1984 Fluoride Abstracts. Part 1. Fluoride Action Network Pesticide Project.

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Acta Physiol Pol 1984 May-Jun;35(3):199-206

Effect of certain agents on subcellular cAMP level in different areas of rat brain.

Janiszewska G, Lachowicz L, Wojtkowiak R.

The influence was studied in vitro of certain agents (adenosine, ADP, ATP, theophylline, together with F- ions) on the cAMP concentrations in the nuclear (N) and mitochondrial (M) fractions from different areas of rat brain. F- ions caused a slight decrease of the cAMP concentrations in nuclear fractions of the thalamus with hypothalamus and a marked decrease of this cyclic nucleotide in M fractions from the cerebral cortex. After incubation with adenosine and F- ions a distinct decrease of cAMP level was observed in N fractions from the midbrain and thalamus with hypothalamus and in mitochondrial fractions obtained from all the investigated regions. The incubation with ATP and F- ions resulted in a distinct decrease of cAMP values in the nuclear fractions from all regions. The concentrations of cAMP in the mitochondria of the midbrain and thalamus with hypothalamus incubated with ATP and F- ions increased 2-3 times. The incubation of the nuclear fraction with theophylline and F- ions caused an increase of cAMP concentration in the cortex and a decrease of cAMP values in the midbrain. The level of cAMP after the incubation with theophylline and fluoride on the mitochondrial fraction is increased in the cortex and decreased in the thalamus with hypothalamus.

PMID: 6100417 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6094742&dopt=Abstract

J Neurosci 1984 Nov;4(11):2764-71
Modification of guanine nucleotide-regulatory components in brain membranes. II. Relationship of guanosine 5'-triphosphate effects on opiate receptor binding and coupling receptors with adenylate cyclase.

Childers SR, LaRiviere G.

Guanine nucleotides couple receptors to stimulate or inhibit adenylate cyclase as well as regulate binding of neurotransmitters. To explore the relationship between these different functions of guanosine 5'-triphosphate (GTP), rat brain membranes were preincubated in 50 mM sodium acetate, pH 4.5, which increased GTP regulation of 3H-opiate agonist binding. Assay of adenylate cyclase in the low pH-pretreated membranes revealed no loss of basal activity but a dramatic loss in fluoride- and guanylyl-5'-imidodiphosphate-stimulated activity, thus suggesting a loss in stimulatory guanine nucleotide coupling function. Manganese stimulation, which presumably occurs on the catalytic subunit of adenylate cyclase directly, was not affected by low pH treatment. In striatum, dopamine-stimulated adenylate cyclase was eliminated, but inhibition of adenylate cyclase by D-Ala2-Met5-enkephalinamide (D-Ala enk) was increased by low pH treatment. The effect of low pH on sodium fluoride-stimulated and D-Ala enk-inhibited adenylate cyclase could be reversed by addition of either cis-vaccenic acid or phosphatidylcholine to treated membranes, but the effect on GTP regulation of binding was not reversed by lipid incorporation. These results suggest that fundamental differences exist between membrane components which couple receptors to adenylate cyclase and those that regulate neurotransmitter binding.

PMID: 6094742 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6728130&dopt=Abstract
Neuropharmacology 1984 Apr;23(4):445-7

Fluoride-stimulated adenylate cyclase activity in rat brain following chronic treatment with psychotropic drugs.

Andersen PH, Klysner R, Geisler A.

Chronic treatment with imipramine and reserpine increased fluoride-stimulated activity of adenylate cyclase in homogenates of cerebral cortex and "limbic" forebrain of the rat. Concomitant treatment with lithium counteracted this effect, while lithium alone had no effect on the activity of adenylate cyclase.

PMID: 6728130 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6692416&dopt=Abstract
Cancer Res 1984 Mar;44(3):938-41

Sodium fluoride-induced morphological and neoplastic transformation, chromosome aberrations, sister chromatid exchanges, and unscheduled DNA synthesis in cultured syrian hamster embryo cells.

Tsutsui T, Suzuki N, Ohmori M.

The effects of exposure of early-passage Syrian hamster embryo cells in culture to sodium fluoride have been studied with respect to induction of morphological and neoplastic transformation, chromosome aberrations, sister chromatid exchanges, and unscheduled DNA synthesis. Exposure of Syrian hamster embryo cells to NaF concentrations between 75 and 125 micrograms/ml for 24 hr caused approximately 90 to 40% cell survival and resulted in a dose-dependent increase in the frequency of morphological transformation of the cells. Mass cultures of cells treated with NaF (75 or 100 micrograms/ml) for 24 hr, followed by continuous cultivation for 35 to 50 passages, developed the ability to grow in soft agar and to produce anaplastic fibrosarcomas when injected into newborn hamsters. In contrast, no morphological and neoplastic transformation was observed in untreated cells. Furthermore, a significant increase in chromosome aberrations at the chromatid level, sister chromatid exchanges, and unscheduled DNA synthesis was induced by NaF in a dose- and time-dependent manner. These results indicate that NaF is genotoxic and capable of inducing neoplastic transformation of Syrian hamster embryo cells in culture. A potential for carcinogenicity of this chemical, which is widely used by humans, is suggested. However, the carcinogenic risk of this chemical to humans may be reduced by factors regulating in vivo dose levels.

PMID: 6692416 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6717484&dopt=Abstract
Mutat Res 1984 Apr;139(4):193-8

Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride.

Tsutsui T, Suzuki N, Ohmori M, Maizumi H.

The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50-150 micrograms/ml) or exposure time (1-24 h). Treatment of the cells with 50 micrograms/ml NaF for 24 h resulted in a lethality (approximately 70%) similar to that obtained with 100 micrograms/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-1 cells treated with 20-50 micrograms/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence of 10 mM hydroxyurea. Treatment with 100-400 micrograms NaF/ml for 4-24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

PMID: 6717484 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6738571&dopt=Abstract
Mutat Res 1984 May;140(1):43-8

Induction of unscheduled DNA synthesis in cultured human oral keratinocytes by sodium fluoride.

