Differential inhibition of multiple cAMP phosphodiesterase isozymes by isoflavones and tyrphostins.

A series of isoflavone and tyrphostin compounds were found to inhibit the degradation of cAMP by several cyclic nucleotide phosphodiesterase (PDE) isozymes. Specific hydroxyl groups on the isoflavone structure were critical for PDE isozyme-selective inhibition. Replacement of the C-7 hydroxyl group of the isoflavone with a methoxy group raised the IC(50) for PDE1, PDE3, and PDE4. The absence of the C-5 hydroxyl group raised the IC(50) from 5 to >100 microM for PDE4, but actually lowered the IC(50) for PDE3 and PDE1. Replacement of the C-4' hydroxyl group with a methoxy group raised the IC(50) for PDE3 and PDE1, yet only slightly changed the IC(50) for PDE4. Various tyrphostins were also potent inhibitors of PDE1, PDE3, and PDE4. The four-carbon side chained tyrphostins were much less potent; however, a very interesting pattern was observed in which removal of phenolic hydroxyls on the tyrphostin structure increased the potency for PDE1 and PDE3, but not PDE4. These results may help to explain some of the therapeutic and intracellular signaling effects of isoflavones and tyrphostins. Moreover, the isozyme selectivity demonstrated by the isoflavones and tyrphostins can serve as a pharmacophore for the design of specific PDE inhibitors.

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.

Ecstasy: 3,4-methylenedioxymethamphetamine (MDMA).

The crystal structure of 3,4-methylenedioxymethamphetamine [systematic name: N-methyl-1-[3,4-(methylenedioxy) phenyl]-2-aminopropane] hydrochloride, C11H15NO2.HCl, also known as 'ecstasy' or MDMA, has been determined by X-ray diffraction.

Modulation of cyclic AMP levels in a clonal neural cell line by inhibitors of tyrosine phosphorylation.

The convergence of tyrosine kinase and cyclic AMP (cAMP) signal transduction pathways was investigated in the HT4.7 neural cell line with inhibitors of tyrosine kinases and tyrosine phosphatases. The protein tyrosine kinase inhibitor genistein inhibited isoproterenol-stimulated cAMP production by 40-60% in whole cells, with no effect on basal cAMP levels. In both whole cells and membranes, genistein also inhibited cAMP produced in response to direct stimulation of adenylyl cyclase with forskolin. However, in the absence of phosphodiesterase inhibitors, genistein presentation resulted in an increase in cAMP levels. Genistein inhibited phosphodiesterase activity by 80-85%, indicating that tyrosine phosphorylation stimulates both cAMP synthesis and degradation. The decrease in cAMP levels by genistein was not merely competitive inhibition of adenylyl cyclase with respect to ATP, since the Km of adenylyl cyclase for ATP remained essentially the same in either the presence or the absence of genistein. Another tyrosine kinase inhibitor, herbimycin A, which inhibits by a different mechanism than genistein, also decreased forskolin-stimulated cAMP in whole cells. As would be expected for the involvement of tyrosine phosphorylation in the control of cAMP production, inhibition of tyrosine phosphatases by vandate increased forskolin-stimulated cAMP production. These results suggest that cAMP production can be regulated by tyrosine phosphorylation, and the simultaneous activation of both cAMP synthesis and degradation may serve to alter the duration of cAMP elevation.

Conditional activation of cAMP signal transduction by protein kinase C. The effect of phorbol esters on adenylyl cyclase in permeabilized and intact cells.

To understand the convergence of cAMP and protein kinase C signal transduction, adenylyl cyclase isozyme identification and biochemical studies were performed on the HT4 neural cell line. In HT4 cells, basal cAMP production by adenylyl cyclase types I and VI were unaffected by phorbol esters, nor did phorbol esters have any effect on forskolin-induced cAMP production. However, phorbol esters synergistically increased cAMP production when adrenaline receptors were simultaneously activated, indicating a conditional activation of cAMP production by phorbol esters. A permeabilized cell preparation was used to analyze the mechanism by which phorbol esters were affecting cAMP production. This preparation enables G-proteins to be activated directly by GTP gamma S or bacterial toxins. In the permeabilized cell preparation, phorbol esters enhanced cAMP produced by GTP gamma S-activated G-protein. A stimulatory G-protein pathway may be involved since phorbol esters synergistically increased cAMP production by cholera toxin, yet had no effect on that produced by pertussis toxin. In this cell culture model, phorbol esters stimulate cAMP production only when some component of the cAMP cascade is simultaneously activated. Moreover, the pattern of modulation suggests that protein kinase C acts on an activated component of the second messenger system, such as the G-protein or the coupling of the G-protein with adenylyl cyclase, and not on the resting state of the protein components.

The plastocyanin-deficient phenotype of Chlamydomonas reinhardtii Ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin.

