen production in human gingival fibroblasts was promoted by AC and AC niosomes, but not CC or CT. The in vivo wound healing properties of AC gel (1 and 10%), AC niosome gel (1 and 10%), fluocinolone acetonide gel, and placebo gel were investigated for incisional wounds in the buccal cavities of Wistar rats. AC gel and AC niosome gel both reduced wound sizes after 3 days. AC niosome gel (10%) gave the highest reduction in wound sizes after day 3 (compared to fluocinolone acetonide gel, p?<?0.05), and resulted in 100% wound healing by day 5. Histological observations of cross-sectioned wound tissues revealed the adverse effects of fluocinolone gel and wound healing potential of AC niosome gel. Topical application of AC niosome gel exhibited an anti-inflammatory effect and promoted oral wound closure in rats, possibly due to the improved mucosal permeability and presence of delivery depots of AC in the niosome gel.     

Regulatory Action of Synthetic Cationic Peptides Containing Residues of Glutamic Acid on Functional Activity of Components of the Adenylyl Cyclase Signal System

One of the most important stages of hormonal signal transduction in cells through the hormone-sensitive adenylyl cyclase signal system (ACS) is functional coupling of receptor of the serpentine type to heterotrimeric GTP-binding protein (G-protein). The main role in realization of such coupling is played by spiralized regions of the receptor cytoplasmic loops proximal in relation to membrane, most of them carrying positive charge. To study molecular mechanisms of interaction of the receptor with G-protein, we compared effects of synthetic cationic peptides containing residues of glutamic acid on the process of regulation of ACS by hormones (biogenic amines) and non-hormonal agents in smooth muscles of the freshwater bivalve mollusc Anodonta cygnea and skeletal muscles of rat. All peptides had the clearly expressed ability to form -helices. Peptides H-(Leu-His-Glu-Lys)₄-Leu-NH₂ (I), H-(Leu-His-Glu-Lys)₃-Lys-His-Glu-Lys-Leu-NH₂ (II), H-(Leu-Lys-Glu-Lys)₄-Leu-NH₂ (III), and H-(Ile-His-Glu-Lys)₄-Ala-NH₂ (IV) at concentrations of 10^–6–10^–3 M reduced dose-dependently the value of stimulating effects of serotonin (in mollusc muscles) and isoproterenol (in rat muscles) on the adenylyl cyclase (AC) and protein kinase A (PKA) activities. Values of concentration of these peptide causing a 50% decrease of the hormone-stimulating effect (IC₅₀) vary from 150 to 750 µM. According to the degree of this inhibitory action on stimulating effects of hormones, they may be arranged in the following series: III II > IV I. The peptides I–IV were more effective than the peptide H-(Glu-Lys)₈-Ala-NH₂ (V) with the charge close to zero, but much less effective than the studied earlier cationic peptides containing only positively charged amino acid residues. The inhibitory effect of the peptides I-IV on stimulation of AC by non-hormonal agents, NaF, Gpp[NH]p, and forskolin, was essentially less pronounced and was marked only at 10^–4–10^–3 M concentrations. Thus, the inclusion of negatively charged amino acid residues in the primary structure of polycationic peptides leads to a decrease in their ability to inhibit hormonal stimulation of AC and PKA, which indicates importance both of the total positive charge of peptides and of distribution of the charged amino acids in the formed helices for realization of the uncoupling action on the ACS components—the receptor and G-protein.