Tsutsui T, Ide K, Maizumi H.

The effect of treatment of cultured human oral keratinocytes with sodium fluoride (NaF) has been investigated with respect to induction of unscheduled DNA synthesis (UDS). Oral keratinocytes were isolated from excised buccal mucosa of normal individuals by trypsinization at 4 degrees C overnight, followed by separation of the epithelium of mucosa from lamina propria mucosae with forceps. Isolated cells were cultured in vitro and all experiments were performed with secondary cultures. For detection of UDS, the keratinocytes were cultivated with medium containing 1% fetal calf serum (FCS) for 2 days and then treated with 100-300 micrograms/ml NaF for 4 h in medium containing 1% FCS and 10 mM hydroxyurea (1% FCS-HU medium). Following treatment with NaF, UDS was measured by direct scintillation counting of [3H]thymidine incorporated into DNA of the cells in 1% FCS-HU medium. Significant levels of UDS were induced in a dose-related fashion by NaF treatment. The results suggest that NaF causes DNA damage in cultured human oral keratinocytes.

PMID: 6738571 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6203198&dopt=Abstract
Tsitologiia 1984 Mar;26(3):299-306

[Morphological and metabolic changes in a HeLa cell culture exposed to fluorine]

[Article in Russian]

Strochkova LS, Zhavoronkov AA, Avtsyn AP.

Fluoride in subtoxic (1.5 mcg/ml) and toxic (12 mcg/ml) concentrations induced characteristic changes in the mitotic regime of HeLa cell culture. Fluctuations of the mitotic index, variations in the duration of division phases and in the spectrum of pathologic mitosis were noticed. It was shown that fluoride inhibited the genome reduplication. In lower concentrations the halogen produced the most expressed effect on transcription and translation, which corresponds to a so called paradoxial action of this trace element. Definite shifts in the ultrastructure of the cell surface and cell organelles were registered, which confirms a connection between morphological and functional changes in the culture during incubation with fluoride.

PMID: 6203198 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6719498&dopt=Abstract
Toxicol Lett 1984 May;21(2):167-72

Alterations in drug metabolising enzymes and lipid peroxidation in different rat tissues by fluoride.

Soni MG, Kachole MS, Pawar SS.

Sodium fluoride at a dose level of 5.0 mg/kg enhanced aminopyrine N-demethylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in rat liver, kidney, lung, intestine and testis, whereas acetanilide hydroxylase activity remained unchanged in kidney and lung and was increased in liver, intestine and testis. Sodium fluoride at 20.0 mg/kg caused a decrease in aminopyrine N-demethylase, acetanilide hydroxylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in all tissues, except for an increase in NADPH cytochrome c reductase activity in the intestine and testis. Fluoride at both dose levels produced only marginal changes in glutathione-S-transferase activity except for a 4-fold increase in the testis at 5.0 mg/kg. Sodium fluoride at 5.0 mg/kg increased lipid peroxidation in all tissues studied. At 20.0 mg/kg there was a decrease in lipid peroxidation in liver, lung and testis and an increase in kidney and intestine.

PMID: 6719498 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6092357&dopt=Abstract
J Biol Chem 1984 Nov 10;259(21):12984-8

The crystal structure of fluoride-inhibited cytochrome c peroxidase.

Edwards SL, Poulos TL, Kraut J.

The three-dimensional crystal structure of yeast cytochrome c peroxidase complexed with fluoride (F- or HF) has been determined by difference Fourier techniques and partially refined at 2.5-A resolution. Fluoride binding induces significant perturbations of the enzyme structure of the distal side of the heme. The major effect occurs at the active-site arginine residue (Arg-48) which moves about 2 A in order to optimize hydrogen-bonded interactions with the fluorine atom. A small readjustment of the distal histidine (His-52), about 0.5 A, is also seen upon fluoride binding. Additionally, a hydrogen-bonded network of 4 water molecules at the active site is reorganized. No significant movements are detectable in either the heme itself or in the proximal histidine ligand. These observations imply that movement of the Arg-48 side chain may play a key role in the enzymic mechanism of cytochrome c peroxidase. Although we cannot unequivocally determine whether fluoride is bound as HF or F-, the hydrogen-bonding pattern around the ligand points to the protonated form. Structural comparison suggests that there is a difference between the tautomeric state of the imidazole side chain of the distal histidine in cytochrome c peroxidase and of the similarly positioned distal histidine in the globins. This difference accounts for the observation that cytochrome c peroxidase preferentially binds the protonated form of ligands, whereas the globins bind the anionic form. The tautomer indicated by the peroxidase structure is the one required for acid base catalysis (Poulos, T.L., and Finzel, B. C. (1984) in Peptide and Protein Reviews (Decker, M., ed) in press).

PMID: 6092357 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6509140&dopt=Abstract
Biol Reprod 1984 Nov;31(4):752-8

Role of ovarian steroid hormones in the regulation of adenylate cyclase during early progestation.

Bekairi AM, Sanders RB, Abulaban FS, Yochim JM.

The hormonal regulation of uterine adenylate cyclase (AC) was measured in the rat by radiochemical analysis. Animals made pseudopregnant by cervical stimulation were ovariectomized on Day 1 (the first appearance of leukocytes in the vaginal smear) and injected for 6 days with sesame oil, 0.1-10.0 micrograms estrone, 2.0 mg progesterone, or 1.0 microgram estrone + 2.0 mg progesterone. AC activity in ovariectomized controls remained at basal levels (2.8-3.3 pmol cAMP formed/min X mg protein). The injection of progesterone did not alter AC activity significantly, but estrone increased AC activity during Days 3-5, and the response (5-17 pmol) was dose dependent. The action of estrone was not inhibited by progesterone. The present experiments revealed:
a) AC from estrone-treated rats was activated 2- to 4-fold by 10 mM NaF;
b) following treatment with estrone + progesterone, AC was activated 2- to 3-fold by a trauma to the uterus;
c) unlike the response to fluoride, the effect of trauma was temporally limited to Day 4; and
d) when AC was activated by trauma, no further increase was elicited by NaF. The data indicated that the transient sensitivity of AC to activation by trauma on Day 4 in E+P-treated rats was identical to that in intact rats and paralleled the normal timing of uterine sensitivity to decidual induction.

