Influence of fluoride on secretory pathway of the secretory ameloblast in rat incisor tooth germs exposed to sodium fluoride

 Fluoride, which is an environmental toxicant, is a potent inducer of mottled enamel in humans and rats. To define the influence of fluoride on the secretory pathway in enamel fluorosis, mottled enamel was induced in the incisor tooth germs of rats by subcutaneous injections of sodium fluoride for 4 days, and then morphological and cytochemical changes of the secretory ameloblast were examined in the tooth germs with HRP-labeled lectin (Con A, GS-I, SBA and PNA) and En3 antibody labeling amelogenins. The accumulation of small vesicles on the route of the secretory pathway between the rER and the Golgi apparatus, disorder of Golgi stacks, and formation of abnormal large granules in distal cytoplasm were seen in the secretory ameloblast. Lectin staining patterns of the secretory ameloblast indicated the disturbance of the vesicular transport between the rER and the Golgi apparatus, and disorganization of the Golgi stack. Immunolabeling of the cell showed disruption of the sorting and fusion process on the secretory pathway. These results suggest that the fluoride disturbs the intracellular transport in the synthesis-secretory pathway of the ameloblast, and that this effect of fluoride on the synthesis-secretory pathway participates in the formation of enamel fluorosis. Received: 9 August 1995 / Accepted: 17 October 1995     

In situ hybridization reveals specific increases in Gαs and Gαo mRNA in discrete brain regions of morphine-tolerant rats

In situ hybridization histochemistry has been used to detect the basal distribution of mRNA encoding the α subunit of G_s, G_o and G_i2 proteins throughout the rat brain. Based on these data we investigated the effect of chronic morphine on the content of these G protein α subunits mRNA. We observed an increase in the expression of α_s and α_o messages of chronically morphine-treated animals, while no changes were seen in α_i2 mRNA. Specifically a 30% increase in expression for α_s was seen only in the paraventricular nucleus of hypothalamus and a 20% elevation for α_o was detected in the claustrum and endopiriform nucleus. Immunoblotting analysis was used to correlate the changes in α_s and α_o messages with equivalent changes in protein levels. Chronic morphine significantly increased α_s amounts in the hypothalamus (70%), and produced a minor elevation (30%) in Gα_o levels in the olfactory area. Our results indicate that in discrete brain regions altered G protein expression is part of the adaptive changes underlying opiate tolerance.     

G Proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors

Expression of functionally active mammalian histamine H₁- and H₂-receptors was recently demonstrated in Sf9 cells. Either receptor elicited phosphoinositide degradation leading to an increased cytoplasmic calcium concentration. In the present study we focussed on identifying the Sf9 guanine nucleotide-binding proteins (G proteins) involved. Immunodetection of Sf9 membranes showed expression of Gα isoforms belonging to all four G protein subfamilies. During prolonged baculovirus infection of Sf9 cells, binding of guanosine 5’-o-(3-thiotriphosphate) as well as the intensities of G protein immunoreactivity, pertussis toxin-mediated ADP-ribosylation, GTP azidoanilide labelling of Gα, and phosphate-labelling of Gβ declined in cell membranes. Some 48h after infection with mammalian histamine receptor-encoding viruses virtually no functional coupling of ligand-activated receptors to insect G proteins was observed despite a high level of expressed receptors. In contrast, Sf9 cells infected only for 28h allowed studies on histamine-induced G protein coupling. In membranes obtained from H₁-receptor-expressing cells, histamine increased incorporation of GTP azidoanilide into G _q/11-like proteins whereas in membranes containing H₂-receptors histamine enhanced GTP azidoanilide-labelling of G _q/11-like and G_s-like proteins. In fura-loaded H₁- and H₂-receptor-expressing cells histamine induced the release of calcium from intracellular stores. This study shows firstly that Sf9 G proteins couple to mammalian histamine receptors and secondly that H₁-receptors activate only G_q/11, whereas H₂-receptors activate G_q/11 and G_s, but neither receptor couples to G_i/o or G₁₂. Finally, the time following baculovirus infection is critical for studying the functional coupling between recombinantly expressed and endogenous signal transduction components. Received: 24 January 1997 /Accepted: 14 April 1997     

