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 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.     

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.     

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Background and purpose

Despite the view that only β₂- as opposed to β₁-adrenoceptors (βARs) couple to G_i, some data indicate that the β₁AR-evoked inotropic response is also influenced by the inhibition of G_i. Therefore, we wanted to determine if G_i exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.

Experimental approach

We used the G_s-selective (R,R)- and the G_s- and G_i-activating (R,S)-fenoterol to selectively activate β₂ARs (β₁AR blockade present) in combination with G_i inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.

Key results

PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC₅₀ values of fenoterol matched published binding affinities. The PTX enhancement of the G_s-selective (R,R)-fenoterol-mediated responses suggests that G_i regulates AC activity independent of receptor coupling to G_i protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of G_i with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.

Conclusions and implications

Together, these data indicate that G_i exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G_i and G_s activity upon AC towards G_s, enhancing the effect of all cAMP-mediated inotropic agents.     

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.     

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.     

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.     

Regulatory Properties of Adenylyl Cyclase Isoforms

Various aspects of mechanisms of the adenylyl cyclase (AC)* activity regulation are considered in the review. Variants of modulation of various AC isoform activity by G-proteins (_s- and _i subunits, -dimer), Ca²⁺-calmodulin, phosphorylation by PKA, PKC or Ca²⁺-CM-activated kinases are presented. Evidence is presented that AC functions as a signaling integrator in the cell by providing propagation and amplification of many both extracellular (with participation of hormones) and intracellular signals (interaction between Ca²⁺ and AC).     

Effects of Biogenic Amines and Polypeptide Hormones on Protein Kinase A and Adenylyl Cyclase Activities in Muscles of the Mollusc Anodonta cygnea

The effects of biogenic amines, glucagon, and insulin on the cAMP-dependent protein kinase A (PKA) activity have been studied in the muscle tissue of the freshwater bivalve mollusc Anodonta cygnea. It was shown that serotonin, glucagon, and insulin both in vivo and in vitro stimulated PKA activity, whereas isoproterenol inhibited it. The stimulating effect of serotonin and inhibiting effect of isoproterenol was blocked by serotoninergic (cyproheptadine) and adrenergic (propranolol) inhibitors, which confirms specificity of the effect of biogenic amines on the PKA activity. Taking into account participation of adenylyl cyclase system in action of the above hormones, the revealed hormonal effects on the PKA activity produce metabolic effects via the following chain reaction. In the case of serotonin and glucagon: receptor G_s-protein AC cAMP PKA phosphorylation of glycogen synthase (GS) and glucose-6-phosphate dehydrogenase (G6PDH) and inhibition of their activity; in the case of isoproterenol: -adrenoreceptor G_i-protein AC inhibition decreasing PKA inhibition of phosphorylase and stimulation of GSI and G6PDH. A participation is suggested of the insulin-stimulated AC signaling system in the mechanism of the mitogenic insulin effect mediated, as shown in this work, via the PKA activation, but not of the metabolic effect of insulin.     

Regulation of Functional Activity of Adenylyl Cyclase Signal System by Synthetic Coil-Forming Peptides in Mouse Fibroblast Culture of Line L (ISM Subline)