PMID: 6509140 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6542430&dopt=Abstract
Biol Reprod 1984 Nov;31(4):742-51

Uterine adenylate cyclase activity during the estrous cycle and early progestation in the rat: responses to fluoride activation and decidual induction.

Bekairi AM, Sanders RB, Yochim JM.

Uterine adenylate cyclase (AC) activity of the rat was measured by radiochemical analysis during the estrous cycle and early pseudopregnancy. During the estrous cycle, AC activity increased from 4.6 to 16.9 pmol cAMP formed/min X mg protein between metestrus and proestrus. Although AC was activated 2- to 3-fold at all cycle stages by 10 mM NaF, the resulting pattern of activity was similar to that measured in the absence of fluoride. The results demonstrated that the pattern of AC activity during the cycle was similar to that of other estrogen-sensitive uterine enzymes and that the ovarian hormones probably altered enzyme biosynthesis and turnover to a greater extent than activation and kinetic properties. Following the induction of pseudopregnancy by cervical stimulation, enzymic activity increased from 3.5 to 9.4 pmol between Days 1-4 (Day 1=leukocytic vaginal smear) and declined thereafter. AC activity was increased 2- to 5-fold by NaF on all days. AC activity was similarly increased by a mechanical trauma to the uterus, but only when the trauma was applied on Day 4. Following trauma to the uterus, AC activity was not increased further by NaF. The similarities between the physicochemical characteristics of AC during the estrous cycle and early progestation suggested that the enzyme during all endocrine states had virtually identical properties. However, the transient sensitivity to activation after trauma on Day 4 was unique to progestational uteri. Because the properties of enzyme were not altered by the endocrine state of the tissue, the transient sensitivity to activation by trauma was suggested to be a result of hormone-induced alterations in the membrane in which AC is sequestered.

PMID: 6542430 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6508819&dopt=Abstract
Biochem Pharmacol 1984 Nov 15;33(22):3573-7

Aging and reactivatability of plaice cholinesterase inhibited by soman and its stereoisomers.

Bucht G, Puu G.

A simple and rapid method to study aging of soman-inhibited cholinesterases was developed. The method was applied to study the aging characteristics of soman-inhibited cholinesterase from the muscles of the plaice (Pleuronectes platessa). The orientation of the soman molecule in the active site is decisive both for the rate of aging and the degree of reactivation of unaged enzyme, a conclusion reached by using soman stereoisomers. Fluoride ions were found to affect reactivatability as well as aging rate.

PMID: 6508819 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6543337&dopt=Abstract
Eur J Pharmacol 1984 Nov 13;106(2):437-40

Activation of adenylate cyclase by dopamine, GTP, NaF and forskolin in striatal membranes of neonatal, adult and senescent rats.

Nomura Y, Makihata J, Segawa T.

Dopamine (DA) caused a significant activation of striatal adenylate cyclase in neonatal and adult but not in senescent rats. GTP activated cyclase at the adult stage but not at both neonatal and senescent stages. NaF and forskolin activated cyclase at every stage. The coupling mechanism between DA1 receptors and catalytic units of cyclase seems to become functional at the neonatal stage but GTP recognition and/or binding sites lack in stimulatory GTP binding protein in neonatal and senescent membranes.

PMID: 6543337 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6525201&dopt=Abstract
Biochem Int 1984 Nov;9(5):659-68

The kinetics of inhibition of human seminal plasma acid phosphatase by sodium fluoride.

NagDas SK, Bhattacharyya AK.

Purified human seminal plasma acid phosphatase is inhibited by sodium fluoride and L(+) tartrate and 50% inhibitions (I50) have been obtained at 7.3 X 10(-5) and 3.0 X 10(-4) M concentrations, respectively. By Lineweaver-Burk plot, sodium fluoride shows non-competitive type of inhibition. Ackermann plot indicates that sodium fluoride is a reversible inhibitor of seminal plasma acid phosphatase. On polyacrylamide activity gel electrophoresis and by densitometric scan, a single molecular form of acid phosphatase has been identified in human seminal plasma. Inhibition patterns by sodium fluoride and L(+) tartrate have also been performed by polyacrylamide activity gel electrophoresis and almost complete inhibition of hydrolysis of alpha-napthol phosphate is observed at 20 mM and 100 mM of sodium fluoride and L(+) tartrate, respectively.

PMID: 6525201 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6487808&dopt=Abstract
Blood 1984 Nov;64(5):986-93

A reassessment of the energy requirements for neutrophil migration: adenosine triphosphate depletion enhances chemotaxis.

Lane TA, Lamkin GE.