The role of G proteins in the activity and mercury modulation of GABA-induced currents in rat neurons

The role of G proteins in the functional modulation and potentiation by mercury chloride of the GABA_A receptor-channel complex in rat dorsal root ganglion neurons was studied by using the whole-cell patch clamp technique. Stimulation of G_s proteins by application of GTP-γ-S in the patch pipette or by incubation of neurons with cholera toxin reduced GABA-induced currents, suggesting modulation of GABA-induced currents via a G_s-protein-coupled pathway. GDP-β-S in the pipette solution or pretreatment of dorsal root ganglion neurons with pertussis toxin suppressed GABA-induced currents, suggesting that basal G_i/G_o-protein activity positively modulates the GABA_A receptor-channel complex. Mercury chloride potentiation of GABA-activated currents was blocked by application of GTP-gg-S in the patch pipette or by incubation of neurons with cholera toxin. Mercury chloride potentiation of GABA-activated currents was blocked by application of GDP-β-S in the patch pipette or by incubation of neurons with pertussis toxin. G proteins, probably G_i/G_o proteins, underlie the mercury chloride potentiation of GABA-induced currents.     

Properties of recombinant adenosine receptors

Four subtypes of adenosine receptors have been cloned in several laboratories. The receptors all interact with G proteins and span the plasma membrane 7 times. They range in size from 319 to 412 amino acids. Conservation of structure among 10 different receptor clones for various species suggests that ligand recognition sites involve amino acids in the 2, 3, and 7 transmembrane segments of receptors. Species differences in ligand binding to A₁ receptors in tissues are very similar; for bovine receptors the affinity is R-PIA ? NECA, for canine receptors NECA ≥ R-PIA. Purified bovine A₁ receptors functionally couple to the pertussis toxin sensitive recombinant G proteins, G_i1, G_i2, G_i3, and G_o. © 1993 Wiley-Liss, Inc.     

Interaction of P2 purinergic receptors with cellular macromolecules

Ionotropic P2X and metabotropic P2Y receptors interact with a number of macromolecules in the cell membrane which may contribute to their functional plasticity. P2X receptors are homomeric or heteromeric assemblies of three subunits. P2Y receptors may form oligomeric complexes either with the same or with other P2Y receptor types. Although the signalling mechanism of P2X receptor channels is fast (within milliseconds) and relatively simple, by originating from the opening of an ion channel permeable to mono- and divalent cations, various macromolecules may modify the trafficking of these receptors to and from the cell membrane, as well as their activation and desensitization kinetics, and the possible opening of membrane pores induced by long-lasting exposure to agonists. P2X and Cys-loop receptors may physically interact with each other, resulting in mutual current occlusion. Heteromeric P2Y receptors may, via G_s, G_q/11 or G_i/o protein-coupling and activation of the respective transduction mechanisms, mediate responses in the range of a few seconds. However, P2Y receptors may also interact with the signalling cascade of, e.g. receptor tyrosine kinases, and thereby mediate responses on a much slower time scale (within hours to days). In addition, P2Y receptors may interact with small, homomeric G proteins, integrins, and PDZ proteins. Eventually, P2Y receptors may cross-talk via Gα-dependent signalling with other G protein-coupled receptors and via Gβγ (or indirectly Gα)-dependent signalling with various ion channels. Thus, the activation of P2X and P2Y receptors by extracellular adenosine triphosphate/adenosine diphosphate or uridine triphosphate/uridine diphosphate may trigger specific chains of events which interact at the level of the individual elements both with each other and with the transduction mechanisms of other receptors, creating a huge diversity of the possible effects.     

