Post-opioid receptor adaptations to chronic morphine; altered functionality and associations of signaling molecules

Opioid desensitization/tolerance mechanisms have largely focused on adaptations that occur on the level of the mu-opioid receptor (MOR) itself. These include opioid receptor phosphorylation and ensuing trafficking events. Recent research, however, has revealed additional adaptations that occur downstream from the opioid receptor, which involve covalent modification of signaling molecules and altered associations among them. These include augmented isoform-specific synthesis of adenylyl cyclase (AC) and their phosphorylation as well as augmented phosphorylation of the G(beta) subunit of G(beta gamma). The aggregate effect of these changes is to shift mu-opioid receptor-coupled signaling from predominantly G(i alpha) inhibitory to (G(i)-derived) G(beta gamma) stimulatory AC signaling. Most recently, chronic morphine has been shown to enhance the association (interaction) between MOR and G(s), which should provide an additional avenue for offsetting inhibitory MOR signaling sequelae. The unfolding complexity of chronic morphine-induced sequelae demands an evolving broader and more encompassing perspective on opioid tolerance-producing mechanisms. This should facilitate understanding tolerance within the context of physiological plasticity that is activated by chronic exposure to drugs of abuse. Additional research is required to integrate the various tolerance-producing adaptations that have been elucidated to date. Specifically, the relative contribution to opioid tolerance of identified adaptations is still unknown as is the extent to which they vary among different regions of the central nervous system.     

The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low K _m and one with a high K _m for GTP. The high-affinity GTPase activity, characteristic of the subunits of the guanine nucleotide binding protein pool, was stimulated by the A₁ adenosine receptor agonists N ⁶(R-phenylisopropyl)adenosine and N ⁶-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N ⁶ (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 mol·1^-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent K _m of GTP is decreased approximately 10% in each of the species, and the V _max values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased K _m of GTP and the increased V _max ranged from-7.0 to-9.9 ml·mol^-1. Increased pressure markedly decreased the efficacy of N ⁶ (R-phenylisopropyl) adenosine, N ⁶-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A₁ adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.Abbreviations [³H] DPCPX ³H cyclopentyl-1, 3-dipropylxanthine - AppNHp 5-adenylylimidodiphosphate - cpm counts per minute - CPA N ⁶-cyclopentyladenosine - EDTA ethylenediaminetetra acetic acid - EGTA ethyleneglycol-bis (-aminoethylether) N, N, N, N-totra-acctic acid - G protein guanine nucleotide binding protein - G_i inhibitory G protein - G_o other G protein, common in brain membranes - G_s stimulatory G protein - GTPase guanosine triphosphatase - K _i inhibition constant - K _m Michaelis constant - pK _a log of the dissociation constant - R-PIA N ⁶ (R-phenylisopropyl) adenosine - TRIS tris[hydroxymethyl]aminomethane - V_max maximal velocity - [-³²P]GTP [-³²P] guanosine 5-triphosphate (tetra (triethylammonium) salt)     

Efficient adenylyl cyclase activation by a beta2-adrenoceptor-G(i)alpha2 fusion protein

The G-protein G(i)alpha can activate adenylyl cyclase (AC), but the relevance of this AC activation is unknown. We used receptor-G protein co-expression and receptor-G protein fusion proteins to investigate G(i)alpha(2) regulation of AC in Sf9 cells. G(i)alpha(2) was fused to the beta(2)-adrenoceptor (beta(2)AR), a preferentially G(s)-coupled receptor, or the formyl peptide receptor (FPR), a G(i)-coupled receptor. The FPR co-expressed with, or fused to, G(i)alpha(2), reduced AC activity. In contrast, the beta(2)AR fused to G(i)alpha(2) was a highly efficient AC activator, while the beta(2)AR co-expressed with G(i)alpha(2) was not. Agonist efficiently stimulated incorporation of [alpha-32P]GTP azidoanilide into beta(2)AR-G(i)alpha(2). We explain AC activation by beta(2)AR-G(i)alpha(2) by a model in which there is interaction of the beta(2)AR and AC, preventing tethered G(i)alpha(2) from interacting with the inhibitory G(i)alpha site of AC. The postulated beta(2)AR/AC interaction brings G(i)alpha(2) into close proximity of the G(s)alpha site of AC, enabling G(i)alpha(2) to activate AC.     