In view of previous studies demonstrating a significant correlation between adenosine triphosphate (ATP) depletion and impairment of chemotaxis (CTX) during granulocyte (PMN) storage, we sought to quantitate the relationship between CTX and PMN energy metabolism. We incubated PMNs at 37 degrees C with 2-deoxyglucose (2-dg) in the presence of 5 mmol/L glucose. As expected, ATP inhibition by 2-dg was time-dependent (T 1/2, 18 minutes) and dose-dependent, with half-maximal inhibition of ATP (ID50) with 1.3 +/- .3 mmol/L 2-dg. Similar concentrations of 2-dg inhibited lactate generation, phagocytosis, superoxide anion generation, and degranulation. The random migration of PMNs was inhibited by somewhat higher concentrations of 2-dg (ID50, 12 mmol/L). In contrast, up to 40 mmol/L 2-dg did not inhibit CTX toward synthetic peptides or activated serum. In fact, 2-dg consistently increased the CTX of PMNs toward 10(-8) mol/L f-Met-Leu-Phe (fMLP), to a maximum of 450% of control CTX using 15 mmol/L 2-dg. Half-maximal stimulation (ED50) of CTX occurred at 6.3 +/- 1.0 mmol/L 2-dg. Although maximal CTX toward optimal concentrations of fMLP was consistently increased with 2-dg, the ED50 of CTX to fMLP was unchanged (ED50 with glucose, 2.0 +/- 0.6 nmol/L fMLP; ED50 with 2-dg 2.2 +/- 0.7 nmol/L fMLP), and 2-dg did not increase fMLP receptors. In the absence of glucose, 2-dg exerted similar effects on ATP and CTX, but at doses 30- to 50-fold lower than in the presence of glucose. Other glycolytic inhibitors (iodoacetamide and sodium fluoride) exerted similar effects. Additional studies indicated that CTX enhancement by 2-dg (a) required Mg++ but not Ca++, (b) occurred with PMNs from a patient with chronic granulomatous disease, (c) was unaltered in the presence of inhibitors of proteolysis, (d) was not due to generation of a soluble agent, (e) was not due to alterations in PMN adherence, and (f) was not due to inhibition of glycosylation. We conclude that the chemotaxis, but not the random migration, of PMNs is surprisingly resistant to inhibition of energy metabolism and depletion of ATP, since concentrations of 2-dg that decreased ATP and other cell functions by more than 50% not only did not inhibit, but actually stimulated, CTX. These studies also indicate that the previously reported correlation between ATP depletion and CTX impairment observed in stored PMNs are not causally related.

PMID: 6487808 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6209573&dopt=Abstract

Mutat Res 1984 Nov;129(2):195-206

The action of anticlastogens in human lymphocyte cultures and its modification by rat liver S9 mix. I. Studies with AET and sodium fluoride.

Gebhart E, Wagner H, Behnsen H.

The action of beta-aminoethylisothiouronium bromide hydrobromide (AET) and sodium fluoride (NaF) on the clastogenic activity of Trenimon, cyclophosphamide, and bleomycin was tested on cultures of human peripheral lymphocytes with and without the addition of rat liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of Trenimon and cyclophosphamide was examined under the same conditions. In the absence of S9 mix both substances displayed the known anticlastogenic action when TR was the standard clastogen but acted coclastogenically in the experiments with BM. Under the influence of rat-liver S9 mix this action on TR-induced chromosome damage was decreased and only a slight anticlastogenic effect was observed in the experiments with activated cyclophosphamide. S9-activated BM lost some of its strong chromosome-damaging effect and AET proved clearly anticlastogenic under these test conditions. AET displayed a slight decreasing effect on SCE induced by TR, but had no effect on CP-induced SCE. No anti-SCE effect at all was found in the experiments with NaF. Detailed analyses revealed different actions of both anticlastogens on the different types of structural chromosome damage.

PMID: 6209573 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6434290&dopt=Abstract
Endocrinology 1984 Oct;115(4):1386-91

Effects of human erythrocyte guanine nucleotide-binding regulatory protein on parathyroid hormone-responsive adenylate cyclase from canine renal cortex.

Levine MA, Greene A, Turner RT, Bell NH.

We studied the effects of the guanine nucleotide-binding regulatory protein (Gs) from human erythrocytes on PTH-responsive adenylate cyclase from partially purified membranes of canine renal cortex (CRC). Extracts of erythrocyte membranes, containing soluble Gs, was obtained by treatment with a detergent (Lubrol PX). Gs did not stimulate adenylate cyclase activity by itself, but amplified the response of adenylate cyclase in CRC membranes to both synthetic bovine PTH-(1-34) [bPTH-(1-34)] and to the hydrolysis-resistant GTP analog 5'-guanylimido-diphosphate [Gpp(NH)p]. Gs increased PTH stimulation of adenylate cyclase activity in both the presence and absence of Gpp(NH)p. In the absence of Gpp(NH)p, the potentiating effect of Gs occurred only when the concentration of bPTH-(1-34) was greater than 10 ng/ml. bPTH-(1-34), Gpp(NH)p, and Gs each enhanced the catalytic activity of adenylate cyclase when added separately or in combination by increasing the apparent maximum velocity (Vmax) of the enzyme without altering the apparent Km for MgATP. The effect of Gs on CRC membrane adenylate cyclase activity in the presence of NaF (10 mM) and forskolin (100 microM) was also examined. NaF- and forskolin-stimulated enzyme activities were significantly increased by Gs in both the presence and absence of Gpp(NH)p (100 microM). Analysis of double reciprocal plots of substrate concentration and enzyme activity revealed that NaF and forskolin increased the Vmax of the catalytic activity and did not alter the apparent Km of the enzyme for MgATP. These data support the role of Gs as a regulator of the response of adenylate cyclase to hormones, guanyl nucleotides, NaF, and forskolin. Our studies address the relative functional stoichiometry between Gs and catalytic unit present in CRC membranes and suggest that the CRC adenylate cyclase system must contain insufficient Gs to couple with all available catalytic units. These results are consistent with the possibility that deficiency of Gs impairs hormonal stimulation by diminishing the apparent Vmax of the catalytic unit and does not alter the apparent affinity of the enzyme for MgATP.

PMID: 6434290 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6477494&dopt=Abstract
Biochem J 1984 Aug 1;221(3):685-95

Conditions that may result in (de-)phosphorylation of hepatic acyl-CoA:cholesterol acyltransferase result also in modulation of substrate supply in vitro.

Mitropoulos KA, Venkatesan S.