G12/13-dependent signaling of G-protein-coupled receptors: disease context and impact on drug discovery

G-protein-coupled receptors (GPCRs) transmit extracellular signals across the plasma membrane via intracellular activation of heterotrimeric G proteins. The signal transduction pathways of G_s, G_i and G_q protein families are widely studied, whereas signaling properties of G₁₂ proteins are only emerging. Many GPCRs were found to couple to G_12/13 proteins in addition to coupling to one or more other types of G proteins. G_12/13 proteins couple GPCRs to activation of the small monomeric GTPase RhoA. Activation of RhoA modulates various downstream effector systems relevant to diseases such as hypertension, artherosclerosis, asthma and cancer. GPCR screening assays exist for G_s-, G_i- and G_q-linked pathways, whereas a drug-screening assay for the G₁₂-Rho pathway was developed only recently. The review gives an overview of the present understanding of the G_12/13-related biology of GPCRs.     

Mechanism of noradrenaline-induced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitory G-protein α-subunits

The mechanism of heterologous desensitization of adenylate cyclase stimulation was studied in cultured neonatal rat heart muscle cells. After culturing of the cells for 3 days in the presence of 1 μM noradrenaline there was in addition to a 52% decrease in isoproterenol-stimulated adenylate cyclase activity, a lessening of the stimulation of ß-adrenoceptor-independent adenylate cyclase by guanosine-5′-O-(thiotriphosphate) and forskolin by 24 and 34%, respectively. The decrease in receptor-independent adenylate cyclase stimulation by forskolin, but not the attenuation of isoproterenol-stimulated adenylate cyclase activity, was abolished by pertussis toxin (PTX) pretreatment of the cells. G_i, the inhibitory G-protein of adenylate cyclase was therefore quantitated. Labelling of the M_r ≈ 40 kDa PTX substrates in membranes of noradrenaline-treated cells was increased by 70% as shown by pertussis toxin-catalyzed ADP ribosylation of heart cell membranes. This increase was also seen in the presence of an excess of purified ßγ-subunits of transducin and of GTP, suggesting that the increased labelling was not due to elevation of the level of ßγ-subunits or increase in the concentration of GTP in the membranes of noradrenaline-treated cells. Analysis of the PTX substrates on high resolution urea/SDS-polyacrylamide gels revealed that at least two distinct PTX substrates (40 and 41 kDa) were present in rat heart cell membranes. The labelling of both substrates was increased in membranes of desensitized cells. Immunoblotting of heart cell membranes with anti-G_iα-antibodies demonstrated a marked increase in the amount of G_iα in membranes of noradrenaline-treated cells. In contrast, immunoblotting with anti-ß-antibodies showed that the level of the ß-subunit of G-proteins (36 kDa) was unchanged after noradrenaline exposure. The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of α-subunits of G_i-proteins. This suggests that this increase is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.     

Fluoride-induced ultrastructural changes in exocrine pancreas cells of rats: fluoride disrupts the export of zymogens from the rough endoplasmic reticulum (rER)

 Influence of fluoride on exocrine pancreas cells was examined morphologically with traditional and prolonged osmium fixation techniques for electron microscopy in the enamel fluorosis model rats injected subcutaneously twice a day with 20 mg/kg body weight of sodium fluoride. Although the rough endoplasmic reticulum (rER) of exocrine pancreas cells in control rats was laminated and oriented parallel to the circumference of the nucleus, the rER of the cells in NaF-treated rats was dilated, disrupted the laminated arrangement, and changed to the globular-shape rER. Many intracisternal granules were formed in these globular-shape rER of the cells exposed to fluoride. Lots of autophagosomes were also seen in the exocrine cells with NaF treatment. The autophagosomes were limited with a double or multiple membranes, and contained cytoplasmic organelles and/or the intracisternal granules. The outer and inner leaflets of double membranes of the autophagosomes were usually separated by a distinct electron-lucent area. In prolonged osmium fixation, the area between the double membranes of the autophagosome was filled with osmiun reaction deposits. Many autophagosomes were encircled with the single or multiple osmiophilic layers. In some cases, the osmium positive saccules also surrounded the free surface of the globular-shape rER containing intracisternal granules. These findings indicate that fluoride disrupts the export of zymogens from the rER, resulting in formation of intracisternal granules and autophagosomes, and that the osmiophilic saccules participate in sequestration of cytoplasmic organelles in forming autophagosomes. Received: 26 August 1999 / Accepted: 18 October 1999     