Ac-208 is a plastocyanin-deficient mutant of Chlamydomonas reinhardtii that contains only 2-3% of the wild-type level of plastocyanin-encoding mRNA and no detectable plastocyanin. Sequence analysis of the ac-208 plastocyanin-encoding gene reveals a single nucleotide insertion in the first exon compared with the wild-type gene; this alters the reading frame and results in a premature nonsense codon. We have introduced the genomic sequence encoding plastocyanin from a wild-type strain into ac-208 by cotransformation with a selectable marker encoding nitrate reductase. Of 22 nit+ transformants characterized, nine contained additional plastocyanin-encoding sequences (compared with untransformed cells) and each of these nine transformants was found to accumulate the protein. Transformants that do not contain newly introduced plastocyanin sequences retain the plastocyanin-deficient phenotype. The introduced plastocyanin-encoding sequences are stable during mitotic growth in liquid culture over a period of several months, as is expression from the introduced sequences. We suggest that the decreased steady state level of plastocyanin-encoding messages is a consequence of the frame-shift mutation in the structural gene. The ability to complement ac-208 with plastocyanin-encoding sequences will allow the introduction and analysis of in vitro mutagenized plastocyanin sequences in vivo in transgenic C. reinhardtii cells.

Identification of cyclic AMP as the response regulator for neurosecretory potentiation: a memory model system.

The neural cell line HT4 serves as a model for memory by exhibiting short- and long-term potentiation of neurotransmitter secretion. Previous studies showed that membrane depolarization elicits secretion and that serotonin and N-methyl-D-aspartate receptors are involved in potentiation of the response. Adrenergic and adenosine receptors, which are coupled to adenylate cyclase, are also found to induce potentiation. In addition, the direct evaluation of cAMP levels by forskolin, or by addition of dibutyryl cAMP, induces potentiation. In these different types of stimuli, it is the level of cAMP that is the common factor allowing prediction of whether potentiation will be observed or not. The cAMP level therefore qualifies as the response regulator for this phenomenon. Repetitive adrenergic receptor stimulation results in short-term potentiation, while repetitive adenosine stimulation results in long-term potentiation. This difference can be explained by assuming that some precursor that determines the cAMP level exceeds a threshold, to produce long-term potentiation. This threshold is exceeded by adenosine stimulation but not by stimulation of the beta-adrenergic receptor.

Molecular cloning of a member of a new class of low-molecular-weight GTP-binding proteins.

We report the cloning of a low-molecular-weight GTP-binding protein that appears to be the first member of a new class of G proteins. This G protein was cloned from the HT4 neural cell line and has the closest homology to the rab, sec4, and ypt1 members of the low-molecular-weight (LMW) G-protein family. The amino acid sequence identity is only 30% with these other LMW G proteins, but in the four conserved GTP-binding domains, amino acid identity increases to 50-100%. A unique feature that distinguishes this G protein from other LMW G proteins is its carboxy-terminal amino acid sequence -Cys-Cys-Pro. In keeping with the current nomenclature for other members of the ras superfamily, we will designate this new class as rah (ras-related homolog). On the basis of sequence homology, rah may function in vesicular trafficking and possibly in neurotransmitter secretion.

Short-term and long-term memory in single cells.

Many approaches have been used to study short- and long-term memory. Bacteria detect chemical gradients using a memory obtained by the combination of a fast excitation process and a slow adaptation process. This model system, which has the advantages of extensive genetic and biochemical information, shows no features of long-term memory. To study long-term memory, neural cell line systems have been developed that exhibit two phenomena associated with learning and memory, habituation and potentiation. The expression of these phenomena in clonal cell lines, devoid of synaptic connections, makes it possible to study the biochemical and molecular mechanisms that contribute to short-term and long-term memory.

Induction and expression of long- and short-term neurosecretory potentiation in a neural cell line.

In a neural cell line, the secretion of excitatory amino acids in response to a depolarizing stimulus is potentiated by the addition of serotonin. The duration of this potentiation is dependent on the strength of the stimulus. Persistent secretory potentiation induced by a strong stimulus requires the activation of both serotonin and NMDA receptors. Inhibiting the NMDA receptor during serotonin presentation prevented the induction of potentiation. The temporal characteristic of the potentiation is correlated with the elevation of cAMP levels. Serotonin exposure while inhibiting NMDA receptors results in a transient elevation of cAMP levels, whereas coactivation with NMDA and serotonin results in a persistent elevation of cAMP. Thus, it is possible to obtain potentiation of secretion in a single cell either transiently or persistently. The timing of potentiated responses in this system is of the same magnitude as that in similar systems used as models for short-term and long-term memory.

Effect of level of dietary protein on arginine-stimulated citrulline synthesis. Correlation with mitochondrial N-acetylglutamate concentrations.