The present experiments were designed to study intervesicular transfer of cholesterol in rat liver microsomal fraction and modulation of the activity of acyl-CoA:cholesterol acyltransferase (ACAT) under conditions that are expected to result in the covalent modification (phosphorylation/dephosphorylation) of the enzyme. Preincubation of rat liver microsomal fraction followed by assay of ACAT showed a time-dependent increase in activity. This rate was temperature-dependent. Preincubation in the presence of cholesterol/phospholipid liposomes resulted in a time-dependent transfer of cholesterol from liposomal to the microsomal vesicles and in an increase in the rate of ACAT change owing to the preincubation. Both these rates were dependent on liposomal cholesterol concentration and on temperature. The presence of cytosol in the preincubation mixture increased the rate of change of ACAT activity in the absence or in the presence of cholesterol/phospholipid liposomes. In the latter case the presence of cytosol also increased the rate of transfer of cholesterol from liposomal to the microsomal vesicles. Activation energies of the rate of this transfer and of the rate of increase of ACAT activity were similar in the presence and in the absence of cytosol. Both in the absence and in the presence of cytosol, the presence of NaF (50 mM) in the preincubation mixture considerably decreased the rate of transfer of cholesterol from liposomal to microsomal vesicles and the rate of increase of ACAT activity. The presence of Mg2+ in the preincubation mixture produced no effect on the rate of transfer of cholesterol from liposomal to the microsomal vesicles, although under most conditions it decreased the rate of increase of ACAT activity caused by the preincubation. These results are discussed in relation to the molecular mechanism involved in this intervesicular transfer of cholesterol and to the modulation of ACAT activity by substrate supply, and also in relation to the hypothesis that ACAT activity can be modulated by a mechanism involving the phosphorylation/dephosphorylation of the enzyme.

PMID: 6477494 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6091663&dopt=Abstract

Biochem Int 1984 Aug;9(2):229-36
Estradiol receptor and cyclic nucleotide phosphodiesterase: functional relationship, possible role of guanine nucleotide binding proteins.

Etingof RN, Dumler IL, Garnovskaja MN, Kalinina SN.

The inhibitory effect of estradiol (10(-9)M - 2 X 10(-5)M) on the cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity of cytosol from rat uterine tissue and human myometrium and endometrium was established. The hormone action was shown to disappear due to preparative separation of the receptor and the enzyme; the effect reappeared after protein mixing. In the presence of GTP, Gpp (NH)p, NaF and cholera toxin the estradiol action on the phosphodiesterase activity was significantly decreased or failed to be revealed. The antibodies raised against G-proteins of retina photoreceptor membranes abolished the hormone action on phosphodiesterase. A conclusion is drawn that in uterine tissue there exists a functional connection between estradiol receptor and phosphodiesterase. The necessary participation of G-proteins in signal transmission is postulated.

PMID: 6091663 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6477124&dopt=Abstract
Arch Toxicol 1984 Jul;55(2):119-22

Modulation of estrogen-induced carcinogenesis by chemical modifications.

Liehr JG.

The mechanism of carcinogenesis by estrogens is still unknown. Uncontrolled stimulation of cell proliferation, an endocrine imbalance, or metabolic activation of estrogens to reactive intermediates capable of tissue injury have previously been proposed. In an attempt to gain insight into mechanistic details of estrogen-induced carcinogenesis in male Syrian hamsters, fluorine substituted estrogens, which were impaired in their capacity to be transformed into catechols, have been tested for their carcinogenic activity. 2-Fluoroestradiol was found to be non-carcinogenic in Syrian hamsters despite its estrogenic potency. In a second unrelated experiment, ascorbic acid, which reduced diethylstilbestrol quinone to cis- and trans-diethylstilbestrol in vitro, was administered to estradiol or diethylstilbestrol-treated hamsters. A lowered incidence of kidney tumors in vivo was found in animals receiving ascorbic acid vs estrogen-treated control animals. These results were taken as evidence for a role of estrogen metabolites (catechols formed from estradiol or quinone formed from diethylstilbestrol) in estrogen-induced tumorigenesis. A mechanistic model of metabolic activation of estrogens followed by damage to cellular macromolecules is proposed.

PMID: 6477124 [PubMed - indexed for MEDLINE]

Fluoride 1984; 17(1):27-35
Microdetermination of total fluoride in serum by aluminum monofluoride molecular absorption spectrometry and its significance

Fujimori S, Itai K, Tsunoda H

Department of Hygiene and Public Health, School of Medicine, Iwate Medical University, Morioka, Japan.

Summary: The AlF molecular absorption spectrometry was applied to the determination of serum fluroide samples of which were made of a mixture of equal volumes of serum, 0.05 M Sr(CH3C)2, and distilled waer. Total fluoride was determined by measuring the molecular absorption intensity of AlF. The sensitivity which gave 1% absorption was 0.028 ng of fluoride and the C.V. was 3 to 9%. The AlF method was used to estimate the normal value of total fluoride in serum.

Determinations were done for 221 healthy individuals who were residing in a rural area. The mean value for total fluoride was 20 +- 9.8 S.D. ng/ml. Fluoride concentrations in serum were determined by both the AlF method and ion electrode method. Values of total fluoride were higher than values of ionic fluoride, which suggests the existence of nonionic fluoride.

Fluoride 1984; 17(2):72-80
The role of calcium in fluoride-activated exocytosis in rabbit polymorphonuclear leukocytes

JGR Elferink

Laboratory of Medical Biochemistry, Sylvius Laboratories, University of Leiden, The Netherland.

Summary: The consecutive exposure of rabbit peritoneal polymorphonuclear leukocytes (PMN) to sodium fluoride and Ca2+ ions result in extensive exocytosis. When Ca2+ is replaced by Sr2+, Mg2+ or Ba2+, no exocytosis occurs. The inorganic calcium antagonistic ions Ni2+, Co2+ and Mn2+ inhibit exocytosis; inhibition can be reversed by an increase of the extracellular Ca2+ concentration. A series of organic calcium antagonists inhibit fluoride-activated exocytosis, but only at relatively high concentrations. Inhibition of fluoride-activated exocytosis by calmodulin inhibitors W-7, chlorpromazine and trifluoperazine suggests the involvement of calmodulin in this type of exocytosis. A hypothesis is presented about the role of Ca2+ in the mechanism of fluroide-activated exocytosis.