The synthesis and biological evaluation of 2-amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophenes as allosteric modulators of the A1 adenosine receptor

A series of 2-amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophenes were prepared and evaluated as potential allosteric modulators of the A₁ adenosine receptor (AR). The structure-activity relationships of the 3-position were explored along with varying the size of the cycloalkyl ring. 2-Aminothiophenes with amide and hydrazide groups in the 3-position were completely inactive in an A₁-AR-mediated ERK1/2 phosphorylation assay, yet most of the 3-benzoyl substituted compounds exhibited allosteric effects on responses mediated by the orthosteric agonist, R-PIA. Despite finding an increase in both agonistic and allosteric activities by going from a cyclopentyl ring to a cyclohexyl ring in the 3-benzoyl series, decreases were observed when further increasing the ring size. Varying the substituents on the phenyl ring of the 3-benzoyl group also affected the activity of these compounds.     

Pressure effects on the GTPase activity of brain membrane G proteins of deep-living marine fishes

In marine fishes, heterotrimeric guanyl nucleotide binding proteins (G proteins), which couple cell surface membrane receptors to their effector elements, are sensitive to hydrostatic pressure. The intrinsic high affinity GTPase activity of the α subunits of G proteins in three signaling systems coupled to adenylyl cyclase, the A₁ adenosine receptor, the muscarinic cholinergic receptor and the β-adrenergic receptor, was tested at pressures up to 340 atm. Brain membrane preparations from four members of the deep-sea teleost fish family Macrouridae were studied. Coryphaenoides armatus, C. filifer, C. rupestris and Macrourus berglax have depth distributions which together span 100–5810 m. Increased pressure inhibited basal GTPase activity only in M. berglax, which of the four species has the shallowest center of abundance. Increased hydrostatic pressure did not alter the response of GTPase activity to the β-adrenergic receptor agonist isoproterenol. Increased pressure decreased the stimulation of GTPase activity by the A₁ adenosine receptor agonist cyclopentyladenosine (CPA) in C. armatus and M. berglax, and by the muscarinic cholinergic receptor agonist carbamyl choline in C. armatus, C. filifer and M. berglax. Decreased agonist-stimulation of the GTPase activity at elevated pressure may result from pressure-induced changes in conformational states or inhibition of agonist binding. The binding of the non-hydrolyzable GTP analog guanosine 5′-[γ-thio]triphosphate (GTP[S]) in response to CPA was determined at 5 °C and atmospheric pressure. Six macrourid species and a morid were studied. The halftime (t_1/2) values for GTP[S] binding, ranging from 20.8 to 40.9 min, are similar to values previously reported for two other cold-adapted fishes.     

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.     

Characterization of the A2AR-D2R interface: focus on the role of the C-terminal tail and the transmembrane helices

A single serine point mutation (S374A) in the adenosine A_2A receptor (A_2AR) C-terminal tail reduces A_2AR-D₂R heteromerization and prevents its allosteric modulation of the dopamine D₂ receptor (D₂R). By means of site directed mutagenesis of the A_2AR and synthetic transmembrane (TM) α-helix peptides of the D₂R we further explored the role of electrostatic interactions and TM helix interactions of the A_2AR-D₂R heteromer interface. We found evidence that the TM domains IV and V of the D₂R play a major role in the A_2AR-D₂R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A_2AR and D₂R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A_2AR-D₂R heteromers. The mutation of two negatively charged aspartates in the A_2AR C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A_2AR-D₂R interaction and lost the ability of antagonistic allosteric modulation over the A_2AR-D₂R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A_2AR and the intracellular loop 3 (IL3) of D₂R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A_2AR-D_2LR heteromer interface together with D_2LR TM segments IV/V interacting with A_2AR TM-IV/V or TM-I/VII.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling

Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model.Compound 8e exhibited the potential growth inhibition of the colon cancer cells in a concentration-dependent manner. The anti-proliferative activity of 8e was also associated with the induction of cell cycle arrest in the G₀/G₁ phase. The 8e-induced cell cycle arrest was well correlated with the suppression of cyclin-dependent kinase 2 (CDK2), CDK4, cyclin D1, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb). The tumor growth in xenograft nude mice bearing HCT 116 cells by compound 8e (10 mg/kg) also significantly inhibited without any overt toxicity. In addition, the down-regulation of epidermal growth factor receptor (EGFR), Akt, and mTOR signalings were associated with the anti-proliferative activity of compound 8e in colon cancer cells. Taken together, these findings suggested that cell cycle arrest and modulation of cell signal transduction pathways might be the plausible mechanisms of actions for the anti-proliferative activity of 8e, and thus 8e might be used as an effective chemotherapeutic agent in human colon cancer.     