Genetic disruption of G proteins,Gi2α or Goα, does not abolish inotropic and chronotropic effects of stimulating muscarinic cholinoceptors in atrium

Background and purpose:

Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved.

Experimental approach:

We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the α subunit of G_i2 protein (G_i2α) or of the α subunit of G_o protein (G_oα). Preparations were stimulated with carbachol alone or after pretreatment with the β-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca²⁺-channels in isolated ventricular cardiomyocytes were studied.

Key results:

The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca²⁺-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied.

Conclusions and implications:

These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G_i2α and G_oα mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.     

Regulation of hormone-sensitive adenylate cyclase

Adenylate cyclases in the membranes of eukaryotes are regulated by stimulatory receptors (R_s) and inhibitory receptors (R_i) which activate the catalyst adenylate cyclase (C) through the heterotrimeric GTP-binding proteins, G_s and G_i. The actions of G_s and G_i are not symmetric, since G_s associates tightly with C, whereas G_i does not. Alexander Levitzki analyses the current knowledge and thinking on the mode of interaction between each of these regulatory units and describes one working model which accounts for all the experimental observations and the kinetics involved.     

G Protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors

The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A_2A receptor agonist 2-[4-(2-p-carboxyethyl[³H])phenyl-amino]-5’-N-ethylcarboxamidoadenosine ([³H]CGS 21680) and of the adenosine A₁ receptor agonist [³H]R-phenylisopropyladenosine ([³H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5’-(β,γ-imido)triphosphate (1–300 μM), which uncouples all G proteins, more effectively inhibited [³H]CGS 21680 (30 nM) binding in the cortex than [³H]R-PIA (2 nM) binding to cortical or striatal membranes or [³H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1–300 μM) or pertussis toxin (1–100 μg/ml), which uncouple G_i/G_o protein-coupled receptors, more effectively inhibited [³H]R-PIA binding to cortical or striatal membranes and [³H]CGS 21680 binding in the cortex than [³H]CGS 21680 binding in the striatum. Cholera toxin (2.5–250 μg/ml), which uncouples G_s protein-coupled receptors, more effectively inhibited [³H]CGS 21680 binding in the striatum than [³H]CGS 21680 binding in the cortex and less effectively inhibited [³H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 μg/ml) decreased [³H]CGS 21680 (30–100 nM) binding which almost fully recovered after reconstitution with G_i/G_o proteins. The K _i for displacement of [2-³H]-(4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol) ([³H]ZM 241385, 1 nM) by CGS 21680 was 110 nM (95%CI: 98–122 nM) in non-treated, 230 (167–292) nM in pertussis toxin (25 μg/ml)-treated and 222 (150–295) nM in cholera toxin (50 μg/ml)-treated cortical membranes; in contrast, the K _i for displacement of [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine ([³H]SCH 58261, 1 nM) by CGS 21680 was 74 (57–91) nM in non-treated, 71 (44–100) nM in pertussis toxin-treated and 147 (100–193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A₁ antagonist, [³H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A₁ agonist, [³H]R-PIA (2 nM), in 2 or 10 mM Mg²⁺-medium, either at 25°C or 37°C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A₁ receptors and that limbic CGS 21680 binding sites differ from striatal-like A_2A receptors since they couple to G_i/G_o proteins, as well as to G_s proteins. Received: 22 July 1998 / Accepted: 18 January 1999     

Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to Gs and Gi

BACKGROUND AND PURPOSE

The cannabinoid CB₁ receptor is primarily thought to be functionally coupled to the G_i form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB₁ receptors and characterized the structural determinants that mediate selective coupling to G_s and G_i.