Introduction: The main task of polymorphonuclear leukocytes (PMN's) is the uptake of invading microorganisms by phagocytosis. Concomitantly with phagocytosis, exocytosis occurs: inracellular granules fuse with the plasma membrane and the content of the granules is liberated extracellular. The physiological relevance of exocytosis by PMN's is not completely clear. It is generally assumed that exocytosis by PMN's plays an important role in inflammations because the granules contain hydrolytic and proteolytic enzymes, and other inflammation-promoting substances (1). These products may damage the surrounding tissues; in this way they are supposed to contribute to the pathogenesis and proliferation of a number of inflammatory conditions, such as rheumatoid arthritis (2).

Apart from phagocytosis it is possible to induce exocytosis in PMN's with certain solube stimuli. these comprise ionophore A 23187, chemotactic peptides, phorbol myristate acetate and concanavalin A (3). Recently we found that fluroide, in combination with calcium ions, could induce exocytosis in rabbit peritoneal PMN's (4,5). A similar observation was made earlier for guinea pig peritoneal PMN's (6). For human PMN's, most authors found little or no exocytosis under comparable conditions (7,8). The combination of fluroide and calcium can also induce exocytosis in mast cells (9) and platelets (10).

Calcium ions play a predominant role in fluoride-activated exocytosis by rabbit PMN's. Without extracellular calcium no exocytosis occurs. In this study we considered the role of calcium, and calcium-dependent targets in fluoride-activated exocytosis.

(1) Weissmann G et al (1979). Leukocytes as secretory organs of inflammation. In: Advances in inflammation research. Vol. 1, ed. G. Weissmann, Raven Press, New York.
(2) Weissmann G (1982). Activation of neutrophils and the lesion of rheumatoid arthritis. J Lab Clin Med, 100:322-333.
(3) Weissmann G et al (1980). Release of inflammatory mediators from stimulated neutrophils. New England Med J, 303:27-34.
(4) Elferink JGJ et al (1980). The interaction of fluoride with rabbit polymorphonuclear leukocytes: induction of exocytosis and cytolysis. Biochem Pharmacol, 29:3051-3057.
(5) Elferink JGR (1982). Fluoride-induced activation and inhibition of granulocyte functions. Fluroide, 15:4-13.
(6) Selvaraj RJ et al (1976). Relationship of glycolytic and oxidative metabolism to particle entry and destruction in phagocytosing cells. Nature, 211:1271-1279.
(7) Harvath L (1978). Chemiluminescence of human and canine polymorphonuclear leukocytes in the absence of phagocytosis. J Clin Invet 61:1145-1154.
(8) Curnutte JT et al (1979). Fluoride-mediated acivation of the respiratory burst in human neutrophils. J Clin Invet, 63:637-647.
(9) Patkar SA et al (1977). Sodium fluoride - a stimulus for a calcium-triggered secretory process. Internat Archs Allergy Appl Immunol, 55:193-200.
(10) Murer EH (1976). Effects of fluoride on blood platelets. Fluoride, 9:173-184.

Fluoride 1984; 17(4):210-217
Stimulation of adenylate cyclase activity by Na2PO3F and NaF in intact rat hepatocytes and plasma membrane fractions

AR Shahed and DW Allmann

Indiana University School of Medicine, Indianapolis, Indiana 46223

Summary: The effect of Na2PO3F (MFP) on cAMP production and adenylate cyclase activity in rat hepatocytes was investigated. MFP (1-10mM) increased cAMP accumulation in hepatocytes isolated from both fed and fasted rats. MFP increased adenylate cyclase activity (a) in plasma membrane frctions and (b) in intact hepatocytes under conditions where hydrolysis of MFP was neglibible. It is proposed that MFP, as a complex, stimulated adenylate cyclase activity and thus increases cAMP accumulation in isolated hepatocytes.

Fluoride 1984; 17(4):217-223
Effect of F- on major salivary glands. The amylase activity, stimulated salivary flow response and cAMP levels in parotid gland of rats consuming F- via drinking water

Boros I, Mozsik G*, Keszler P

Research Group of Oral Biology, Semmelweis University Medical School, Budapest, and *First Department of Internal Medicine, University Medical School, Pecs, Hungary

Summary: This study examined the effect of intakes of 25 or 50 ppm fluoride via drinking water for four weeks on the amylase activity of the parotid gland, isoproterenol-stimulated (1 mg/100 g b.w., i.p.) salivary flow and on the amylase activity in saliva fractions. A significant elevation of the tissue amylase activity was seen in the F25 and F50 groups compared to control. The volume of saliva collected for 30 min after isoproterenol injection was higher in the fluoride-treated groups; the amylase activity was also increased. In the glandular tissue cAMP level was augmented. It appears that fluoride may affect the parotid function; it may also influence the salivary amylase activity, presumably by acting on the adenyl cyclase activity.

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6717484&dopt=Abstract
Mutat Res 1984 Apr;139(4):193-8

Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride.

Tsutsui T, Suzuki N, Ohmori M, Maizumi H.

The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50-150 micrograms/ml) or exposure time (1-24 h). Treatment of the cells with 50 micrograms/ml NaF for 24 h resulted in a lethality (approximately 70%) similar to that obtained with 100 micrograms/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-1 cells treated with 20-50 micrograms/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence of 10 mM hydroxyurea. Treatment with 100-400 micrograms NaF/ml for 4-24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

PMID: 6717484 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6707909&dopt=Abstract
J Pharmacol Exp Ther 1984 Mar;228(3):579-87

Activation of adenylate cyclase by alcohols requires the nucleotide-binding protein.

Luthin GR, Tabakoff B.