Loss of myocardial protection from ischemic preconditioning following chronic exposure to R(-)-N6-(2-phenylisopropyl)adenosine is related to defect at the adenosine A1 receptor

Exogenously administered adenosine agonist will protect myocardium against infarction during ischemia. However, long-term exposure to adenosine agonists is associated with loss of this protection. To determine why this protection is lost, isolated, perfused rabbit hearts were studied after administration of R(-)-N⁶-(2-phenylisopropyl)adenosine (PIA), 0.25 mg/h IP, for 3-4 days to intact animals. All hearts experienced 30 min of regional ischemia and 120 min of reperfusion. Control groups 1 and 2 were untreated. In group 1 this ischemia/reperfusion was the only intervention, whereas group 2 hearts were preconditioned with a cycle of 5 min global ischemia/10 min reperfusion preceding the 30 min regional ischemia. Groups 3-5 had been chronically exposed to PIA. Group 3 hearts had 1 preconditioning ischemia/reperfusion cycle before the prolonged ischemia. Group 4 received a 5 min infusion of 0.1 mol/L phenylephrine in lieu of global ischemia, whereas group 5 was instead treated with 1 mol/L carbachol. Infarct size averaged 32% of the risk zone in group 1, whereas ischemic preconditioning limited infarction to 8.2 in group 2. Prolonged exposure of group 3 hearts to PIA resulted in the inability of preconditioning with 5 min global ischemia to protect (28.7 ± 4.4% infarction). However, protection was restored by either phenylephrine, an agonist of ₁-adrenergic receptors which couple to G_q and stimulate PKC, or carbachol, an agonist of M₂-muscarinic receptors which couple instead to G_i as do adenosine A₁ receptors (5.2 ± 1.7% and 9.2 ± 2.1% infarction, resp.). Therefore, cross tolerance to ischemic preconditioning develops after chronic PIA infusion. Since both the G_i and the PKC components of the preconditioning pathway were shown to be intact, tolerance must have been related to downregulation or desensitization of the A₁ adenosine receptor.     

Positive coupling of atypical adenosine A3 receptors on human eosinophils to adenylyl cyclase

Adenosine A(3) receptors are reported to couple negatively to adenylyl cyclase (AC) but their mediation of anti-inflammatory effects in human eosinophils prompted us to investigate their coupling to AC. The A(3)-selective agonists IB-MECA and Cl-IB-MECA evoked a concentration-dependent generation of cAMP (EC(50), 3.2 and 1.8 microM, respectively) and were more potent than the A(2A) agonist CGS 21680 (EC(50)=15.4 microM) and adenosine (EC(50)=19.2 microM). The cAMP response was additive to that produced by forskolin (10 microM). The effect of IB-MECA was insensitive to A(1) and A(2A) receptor antagonists, but was antagonized by the A(3)-selective antagonist MRS 1220 (0.1-2.5 microM) in a competitive manner. The estimated K(B) of 190 nM was, however, atypical. The cyclo-oxygenase inhibitor, indomethacin, had no effect on the cAMP response. A general inverse relationship between cAMP generation and inhibition of degranulation was seen. We conclude that in human eosinophils, an atypical form of A(3) receptors positively coupled to AC may exist. The resulting cAMP generation may underlie the anti-inflammatory actions of A(3) agonists in eosinophils.     

