Isolation and characterization of the protein phosphoamidates formed by a membrane bound adenylyl transferase reaction in Dictyostelium discoideum.

1. In this paper we report the results of studies on an adenylyl transferase (AyTase) activity from Dictyostelium discoideum. 2. Previous studies suggested that this activity catalyzed the transfer of AMP from ATP to a membrane protein to form a phosphoamidate reaction product. 3. In the present study we have isolated and characterized the product of the AyTase reaction and surprisingly found two AMP labeled products by SDS-gel-filtration HPLC. 4. The apparent molecular weights of these phosphoamidates were 13.5 and 1.5 kDa. 5. In addition, because the reaction product was rapidly degraded by a phosphoamidase, experiments were undertaken using AVAMP, a synthetic phosphoamidate, as an alternative phosphoamidase substrate, to trap the reaction products. 6. As the phosphoamidase activity could be inhibited by cAMP, cyclic formycin monophosphate, an analog of cAMP resistant to hydrolysis by cAMP phosphodiesterase, was also used to trap the products. 7. Both attempts at trapping failed. 8. A model for the AyTase reaction was developed to account for the failure to trap the products and the formation of two phosphoamidates.     

Adenosine 5'-monophosphate is a selective inhibitor of the brown adipocyte nonselective cation channel

Calcium-activated nonselective cation channels (NSC(Ca)) in brown adipocytes are inhibited by several nucleotides acting on the cytosolic side of the membrane. We used excised inside-out patches from rat brown adipocytes to identify important nucleotide structures for NSC-channel inhibition. We found that 100 microM 5'-AMP inhibited NSC-channel activity more than did ATP or ADP. Adenosine was a weak inhibitor, whereas adenine and ribose-5-phosphate had no effect. The channel activity was effectively blocked by 10 microM AMP, but it was unaffected by 10 microM cAMP, CMP, GMP, IMP, TMP or UMP. Dose-response studies yielded IC(50)-values of 4 microM for AMP and 32 microM for cAMP. dAMP was as effective as AMP, but all 5'-phosphate group modifications on AMP dramatically lowered the inhibitory effect. 10 microM of the AMP precursor adenylosuccinate weakly inhibited the channel activity. An increase in AMP concentration from 1 to 10 microM shifted the EC(50) for Ca(2+) activation almost 1 order of magnitude; a Schild plot analysis yielded a K(B) value of 0.3 microM for AMP. We conclude that AMP is the most efficacious endogenous nucleotide inhibitor of the brown adipocyte nonselective cation channel (NSC(Ca/AMP)) yet identified and that there is functional competition between Ca(2+) and AMP. The brown adipocyte NSC(Ca/AMP) thus appears to be functionally different from the NSC(Ca,PKA) in the exocrine pancreas and the NSC-(Ca,cAMP) in the endocrine pancreas, but similar to the NSC(Ca/AMP) in the endocrine pancreas.     

Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds

Three distinct enzymes hydrolyzing either ApppA or AppppA, or both, were separated and purified from yellow lupin seed extracts. Two of the enzymes were purified to homogeneity. These enzymes differ greatly in their catalytic and physical properties. One hydrolase, with a native molecular weight of 41,000, exhibits broad pH (from 5-8) optimum for activity, requires Mg2+ for activity, is inhibited by zinc ions (I0.5 = 25 microM) and hydrolyses ApppA (V = 1), ApppC (V = 0.38), ApppG (V = 0.2), and ribose(5')pppA (V = 0.2). The enzyme exhibits much lower activity with AppppA (V = 0.1), and ApppppA, AppppppA, ppppA, and ATP are hydrolyzed 25- to 100-fold slower then ApppA. ADP was always one of the products of the reactions catalyzed by the enzyme. AppA, NAD, NADP, FAD, cAMP, and p-nitrophenyl-thymidine 5'-phosphate were not hydrolyzed by the enzyme. The enzyme is diadenosine 5',5"'-P1, P3-triphosphatase. The second hydrolase, composed of one polypeptide chain of a molecular weight 18,000-18,500, exhibits optimal activity in the pH range from 7.5-9, requires Mg2+ for activity, is inhibited by calcium ions (I0.5 for calcium depends on the concentration of Mg2+ and is 35-180 microM in the presence of 0.5-10 mM Mg2+, respectively), and hydrolyzes AppppA (V = 1, Km = 1 microM), ApppppA (V = 0.42, Km = 1.8 microM), AppppppA (V = 0.34), AppppU (V = 0.73), AppppC (V = 0.67), AppppG (V = 0.27), and ppppA. ATP was always one of the products of the reactions catalyzed by the enzyme. Dinucleoside di- and triphosphates, ATP, cAMP, and p-nitrophenylthymidine 5'-phosphate were not hydrolyzed by the enzyme. This enzyme is diadenosine 5',5"'-P1,P4-tetraphosphatase (EC 3.6.1.17). The third hydrolase, composed of one polypeptide chain of a molecular weight of 56,000, exhibits maximal activity at pH 9-10.5, does not require Mg2+ ions for activity, is inhibited neither by divalent cations (Mg2+, Ca2+, Zn2+, Co2+, Mn2+, or Ni2+) nor by EDTA, and uses as substrates all compounds which are substrates for the diadenosine 5',5"'-P1,P3-triphosphatase and diadenosine 5',5"'-P1,P4-tetraphosphatase. In addition, the enzyme hydrolyzes p-nitrophenyl-thymidine 5'-phosphate, p-nitrophenylthymidine 3'-phosphate, bis-p-nitrophenylphosphate, ADP, AppA, NAD, NADP, and FAD, but not cAMP. With the exception of p-nitrophenylphosphate derivatives all other substrates of the enzyme yield AMP as one of the products of hydrolysis. This enzyme has a specificity similar to that of phosphodiesterases (EC 3.1.4.1) from other sources. With the lupin phosphodiesterase, ApppA (V = 1, Km = 2.2 microM) and AppppA (V = 1, Km = 2.0 microM) are better substrates than NAD (V = 0.8, Km = 9.6 microM), AppA (V = 0.4), ApppppA (V = 0.6), and AppppppA (V = 0.34).     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

Effects of temperature on allosteric and catalytic properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.     

cGMP-activated phosphodiesterase from human brain: kinetic and regulatory properties]

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.     

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.     

Brain glucose bisphosphatase requires inosine monophosphate

Glucose bisphosphate phosphatase has been partially purified from the cytosol of mouse brain. Enzyme activity required Mg2+ and a heat-stable cofactor. The activator was present in boiled extracts of mouse brain mitochondrial-nuclear fraction, of red blood cells, or of rabbit muscle. The chemical properties of the activator are consistent with its identification as inosine monophosphate (IMP), including its mobility in a high pressure liquid chromatography (HPLC) system capable of resolving all of the biologically important mononucleotides. A large number of other biologically important compounds were not effective, including AMP, cAMP, cGMP, and UMP, GMP, purified by HPLC, (50 or 74 microM), gave a rate about 35% of that obtained with IMP (5 microM). The enzyme was separated completely from phosphoglucomutase and significantly from glucose bisphosphate synthase. The products of the reaction are glucose-P and Pi. Fructose bisphosphate at 500 microM inhibited only 40% in the presence of 20 microM glucose bisphosphate. The activation by IMP follows hyperbolic kinetics with an apparent Ka of 5 microM in the presence of 12 microM glucose bisphosphate. The apparent Km of glucose bisphosphate was 10 microM in the presence of 50 microM IMP. There was no inhibition by 5 or 50 microM AMP or ADP. The possible regulatory importance of glucose bisphosphate in carbohydrate metabolism and the significance of the regulation of the phosphatase by the nucleotide are discussed.     

Stimulation of hepatic inositol 1,4,5-trisphosphate kinase activity by Ca2+-dependent and -independent mechanisms.