Increases in dietary protein have been reported to increase the rate of citrulline synthesis and the level of N-acetylglutamate in liver. We have confirmed this effect of diet on citrulline synthesis in rat liver mitochondria and show parallel increases in N-acetylglutamate concentration. The magnitude of the effect of arginine in the suspending medium on citrulline synthesis was also dependent on dietary protein content. Mitochondria from rats fed on a protein-free diet initially contained low levels of N-acetylglutamate, and addition of arginine increased the rate of its synthesis. Citrulline synthesis and acetylglutamate content in these mitochondria increased more than 5-fold when 1 mM-arginine was added. A diet high in protein results in mitochondria with increased N-acetylglutamate and a high rate of citrulline synthesis; 1 mM-arginine increased citrulline synthesis in such mitochondria by only 36%. The concentration of arginine in portal blood was 47 microM in rats fed on a diet lacking protein, and 182 microM in rats fed on a diet containing 60% protein, suggesting that arginine may be a regulatory signal to the liver concerning the dietary protein intake. The rates of citrulline synthesis were proportional to the mitochondrial content of acetylglutamate in mitochondria obtained from rats fed on diets containing 0, 24, or 60% protein, whether incubated in the absence or presence of arginine. Although the effector concentrations are higher than the Ka for the enzymes, these results support the view that concentrations of both arginine and acetylglutamate are important in the regulation of synthesis of citrulline and urea. Additionally, the effects of dietary protein level (and of arginine) are exerted in large part by way of modulation of the concentration of acetylglutamate.

Excitatory amino acid uptake and N-methyl-D-aspartate-mediated secretion in a neural cell line.

A functional N-methyl-D-aspartate (NMDA) receptor has been identified on HT-4 cells, a clonal neural cell line, in which glutamate activates the receptor to elicit neurotransmitter secretion. Specific inhibitors of the NMDA receptor block glutamate-mediated secretion, and the characteristics of NMDA-mediated secretion parallel the reported properties of the NMDA receptor. Excitatory amino acid secretion can be elicited by potassium-evoked depolarization and is not the simple reversal of the uptake system. 2-Amino-4-phosphonobutyrate (APB) inhibits depolarization-induced secretion of excitatory amino acids but has no effect on excitatory amino acid uptake, suggesting that the APB binding protein in the brain represents a component involved in the secretion of excitatory amino acids.

Metabolism of S-adenosyl-L-methionine in intact human erythrocytes.

Freshly isolated human erythrocytes contain S-adenosyl-L-methionine (AdoMet) at a concentration of about 3.5 mumol/l cells. When such cells are incubated in a medium containing 30 microM L-methionine, 18 mM D-glucose and 118 mM sodium phosphate (pH 7.4), intracellular AdoMet levels continuously decrease to a value of about 0.1 microM after 24 h. This occurs in spite of the fact that the cellular concentrations of the substrates for the AdoMet synthetase reaction, ATP and L-methionine, remain relatively constant. In a search for incubation conditions that lead to stable levels of AdoMet in incubated cells, we have developed a sodium-Hepes-buffered medium which includes 1 mM adenine and a stoichiometric excess of MgCl2 over its ligand, phosphate. The inclusion of magnesium ion (and a reduction in phosphate) appears to increase intracellular free Mg2+, which is required for full activity of the erythrocyte AdoMet synthetase. Even in the presence of MgCl2, however, the AdoMet pool level can drop 4-6-fold within the first 2 h of incubation. We present evidence that suggests that this initial fall in the cellular AdoMet level may be due to the activation of AdoMet-dependent protein carboxyl methyltransferase, an enzyme which accounts for a large fraction of the total cellular AdoMet utilization. Adenine, or related compounds in the medium may prevent this activation, although the mechanism of this action is not clear at present.

Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages.

Ordinarily packaged in DNA, adenine deoxyribonucleotides are preferentially concentrated in erythrocyte and lymphocyte cytosol in adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. A spectrum of cytosol enzyme activities are defined in terms of reaction velocities, K0.5s, and nucleotide partition after incubation with ribo- and deoxyribonucleotides. AMP and dAMP were dephosphorylated, but only AMP was deaminated in vitro. Although nucleotidase activity is much stronger in lymphocytes, AMP deaminase was the dominant degradative reaction in all erythrocyte and lymphocyte lysates under the conditions specified. For most cytosolic enzymes, ribonucleotides were preferred cofactors, implying that dADP and dATP often may be bystanders at metabolic events. The adenylate kinase-mediated partition of approximately equimolar ribo- and deoxyribonucleotide substrates yielded a very large preponderance of AMP in the monophosphate compartment, the monophosphates alone being directly vulnerable to degradative loss. The adenylate kinase(s) of lymphocytes differed strikingly from those of erythrocytes in reaction velocities with nucleotide cofactors, K0.5s, and in susceptibility to substrate inhibition.