The signal transduction process via adenylyl cyclase system (ACS) requires coordinated functioning of signal proteins—components of ACS. It is suggested that functional coupling between them, together with other molecular mechanisms, is based on coiled-coil interactions. To study role of these interactions in functioning of ACS, we synthesized cationic coiled-forming peptides with a regular structure Ac–Ala–His– (Ala)₂–His–Ala–NH₂ (I), Ac–Ala–His–(Ala)₃–His– (Ala)₂–His–Ala–NH₂ (II), and Ac–(Pro(₂–His– (Ala)₂–His– (Ala)₂–His– (Ala)₂–His–Ala–NH₂ (III). Using circular dichroism (CD) spectroscopy, a portion of -helix conformation in their secondary structure was determined, and effects of these peptides on basal adenylyl cyclase (AC) activity as well as on the activity stimulated by non-hormonal (NaF and Gpp[NH]p) and hormonal (serotonin) agents was studied in homogenate of mouse fibroblasts, line L (subline LSM). The synthetic peptides were shown to inhibit in a dose-dependent manner both basal and induced AC activity, which indicates their uncoupling action on ACS. The biological effect of these peptides correlated with their length (I < II < III), but not with coiled-coil structure, which was 20, 7, and 21%, respectively, according to data of circular dichroism spectroscopy in 3-fluoroethanol. However, there are reasons to believe that the coiled-coil structure of peptides, first place extended ones, increases at interaction with plasma membrane and signal proteins, which affects the degree of their effect on functional ACS activity. At micromolar concentrations, peptides II and III were established to markedly stimulate the basal AC activity, thereby mimicking G-protein-binding sites of cytoplasmic receptor loops. The data obtained indicate participation of the coiled-coil interactions in functional coupling of ACS components, and the methodology itself of the use of model peptides with different coiled-coil structure and distribution of charged amino acids is an efficient approach for studying molecular bases for functioning of signal systems.     

Insulin-Regulated Adenylyl Cyclase Signaling System in Cell Culture of Mouse Fibroblasts of L Line

In terms of the subsequent study of the earlier discovered adenylyl cyclase signaling mechanism of insulin action [1, 2], we studied participation in it of various isoforms of protein kinase C. As object of study, a culture of mouse fibroblast cells of the L line (LSM subline) was chosen. It has been shown that insulin and a non-hydrolyzed analog of GTP, Gpp[NH]p, stimulate the adenylyl cyclase (AC) activity in these cells both individually and in combination. Activators of phorbol-sensitive isoforms of protein kinase C, diacylglycerol and phorbol ester (phorbol-12-myristate-13-acetate) at their concentrations of 1–100 nM stimulate basal activity of AC. In their presence, a significant decrease of stimulating effects of insulin and Gpp[NH]p or their complete disappearance are observed. Calphostin C (1–100 nM), an inhibitor of both phorbol-sensitive and atypical, phorbol-insensitive isoforms of protein kinase C, somewhat increased the basal AC activity. However, the stimulating effects of insulin and Gpp[NH]p in the presence of calphostin C decreased markedly. On the whole, the obtained data allow us to suggest participation of various isoforms of protein kinase C (sensitive and insensitive to the phorbol esters) in regulation of the process of the insulin signal transduction in mouse fibroblasts through the adenylyl cyclase signaling mechanism. Thus, mechanisms of functioning of the insulin-regulated AC-system in fibroblasts, representatives of connective tissue cells, are similar to those that we described earlier in muscle tissues of vertebrate and invertebrate animals. Taken together, these data indicate the absence of tissue- and species-specificity in functioning of the insulin-regulated AC system and its wide spread in tissues of different animals.     

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptus anser

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 microM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 microM 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 microM Ca2+ with IC50 values of 35, 137, and 174 microM, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 microM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 microM calcium ions, but were markedly inhibited by 100 microM 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 were 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 does not D. depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

Role of Sulfhydryl Groups in Functioning of Adenylyl Cyclase Signaling System

This review considers the literature data and author's own results on the role of SH-groups in functioning of the hormone-sensitive adenylyl cyclase system (ACS). It has been shown that the state of SH-groups affects crucially all main stages of the hormonal signal transudation: the ligand-binding properties of receptor and its coupling to G-proteins, interaction of G-proteins with adenylyl cyclase (AC) and its catalytic activity. It is noted that for the receptors, coupled to AC by a stimulating mode, the central aspect of the SH-dependent regulation of ACS is shifted to the receptor, while for the receptors coupled to AC by an inhibiting mode, it coincides with G-protein of the inhibiting type, which is sensitive to the SH-group state. Based on the performed comparative analysis of primary structures of signalling proteins—ACS components and of literature data, there are revealed the cysteine residues determining the functional activity of these proteins in the process of the hormonal signal transudation. The conclusion is made that the SH-group state (the ratio of free SH-groups and disulfide bonds) is the main factor determining the ACS reactivity to hormonal effects and selectivity of process of the signal transudation.     