EXPERIMENTAL APPROACH

A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB₁/CB₂ receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G_s and G_i.

KEY RESULTS

CB₁ receptors could couple to both G_s-mediated cAMP accumulation and G_i-induced activation of ERK1/2 and Ca²⁺ mobilization, whereas CB₂ receptors selectively coupled to G_i and inhibited cAMP production. Using CB₁/CB₂ chimeric receptors, the second intracellular loop (ICL2) of the CB₁ receptor was identified as primarily responsible for mediating G_s and G_i coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G_s to G_i, while to Ile or Val led to balanced coupling of the mutant receptor with G_s and G_i.

CONCLUSIONS AND IMPLICATIONS

The ICL2 of CB₁ receptors and in particular Leu-222, which resides within a highly conserved DRY(X)₅PL motif, played a critical role in G_s and G_i protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB₁ receptors to G proteins and cannabinoid-induced tolerance.     

Antiarrhythmic effect of prolonged morphine exposure is accompanied by altered myocardial adenylyl cyclase signaling in rats

BackgroundMorphine is often administered to patients for pain management, but it is also recommended to ameliorate some types of cardiovascular diseases. Nevertheless, there is a lack of information regarding the effect of prolonged morphine treatment on myocardial adenylyl cyclase (AC) signaling, which plays an important role in regulating heart function.MethodsThe present work has investigated the consequences of 10-day administration of high morphine doses (10 mg/kg per day) to adult Wistar rats for functioning of the G-protein-mediated AC signaling system.ResultsMorphine treatment appreciably affected neither the number of myocardial β-adrenoceptors nor the content of selected subunits of trimeric G-proteins (G_sα, G_i/oα, G_zα, G_q/11α and Gβ) but the amount of the dominant myocardial AC isoform V/VI almost doubled. These alterations were accompanied by a marked AC supersensitization: the enzyme activity stimulated by manganese, fluoride, forskolin or isoproterenol was considerably increased (by about 50–100%). In contrast, the ability of opioid agonists to inhibit forskolin-stimulated AC activity was slightly but significantly decreased in both groups. Besides that, morphine markedly decreased the incidence of ischemic ventricular arrhythmias induced by coronary artery occlusion, but did not significantly influence infarct size and arrhythmias occurring during reperfusion.ConclusionsOverall, these results indicate that prolonged treatment of rats with high doses of morphine substantially alters the function of myocardial G-protein-regulated AC signaling. These alterations are accompanied by a reduced susceptibility to ischemia-induced ventricular arrhythmias.     

Tumor necrosis factor α up-regulates Giα and Gß proteins and adenylyl cyclase responsiveness in rat cardiomyocytes

Treatment of cultured rat cardiomyocytes in serum-free medium for 48 h with recombinant human tumor necrosis factor α (TNFα) led to a concentration-dependent increase in the level of membrane-inhibitory guanine nucleotide-binding protein (G₁) α-subunits and in pertussis toxin-catalyzed [³²P]ADP ribosylation of 40 kDa proteins. Both G_iα protein subtypes present in rat cardiac myocyte membranes, G_iα40 and G_iα41, were up-regulated by the cytokine, with the maximal increase occurring at 10 U/ml TNFα. In contrast to noradrenaline exposure, which causes a similar, but apparently exclusive, increase in α₁-subunits, treatment with TNFα in addition increased the level of membrane G protein ß₃₆-subunits. Furthermore, while noradrenaline exposure led to a decrease in receptor-dependent and -independent adenylyl cyclase activity, treatment of cardiomyocytes with TNFα caused a concentration-dependent increase in cyclase responsiveness to either forskolin, guanosine 5′-O-(3-thiotriphosphate) or isoproterenol, even though ß-adrenoceptor density was decreased by TNFα. The increase in adenylyl cyclase activity induced by TNFα was completely suppressed when the cells were cocultured with noradrenaline, a condition leading to an additive increase in G_iα level. The data indicate that the cytokine TNFα can potently modulate G protein-mediated signal transduction in rat cardiac myocytes. Although TNFα, like noradrenaline, exposure of the cells increased the level of membrane G_iα proteins, it did not decrease but rather caused an increase in adenylyl cyclase responsiveness. This hypertensitivity may be due to concomitant alterations of other components, e.g. G_ß, G_sα and/or the cyclase itself, of this multi-subunit signal transduction system following TNFα exposure.     