Cellular models for the study of the pharmacology and signaling of melanin-concentrating hormone receptors

Cellular models for the study of the neuropeptide melanin-concentrating hormone (MCH) have become indispensable tools for pharmacological profiling and signaling analysis of MCH and its synthetic analogues. Although expression of MCH receptors is most abundant in the brain, MCH-R₁ is also found in different peripheral tissues. Therefore, not only cell lines derived from nervous tissue but also from peripheral tissues that naturally express MCH receptors have been used to study receptor signaling and regulation. For screening of novel compounds, however, heterologous expression of MCH-R₁ or MCH-R₂ genes in HEK293, Chinese hamster ovary, COS-7, or 3T3-L1 cells, or amplified MCH-R₁ expression/signaling in IRM23 cells transfected with the G_q protein gene are the preferred tools because of more distinct pharmacological effects induced by MCH, which include inhibition of cAMP formation, stimulation of inositol triphosphate production, increase in intracellular free Ca²⁺ and/or activation of mitogen-activated protein kinases. Most of the published data originate from this type of model system, whereas data based on studies with cell lines endogenously expressing MCH receptors are more limited. This review presents an update on the different cellular models currently used for the analysis of MCH receptor interaction and signaling.     

Structural and functional characterization of the first intracellular loop of human thromboxane A2 receptor

The conformation of a constrained peptide mimicking the putative first intracellular domain (iLP1) of thromboxane A(2) receptor (TP) was determined by (1)H 2D NMR spectroscopy. Through completed assignments of TOCSY, DQF-COSY, and NOESY spectra, a NMR structure of the peptide showed a beta-turn in residues 56-59 and a short helical structure in the residues 63-66. It suggests that residues 63-66 may be part of the second transmembrane domain (TM), and that Arg60, in an exposed position on the outer surface of the loop, may be involved in signaling through charge contact with Gq protein. The sequence alignment of Lys residue in the same position of other prostanoid receptors mediates different G protein couplings, suggesting that the chemical properties of Arg and Lys may also affect the receptor signaling activity. These hypotheses were supported by mutagenesis studies, in which the mutant of Arg60Leu completely lost activity in increasing intracellular calcium level through Gq coupling, and the mutant of Arg60Lys retained only about 35% signaling activity. The difference between the side chain functions of Lys and Arg in effecting the signaling was discussed.     

Pertussis toxin-sensitive G proteins mediate the inhibition of basal phosphoinositide metabolism caused by adenosine A1 receptors in rat hippocampal slices

The adenosine A₁ receptor selective agonist, N ⁶-cyclopentyladenosine (CPA, 300 nM) inhibited basal accumulation of [³H]inositol phosphates ([³H]InsPs), but not the total levels of membrane [³H]-phosphoinositides, in rat hippocampal slices. This action of CPA was not significantly modified when synaptic transmission was blocked with tetrodotoxin (TTX, 200 nM) but was prevented in slices pre-incubated with pertussis toxin (PTX, 5 g/mL) for 12-16 hr. Neither PTX nor TTX, when applied in the absence of CPA, influenced basal [³H]InsPs accumulation. It is concluded that the inhibition of the basal phosphatidylinositol metabolism by adenosine A₁ receptor activation is independent of neurotransmission and involves a PTX-sensitive G protein, probably of the G_i/G_o family.     

Characterization of the prostaglandin H2 mimic: binding to the purified human thromboxane A2 receptor in solution

For decades, the binding of prostaglandin H₂ (PGH₂) to multiple target proteins of unrelated protein structures which mediate diverse biological functions has remained a real mystery in the field of eicosanoid biology. Here, we report that the structure of a PGH₂ mimic, U46619, bound to the purified human TP, was determined and compared with that of its conformation bound to the COX-downstream synthases, prostacyclin synthase (PGIS) and thromboxane A₂ synthase (TXAS). Active human TP protein, glycosylated and in full length, was expressed in Sf-9 cells using a baculovirus (BV) expression system and then purified to near homogeneity. The binding of U46619 to the purified receptor in a nonionic detergent-mimicked lipid environment was characterized by high-resolution NMR spectroscopy. The conformational change of U46619, upon binding to the active TP, was evidenced by the significant perturbation of the chemical shifts of its protons at H3 and H4 in a concentration-dependent manner. The detailed conformational changes and 3D structure of U46619 from the free form to the TP-bound form were further solved by 2D ¹H NMR experiments using a transferred NOE (trNOE) technique. The distances between the protons of H11 and H18, H11 and H19, H15 and H18, and H15 and H19 in U46619 were shorter following their binding to the TP in solution, down to within 5 Å, which were different than that of the U46619 bound to PGIS and U44069 (another PGH₂ mimic) bound to TXAS. These shorter distances led to further separation of the U46619 α and ω chains, forming a unique “rectangular” shape. This enabled the molecule to fit into the ligand-binding site pocket of a TP model, in which homology modeling was used for the transmembrane (TM) domain, and NMR structures were used for the extramembrane loops. The proton perturbations and 3D conformations in the TP-bound U46619 were different with that of the PGH₂ mimics bound to PGIS and TXAS. The studies indicated that PGH₂ can adopt multiple conformations in solution to satisfy the specific and unique shapes to fit the different binding pockets in the TP receptor and COX-downstream enzymes. The results also provided sufficient information for speculating the molecular basis of how PGH₂ binds to multiple target proteins even though unrelated in their protein sequences.     