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.     

Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

Similarly Potent Inhibition of Adenylyl Cyclase by P-Site Inhibitors in Hearts from Wild Type and AC5 Knockout Mice

Adenylyl cyclase type 5 (AC5) was described as major cardiac AC isoform. The knockout of AC5 (AC5KO) exerted cardioprotective effects in heart failure. Our study explored the impact of AC5KO on mouse heart AC activities and evaluated putative AC5-selective inhibitors. In cardiac membranes from AC5KO mice, basal AC activity was decreased, while AC stimulation was intact. The putative AC5-selective P-site inhibitors SQ22,536 [9-(tetra-hydro-2-furanyl)-9H-purin-6-amine], vidarabine (9-β-D-arabinosyladenine) and NKY80 [2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone] inhibited recombinant AC5 more potently than AC2 and AC1, but selectivity was only modest (∼4-40-fold). These compounds inhibited cardiac AC from WT and AC5KO mice with similar potencies. In conclusion, AC regulation in AC5KO hearts was unimpaired, questioning the supposed dominant role of AC5 in the heart. Moreover, the AC inhibitors SQ22,536, NKY80 and vidarabine lack adequate selectivity for AC5 and, therefore, do not present suitable tools to study AC5-specific functions.     

Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture

Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E₁ and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocotisone, glucagon and growth hormone, that can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.     

Histamine H1-Receptors Modulate Somatostatin Receptors Coupled to the Inhibition of Adenylyl Cyclase in the Rat Frontoparietal Cortex

Puebla, L., A. OcaÑa and E. Arilla. Histamine H₁-receptors modulate somatostatin receptors coupled to the inhibition of adenylyl cyclase in the rat frontoparietal cortex. Peptides 18(10) 1569–1576, 1997.—Since exogenous histamine has been previously shown to increase the somatostatin (SS) receptor-effector system in the rat frontoparietal cortex and both histamine H₁-receptor agonists and SS modulate higher nervous activity and have anticonvulsive properties, it was of interest to determine the participation of the H₁-histaminergic system in this response. The intracerebroventricular (i.c.v.) administration of the specific histamine H₁-receptor agonist 2-pyridylethylamine (PEA) (10 μg) to rats 2 h before decapitation increased the number of SS receptors (599 ± 40 vs 401 ± 31 femtomoles/mg protein, p< 0.01) and decreased their apparent affinity for SS (0.41 ± 0.03 vs 0.26 ± 0.02 nM, p < 0.01) in rat frontoparietal cortical membranes. No significant differences were seen for the basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activities in the frontoparietal cortex of PEA-treated rats when compared to the control group. In the PEA group, however, the capacity of SS (10⁻⁴ M) to inhibit basal and FK (10⁻⁵ M)-stimulated AC activity in frontoparietal cortical membranes was significantly higher than in the control group (34 ± 1% vs 20 ± 2%, p < 0.001). The ability of low concentrations of the stable GTP analogue 5′-guanylylimidodiphosphate [Gpp(NH)p] to inhibit FK-stimulated AC activity in frontoparietal cortical membranes was similar in the PEA-treated and control animals. These results suggest that the increased SS-mediated inhibition of AC activity in the frontoparietal cortex of PEA-treated rats may be due to the increase of the number of SS receptors induced by PEA. Pretreatment with the H₁-receptor antagonist mepyramine (30 mg/kg, intraperitoneally (IP) prevented the PEA-induced changes in SS binding and SS-mediated inhibition of AC activity. Mepyramine (30 mg/kg, IP) alone had no observable effect on the somatostatinergic system. The in vitro addition of PEA or mepyramine to frontoparietal cortical membranes obtained from untreated rats did not affect the SS binding parameters. Altogether, these results suggest that the H₁-histaminergic system modulates the somatostatinergic system in the rat frontoparietal cortex.