Ethanol was shown to activate adenylate cyclase in mouse striatal membranes, but significant activation of adenylate cyclase by ethanol concentrations below 500 mM was found only in the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p] or other guanine nucleotides. Ethanol did not alter the amount of guanine nucleotide required for half-maximal activation of adenylate cyclase, but was found to further increase adenylate cyclase activity under conditions wherein the nucleotide binding protein was preloaded with Gpp(NH)p or when hydrolysis of added GTP was blocked using cholera toxin. The stimulation of adenylate cyclase activity by sodium fluoride was also accentuated by ethanol. Ethanol, propanol and butanol all increased adenylate cyclase activity in the presence of Gpp(NH)p, and their effects on adenylate cyclase activity were linearly correlated with their respective carbon chain lengths. Equivalent membrane concentrations of ethanol and chloroform produced similar increases in adenylate cyclase activity under conditions where hydrolysis of added GTP was inhibited. However, chloroform and ethanol had opposite effects on adenylate cyclase activity in assays containing GTP and membranes not treated with cholera toxin. The apparent Km of adenylate cyclase for Mg-ATP and the Arrhenius activation energy for the enzyme in membranes incubated with Gpp(NH)p were similar in the presence and absence of ethanol. Ethanol, in concentrations up to 750 mM, did not alter the pattern of stimulation of adenylate cyclase by calcium and calmodulin. Our results suggest that ethanol modifies the equilibrium for the interaction of the nucleotide-loaded G-protein with the catalytic unit of adenylate cyclase to favor formation of the active nucleotide-G-protein-catalytic unit complex.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 6707909 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6704183&dopt=Abstract
Biochem Pharmacol 1984 Feb 15;33(4):663-9

Formation of soman (1,2,2-trimethylpropyl methylphosphonofluoridate) via fluoride-induced reactivation of soman-inhibited aliesterase in rat plasma.

de Jong LP, van Dijk C.

After incubation (37 degrees) of rat blood or plasma with the nerve agent soman, (CH3)3C(CH3)C(H)O(CH3)P(O)F (7.7 microM), for 10 min, only a small amount of this organophosphate (7 or 1%, respectively) is left, as determined enzymatically (acetylcholinesterase) and gas chromatographically. Comparison of the results obtained with both analyses shows that this residual soman consists only of its P(-)-isomers. Incubation (25 degrees) at pH 4.8-6.1 of such soman-treated rat blood or plasma with sodium fluoride (2.5 mM) for 0.5 min leads to
(i) a substantial increase of the P(-)-soman concentration, and
(ii) a (partial) reactivation of the soman-inhibited aliesterase, proportional to the amount of generated P(-)-soman.
These results indicate strongly that added fluoride ions regenerate soman by a reversal of the inhibition reaction. From the relationship between percentage of reactivation and increase of soman concentration the aliesterase concentration in rat plasma is calculated as 2.6 microM. Sodium fluoride has a similar effect in blood taken from rats to which soman was administered intravenously. The increase of the P(-)-soman concentration is higher with higher sodium fluoride concentrations and at lower pH values. In accordance with the absence of aliesterase, addition of sodium fluoride does not induce an increase of the P(-)-soman concentration in soman-treated human plasma.

PMID: 6704183 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6587580&dopt=Abstract
Shikwa Gakuho 1984 Feb;84(2):229-51

No Abstract available

[Effects of sodium fluoride on blastogenesis in mice lymphocytes, with special reference to uptake of 3h-thymidine, 3h-uridine or 3h-leucine]

[Article in Japanese]

Kataoka M.

PMID: 6587580 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6533415&dopt=Abstract
Med Biol 1984;62(6):326-30

Inhibition of phagocytosis of polymorphonuclear leucocytes by adenosine and HoCl3 in vitro.

Mircevova L, Viktora L, Hermanova E.

Phagocytosis of polymorphonuclear leucocytes treated with NaF, HoCl3 and adenosine were studied. The highest concentration used was 25 mM of NaF, 25 mM of adenosine and 5 mM of HoCl3. It was ascertained that these substances, inhibitors of erythrocyte contractile protein, inhibit both phagocytosis and ability of polymorphonuclear leucocytes to change their shape. These unfavourable effects may be induced by the chemicals interfering with polymorphonuclear leucocytes contractile protein. NaF, HoCl3 and adenosine are also responsible for morphological changes in the cell nucleus.

PMID: 6533415 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6364322&dopt=Abstract
Scand J Haematol 1984 Jan;32(1):25-32

Differential effect of the serine protease inhibitor phenyl methyl sulfonyl fluoride on cytochemically detectable esterases in human leucocytes and platelets.

Dufer J, Trentesaux C, Desplaces A.

Esterases of human leucocytes and platelets were studied by cytochemical methods. The aim of the study was to clarify the cellular distribution and possible nature of esterases types differing in their substrate specificity and/or their inhibitor sensitivity. 3 substrates (alpha-naphthyl acetate: ANA; naphthol AS-D chloroacetate: NASDCA; and N-acetyl DL-alanine alpha-naphthyl ester: NACALA) were used and the effects of 2 inhibitors (sodium fluoride and the serine protease inhibitor phenyl methyl sulfonyl fluoride: PMSF) were evaluated. 4 enzyme types were described: Type I, present in granulocytes, was detected using NASDCA and NACALA and was resistant to fluoride but sensitive to PMSF. Other types were detected using ANA as substrate. Type II, present in monocytes, was inhibited by both fluoride and PMSF. Type III, present in platelets and plasma cells, was inhibited by fluoride but resistant to PMSF. Type IV, present in lymphocytes, was resistant to both fluoride and PMSF. The specific aims and possible areas for application of these results are discussed.

PMID: 6364322 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6328553&dopt=Abstract
Physiol Bohemoslov 1984;33(2):97-103

Postnatal development of the beta-adrenergic receptor complex in brown adipose tissue of the rat.

Svoboda P, Skobisova E, Drahota Z.

The amount of specific (-)-3H-dihydroalprenolol binding sites in the crude membranes of brown adipose tissue is constant in the course of postnatal development of the rat. Contrarily, the activity of the isoprenaline and fluoride-stimulated adenylate cyclase undergoes rather pronounced changes with maximum enzyme activity measured shortly after birth and minimum activity between the 10th and 20th day. These data may be interpreted to mean that the binding and catalytical moiety of the beta-adrenergic receptor complex represents two functionally distinct entities which develop independently of each other.