Metabolic depletion of sphingolipids enhances the mobility of the human serotonin1A receptor

Sphingolipids are essential components of eukaryotic cell membranes. We recently showed that the function of the serotonin_1A receptor is impaired upon metabolic depletion of sphingolipids using fumonisin B₁ (FB₁), a specific inhibitor of ceramide synthase. Serotonin_1A receptors belong to the family of G-protein coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral and developmental functions. Since function and dynamics of membrane receptors are often coupled, we monitored the lateral dynamics of the serotonin_1A receptor utilizing fluorescence recovery after photobleaching (FRAP) under these conditions. Our results show an increase in mobile fraction of the receptor upon sphingolipid depletion, while the diffusion coefficient of the receptor did not exhibit any significant change. These novel results constitute the first report on the effect of sphingolipid depletion on the mobility of the serotonin_1A receptor. Our results assume greater relevance in the broader context of the emerging role of receptor mobility in understanding cellular signaling.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.     

Organelle motility regulated by the cell's environment: dissection of signaling pathways regulating movements of peroxisomes

Summary Chloroplasts and pigment granules are known to be intracellularly translocated upon discrete extracellular stimuli. The machineries transducing these signals inside cells are yet not understood. In studies investigating the motility of peroxisomes, we were able to identify both extracellular and intracellular signaling steps regulating movements of these organelles. Following simultaneous stimulation of CHO cells with both extracellular ATP and lysophosphatidic acid, an arrest of peroxisomes was observed. This block of motility was shown to be dependent on signaling cascades involving heterotrimeric G proteins of the class G_i/G_o, phospholipase C, calcium influx, and activation of protein kinase C as well as of mitogen-activated protein kinase. Cytosolic phospholipase A₂ is a point of convergence for these pathways, resulting in the release of arachidonic acid. This signaling pathway is specific for peroxisomes and does not influence motility of mitochondria, lysosomes, or endosomes. However, since the cytoskeleton and its associated proteins including the motor proteins play an important role in mediating motility of all cell organelles, it may well be that variant signaling cascades exist ensuring specific regulation of each distinct compartment.Abbreviations AA arachidonic acid - ATPS adenosine-5-O-(3-thiotriphosphate) - cAMP cyclic adenosine monophosphate - CaM-PK calmodulin-dependent protein kinase - CLIP cytosolic linker protein - DAG diacylglycerol - DiC₈ 1,2-dioctanoyl-sn-glycerol - GFP green-fluorescent protein - GTPS guanosine-5-O-(3-thiotriphosphate) - IP₃ inositol trisphosphate - LPA lysophosphatidic acid - MAPK mitogen-activated protein kinase - MEK MAPK kinase - PKA protein kinase A - PKC protein kinase C - cPKC classical PKC isoforms - PLA₂ phospholipase A₂ - PLAP PLA₂-activating proteinpeptide - PLC phospholipase C - PP2A protein phosphatase 2A     

Effect of pertussis toxin on hormonal responsiveness of rat hepatocytes

The ureogenic action of epinephrine in hepatocytes from normal adult rats is mediated through activation of alpha 1-adrenoceptors. beta-Adrenoceptors in addition to alpha 1-adrenoceptors, became involved in mediating this effect in cells from animals treated with pertussis toxin. The accumulation of cyclic-AMP in response to epinephrine or isoproterenol was markedly increased in hepatocytes from pertussis-treated rats as compared to that observed in control cells. The accumulation of cyclic-AMP due to glucagon was also increased. It is suggested that pertussis toxin may release a constraint on adenylate cyclase activity by blocking the inhibitory coupling mechanism (Ni) or some other entity involved in the regulation of the activity of this enzyme.