Lateral Diffusion of Gαs in the Plasma Membrane Is Decreased after Chronic but not Acute Antidepressant Treatment: Role of Lipid Raft and Non-Raft Membrane Microdomains

GPCR signaling is modified both in major depressive disorder and by chronic antidepressant treatment. Endogenous Gα_s redistributes from raft- to nonraft-membrane fractions after chronic antidepressant treatment. Modification of G protein anchoring may participate in this process. Regulation of Gα_s signaling by antidepressants was studied using fluorescence recovery after photobleaching (FRAP) of GFP-Gα_s. Here we find that extended antidepressant treatment both increases the half-time of maximum recovery of GFP-Gα_s and decreases the extent of recovery. Furthermore, this effect parallels the movement of Gα_s out of lipid rafts as determined by cold detergent membrane extraction with respect to both dose and duration of drug treatment. This effect was observed for several classes of compounds with antidepressant activity, whereas closely related molecules lacking antidepressant activity (eg, R-citalopram) did not produce the effect. These results are consistent with previously observed antidepressant-induced translocation of Gα_s, but also suggest an alternate membrane attachment site for this G protein. Furthermore, FRAP analysis provides the possibility of a relatively high-throughput screening tool for compounds with putative antidepressant activity.     

Chronic exposure of rat glioma C6 cells to cholera toxin induces loss of the α-subunit of the stimulatory guanine nucleotide-binding protein (Gs)

Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [³²P]NAD⁺ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of G_sα. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenouss NAD⁺ as substrate. However, immunological studies with anti-peptide antisera which identify all forms of G_sα demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes. This effect was specific for G_sα as neither the α-subunits of the pertussis toxin-sensitive G-proteins G_i2 and G_i3, nor the β subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of G_sα nor of the α-subunits of G_i2 or G_i3 although it did cause ADP ribosylation of the entire complement of both G_i2 and G_i3 in the membranes. Despite the reduction in levels of immunoreactive G_sα from the membranes of cholera toxin-treated cells, no alterations in levels of MRNA corresponding to this G-protein were noted.     

Ultrastructural study in canine prostatic hyperplasia--effect of antiandrogen.

Ultrastructural changes in canine prostates after treatment with chlormadinone acetate (CMA) were investigated. Old male beagle dogs (5-8 years old ) were divided into two experimental groups; group 1 consisted of benign prostatic hyperplasia (BPH) controls, and group 2 received 0.3 mg/kg/day CMA orally for 6 months. In group 1 animals, the most striking ultrastructural changes were detected in the rough endoplasmic reticulum (rER) and Golgi complexes. The secretory granules were lined up along the apical plasma membrane, and exocytosis was frequently seen. In group 2 animals, the cytoplasm was electron-lucent and contained relatively few, poorly developed organelles. The rER was sparse and consisted of a few scattered, short profiles studded with ribosomes. The Golgi complexes were inconspicuous. The secretory granules were markedly decreased in both number and size. Furthermore, mitochondrial degeneration such as swollen or disappeared mitochondrial cristae or decreased electron density of the matrix were frequently seen in the smooth muscle cells. Based on our data, atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.     