A cytosolic fraction derived from rat hepatocytes was used to investigate the regulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] kinase, the enzyme which converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. The activity was doubled by raising the free Ca2+ concentration of the assay medium from 0.1 microM to 1.0 microM. A 5 min preincubation of the hepatocytes with 100 microM-dibutyryl cyclic AMP (db.cAMP) plus 100 nM-tetradecanoylphorbol acetate (TPA) resulted in a 40% increase in Ins(1,4,5)P3 kinase activity when subsequently assayed at 0.1 microM-Ca2+. This effect was smaller at [Ca2+] greater than 0.5 microM, and absent at 1.0 microM-Ca2+. Similar results were obtained after preincubation with 100 microM-db.cAMP plus 300 nM-vasopressin (20% increase at 0.1 microM-Ca2+; no effect at 1.0 microM-Ca2+). Preincubation with vasopressin, db.cAMP or TPA alone did not alter Ins(1,4,5)P3 kinase activity. It is proposed that these results, together with recent evidence implicating Ins(1,3,4,5)P4 in the control of Ca2+ influx, could be relevant to earlier findings that hepatic Ca2+ uptake is synergistically stimulated by cyclic AMP analogues and vasopressin.     

Further characterization of the inhibition of casein production in a primary mouse mammary epithelial cell culture by epidermal growth factor. Antagonism by cyclic AMP

Epidermal growth factor (EGF) inhibited casein production and the accumulation of casein mRNA activity induced by insulin (I), cortisol (F) and prolactin (P) in a primary culture of mammary epithelial cells from pregnant mice. The inhibitory effects of EGF were blocked by 8-bromo cyclic AMP (8-br-cAMP) in a dose-dependent manner. The effect of 8-br-cAMP was observed at a concentration as low as 20 microM and was maximal at 500 microM. Dibutyryl cyclic AMP (db-cAMP), cAMP, and 3-isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, also antagonized the inhibitory effect of EGF on casein production. 8-Br-cAMP had, however, no effect on the mitogenic activity of EGF in this system. These results suggest a possible modulatory role of cAMP in EGF-induced inhibition of casein production in cultured mammary epithelial cells.     

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.     

Dissociation of the Stellate Morphology from Intracellular Cyclic AMP Levels in Cultured Rat Brain Astroglial Cells: Effects of Ganglioside GMl and Lysophosphatidylserine

Secondary microcultures of newborn rat cerebrum astroglial (AG) cells, maintained in a serum-free, chemically defined medium, were treated with various agents known to elevate intracellular cyclic AMP (cAMP) levels. Earlier studies had shown these drugs to induce a process-bearing (stellate) morphology in the AG cells, a response that was antagonized by the presence of gangliosides. One millimolar dibutyryl cyclic AMP (dBcAMP), 10 microM forskolin, 12 nM cholera toxin, and 30 microM isoproterenol all raised intracellular cAMP levels, from basal values of 3 pmol/10(6) cells to 30-30,000 pmol/10(6) cells, depending on the agent tested. dBcAMP caused the greatest elevation, and forskolin the least. The timing and/or the level of the AMP response did not precisely correlate with those of the stellation response. Values of ED50 with the four agents, as determined for the cAMP response, were always higher than stellation ED50 values in all treatments, and ED50 did not correlate with the maximal levels of cyclic AMP induced by the four agents. The capacity of ganglioside GM1 to block the stellation response to the four agents was not accompanied by a similar capacity to block the cAMP responses. Lysophosphatidylserine (lysoPS) had the capacity to induce AG cell stellation as well, without altering the basal level of cAMP. Both lysoPS and gangliosides, therefore, may act directly on the cellular machinery underlying the stellation response without involving changes in intracellular AMP.     

Forskolin Stimulation of Cyclic AMP Accumulation in Rat Brain Cortex Slices Is Markedly Enhanced by Endogenous Adenosine

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.     