PMID: 6328553 [PubMed - indexed for MEDLINE]

NOTE: AG Gilman, co-author of this report, shared the 1994 Nobel Prize for the discovery of "G-proteins and the role of these proteins in signal transduction in cells."

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6323430&dopt=Abstract
J Biol Chem 1984 Mar 25;259(6):3568-77

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein.

Katada T, Bokoch GM, Northup JK, Ui M, Gilman AG.

Treatment of membranes with islet activating protein (IAP), a toxin from Bordetella pertussis, results in abolition of GTP-dependent, receptor-mediated inhibition of adenylate cyclase. This appears to result from IAP-catalyzed ADP-ribosylation of a 41,000-Da membrane-bound protein. A protein with 41,000- and 35,000-Da subunits has been purified from rabbit liver membranes as the predominant substrate for IAP. This protein has now been shown to be capable of regulating membrane-bound adenylate cyclase activity of human platelets under various conditions. The characteristics of the actions of the IAP substrate are as follows.
1) Purified 41,000/35,000-Da dimer is capable of restoring the inhibitory effects of guanine nucleotides and the alpha 2-adrenergic agonist, epinephrine, on the adenylate cyclase activity of IAP-treated membranes.
2) The subunits of the dimer dissociate in the presence of guanine nucleotide analogs or A1(3+), Mg2+, and F-. The 41,000-Da subunit has a high affinity binding site for guanine nucleotides.
3) The resolved 35,000-Da subunit of the dimer mimics guanine nucleotide- and epinephrine-induced inhibition of adenylate cyclase.
4) The resolved (unliganded) 41,000-Da subunit stimulates adenylate cyclase activity and relieves guanine nucleotide- +/- epinephrine-induced inhibition of the enzyme. In contrast, the GTP gamma S-bound form of the 41,000-Da subunit inhibits adenylate cyclase activity, although with lower apparent affinity than does the 35,000-Da subunit.
5) The 35,000-Da subunit increases the rate of deactivation of Gs, the stimulatory regulatory protein of adenylate cyclase. In contrast, the 41,000-Da subunit can interact with Gs and inhibit its deactivation. These data strongly suggest that the IAP substrate is another dimeric, guanine nucleotide-binding regulatory protein and that it is responsible for inhibitory modulation of adenylate cyclase activity.

PMID: 6323430 [PubMed - indexed for MEDLINE]

NOTE: AG Gilman, co-author of this report, shared the 1994 Nobel Prize for the discovery of "G-proteins and the role of these proteins in signal transduction in cells."
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6323431&dopt=Abstract

J Biol Chem 1984 Mar 25;259(6):3578-85

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition.

Katada T, Northup JK, Bokoch GM, Ui M, Gilman AG.

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta (35,000 Da) subunits are functionally indistinguishable. Gi and Gs both dissociate in the presence of guanine nucleotide analogs or Al3+, Mg2+, and F- in detergent-containing solutions. Several characteristics of Gi- and Gs-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes. The nonhydrolyzable analog of GTP, guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin E1 with GTP gamma S results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin E1-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTP gamma S plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit. Detergent extracts of membranes that have been treated with GTP gamma S plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi contained in such extracts has a reduced ability to be ADP-ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTP gamma S-bound form. The irreversible inhibition of adenylate cyclase caused by GTP gamma S (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate cyclase activity in GTP gamma S (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 6323431 [PubMed - indexed for MEDLINE]

NOTE: AG Gilman, co-author of this report, shared the 1994 Nobel Prize for the discovery of "G-proteins and the role of these proteins in signal transduction in cells."
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6327672&dopt=Abstract

J Biol Chem 1984 May 25;259(10):6235-40

ADP-ribosylation of Gs promotes the dissociation of its alpha and beta subunits.

Kahn RA, Gilman AG.

We have utilized purified reactants and cofactors to examine the form of the stimulatory guanine nucleotide-binding regulatory component (Gs) of adenylate cyclase that serves as a substrate for ADP-ribosylation by cholera toxin; we have also investigated some of the consequences of that covalent modification. Activation of Gs with nonhydrolyzable analogs of GTP, which causes dissociation of its subunits, completely inhibits the toxin-catalyzed covalent modification. However, this effect cannot be explained by subunit dissociation, since activation of Gs by fluoride is not inhibitory and ADP ribosylation of the alpha (45,000-Da) subunit of Gs proceeds equally well in the presence and absence of the beta (35,000-Da) subunit. ADP-ribosylation of the alpha subunit of Gs decreases its apparent affinity for the beta subunit; however, the affinity of alpha and ADP-ribosyl-alpha for GTP appear to be approximately the same. ADP-ribosylation of Gs thus promotes the dissociation of its alpha and beta subunits. This effect may account for or contribute to the activation of adenylate cyclase by cholera toxin.

PMID: 6327672 [PubMed - indexed for MEDLINE]

NOTE: AG Gilman, co-author of this report, shared the 1994 Nobel Prize for the discovery of "G-proteins and the role of these proteins in signal transduction in cells."
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6328910&dopt=Abstract

Adv Cyclic Nucleotide Protein Phosphorylation Res 1984;17:1-18

Mechanisms of guanine nucleotide-mediated regulation of adenylate cyclase activity.

Smigel M, Katada T, Northup JK, Bokoch GM, Ui M, Gilman AG.

Publication Types: Review

PMID: 6328910 [PubMed - indexed for MEDLINE]

http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6323481&dopt=Abstract
J Biol Chem 1984 Apr 10;259(7):4629-33

Cell-free desensitization of catecholamine-sensitive adenylate cyclase. Agonist- and cAMP-promoted alterations in turkey erythrocyte beta-adrenergic receptors.

Nambi P, Sibley DR, Stadel JM, Michel T, Peters JR, Lefkowitz RJ.

Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.

PMID: 6323481 [PubMed - indexed for MEDLINE]

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