Phosphorylation of G-protein α-subunits in intact adipose cells: evidence against a mediating role in insulin-dependent metabolic effects

The phosphorylation of G-protein α-subunits was studied in plasma membranes prepared from isolated, intact adipocytes equilibrated with [³²P]phosphate and subsequently incubated in the presence or absence of insulin. In iodinated or unlabeled plasma membranes, antiserum generated against a peptide corresponding to a region common to G-protein α-subunits immunoprecipitated two major proteins of 45 and 40 kDa, which were identified as G_s and G_i α-subunit, respectively, by comparison with [³²P]ADP-ribosylated G-proteins. In membranes prepared from cells equilibrated with [³²P]phosphate, the antiserum precipitated a 45 kDa phosphoprotein. Pre-immune serum failed to immunoprecipitate the phosphoprotein. Insulin stimulated [³²P]phosphate incorporation into the 45 kDa protein approximately 2-fold. Control experiments suggested that the 45 kDa phosphoprotein was not identical with Gα_s, since (1) the peptide used to raise the antiserum failed to inhibit significantly immunoprecipitation of the 45 kDa phosphoprotein with the antiserum, (2) in contrast to the G_s α-subunit, the phosphorprotein was readily removed from the immunocomplex by washing with sodium dodecyl sulfate (SDS), and (3) the subcellular localization of the phosphoprotein differed considerably from that of the G_s α-subunit. No phosphate was detected in immunoprecipitates from either basal or insulin-treated cells after the 45 kDa phosphoprotein had been removed. These data argue against a mediating role of phosphorylated G-protein α-subunits in the action of insulin.     

Terminal serotonin autoreceptor function in the rat hippocampus is not modified by pertussis and cholera toxins

Summary The possibility that the terminal serotonin (5-HT) autoreceptor in the rat hippocampus is coupled to G_i, G_o or G_s regulatory proteins was investigated using the electrically evoked overflow of [³H]5-HT from preloaded slices. Pertussis toxin, which inactivates G_i/o or cholera toxin, which stimulates GS, was injected directly in the hippocampus 3 to 11 days prior to the experiments. Hippocampus slices were prepared, loaded with [³H]5-HT, superfused continuously, and stimulated electrically 72 min (S₁) and 116 min (S₂) after the beginning of superfusion. In the absence of any drug, the evoked overflow of [³H]5-HT in S₁ was not altered by either toxin. The enhancing effect of the 5-HT reuptake blocker paroxetine (1 mol/I) on the evoked [³H]5-HT overflow was also unaltered by these toxins. 5-Carboxyamidotryptamine, a 5-HT autoreceptor agonist, inhibited in a concentration-dependent manner the stimulation-evoked release of [³H]5-HT The concentration-effect curve (0.001–0.1 mol/I) for this drug was not altered by pretreatment with either pertussis or cholera toxin. Similarly, the effect of another 5-HT autoreceptor agonist, 5-methoxytryptamine (0.1 and I mol/I), was not altered in the pretreated rats. In addition, the reduction of [³H]5-HT overflow obtained by increasing the stimulation frequency from 1 Hz to 5 Hz, which is due to an increase in terminal 5-HT autoreceptor activation at the higher frequency, was not altered by either toxin. The enhancing effect of the 5-HT autoreceptor antagonist methiothepin (1 mol/I) on stimulation-evoked [³H]5-HT overflow was not changed by either pretreatment. N-Ethylmaleimide inactivates G_i/o proteins by alkylation. Preincubation with 30 mol/I N-ethylmaleimide for 30 min did not alter the efficacy of the 5-HT reuptake blocker paroxetine to enhance [³H]5-HT overflow nor did it alter the attenuating effect of 5-methoxytryptamine and the differential effectiveness of 1 and 5 Hz stimulations on the overflow of [³H]5-HT In conclusion, the results suggest that the terminal 5-HT autoreceptor in the rat hippocampus may not be coupled to G_i, G_o or G_s proteins.