HLAMP – a conjugate of hippuryllysine and AMP which contains a phosphoamide bond – stimulates chemotaxis in Dictyostelium discoideum

A conjugate of hippuryllysine (HP) and adenylic acid was synthesized and purified. The structure of the conjugate, hippuryllysyl(N-epsilon-5'-phospho)adenosine (HLAMP) was established using 31P nuclear magnetic resonance, UV spectroscopy, acid/base lability, and enzyme digestion with AMP deaminase, alkaline phosphatase, 5'-nucleotidase, and a phosphoamidase activity recently identified in Dictyostelium discoideum. The results indicate that HLAMP contains a phosphoamide bond between the phosphate of AMP and the epsilon amino group of HL. Employing a microdroplet assay to assess chemotactic activity, HLAMP was found to be a potent chemoattractant of 7-h developing amoebae of D. discoideum. Other conjugates, including lysine-AMP (LAMP), tuftsin-AMP (TAMP) and avidin-AMP (AVAMP), as well as the degradation products of HLAMP (HL, AMP, and lysine) exhibited no chemotactic activity. The molecular structure of HLAMP is compared to that of other known chemoattractants of the cellular slime molds, and possible chemotactic receptors for HLAMP are considered.     

Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea

The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 microM each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-DL-homoserine lactone at a low concentration of 10 microM. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.     

Changes in adenylate cyclase and phosphodiesterase activities during the growth cycle of adult rat hepatocytes in primary culture

Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.     

Diazepam reduces action potential duration in guinea-pig papillary muscle by a cAMP-dependent mechanism.

In this study, we have analyzed the role of cyclic AMP (cAMP) as the mediator of the decrease in action potential duration induced by diazepam. Diazepam (1-100 microM) reduced, in a dose-dependent manner, the duration of intracellular action potential recorded in the papillary muscle obtained from the right ventricle of the guinea pig heart. This effect was mimicked by the analog of cyclic AMP, 8-Br-cAMP (100 microM), but not by gamma-amino-butyric acid (GABA). Also, the selective antagonist of the benzodiazepine receptors, flumazenil did not modify the effect of diazepam. The diazepam-induced shortening of action potential duration was partially antagonized by the inhibitor of cAMP synthesis carbachol (1 microM) or the blocker of the cAMP-dependent protein kinase A, Rp-cAMP[S] (1 microM). These results indicate that cyclic AMP is involved in the diazepam-induced shortening of the action potential duration of the guinea pig papillary muscle.     

Study on the role of endogenous polyamines in glucagon, isoproterenol or serum-mediated induction of tyrosine aminotransferase in cultured heart cells

In confluent and serum-starved embryonic heart cell cultures, the addition of serum (10%), glucagon (GLU, 0.1 microM) or isoproterenol (ISO, 10 microM), causes the onset of ornithine decarboxylase (ODC) activity, with a maximum after 5-6 hr. This is paralleled by polyamine accumulation and by the induction of TAT, which, in the case of GLU and ISO, exhibits maximal activity at 4-3 hr respectively, followed by a net decline. Cyclic AMP (cAMP) also accumulates after exposure to GLU or ISO. However, under different conditions of ODC inhibition, serum fails to induce TAT, thus supporting a relevant role of cellular polyamines in serum action. Conversely, cAMP and TAT responses to GLU or ISO are markedly improved under prevention of polyamine accumulation, which also leads to a longer lasting TAT inducibility. The suggestion is made that polyamines are not required in the cAMP-dependent mechanism of TAT induction, but rather in the restoration of the basal activity of the enzyme.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Purification and characterization of palmitoyl-CoA ligase from rat brain microsomes

We have previously shown the existence of two separate enzymes for the synthesis of palmitoyl-CoA and lignoceroyl-CoA in rat brain microsomal membranes (1). Palmitoyl-CoA ligase activity was solubilized from brain microsomal membranes with 0.3% Triton X-100 and purified 93-fold by a combination of protein purification techniques. The Km values for the substrates palmitic acid, CoASH and ATP were 11.7 microM, 5.88 microM and 3.77 mM respectively. During activation of palmitic acid ATP is hydrolyzed to AMP and pyrophosphate, as evidenced by the inhibition of this activation by 5 mM concentrations of AMP, pyrophosphate or AMP and pyrophosphate to 70%, 60% and 85% respectively. The divalent metal ion Mg2+ was required for activity; its replacement with Mn2+ resulted in a 35% decrease in activity. Palmitoyl-CoA ligase activity was inhibited by the addition of oleic or stearic acids whereas addition of lignoceric acid or behenic acid had no effect. This supports our previous observation that palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes in rat brain microsomal membranes.