GMP Kinase Research Articles

Cyclic GMP kinase I is involved in the regulation of cytosolic calcium transients in murine neutrophils

Cyclic GMP kinase I is involved in the regulation of cytosolic calcium transients in murine neutrophils

Regulation of splanchnic perfusion

In splanchnic arteries, vasodilators decrease the cytosolic free calcium concentration ([Ca2+]i) by inducing Ca2+sequestration and extrusion, by limiting the entry of extracellular Ca2+via L-type Ca2+channels and by decreasing the agonist-induced mobilization of intracellular Ca2+. Cyclic GMP kinase and membrane hyperpolarization are important mediators of the decrease in [Ca2+]i. Vasoconstrictors increase [Ca2+]i by mobilizing intracellular Ca2+from the sarcoplasmic reticulum and by stimulating extracellular Ca2+entry via L-type Ca2+channels. Activation of G proteins, inositol trisphosphate, diacylglycerol and membrane depolarization are important mediators of the increase in [Ca2+]i.

Interaction of guanine phosphonomethoxyalkyl derivatives with GMP kinase isoenzymes

Substrate activity and inhibitory potency of guanine phosphonomethoxyalkyl derivatives towards GMP kinase isoenzymes from L1210 cells were studied. 9-[2-(Phosphonomethoxy)ethyl]guanine (PMEG) and the ( R)- and ( S)-enantiomers of both 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG) and 9-[2-(phosphonomethoxy)propyl]guanine (PMPG) were phosphorylated to the first step. Kinetic data showed that ( R)-PMPG was a good substrate with a relative phosphorylation efficacy of 12% compared with the natural substrate GMP, whereas PMEG was a poor substrate with a relative phosphorylation efficacy of 1.1%. The structurally related 2,6-diaminopurine analogues 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) and ( R)- and ( S)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP) were not phosphorylated by any of the GMP kinase isoenzymes tested. The inhibitory activities of the individual compounds on GMP kinase isoenzymes decreased in the following order: ( S)-HPMPG > ( R)-PMPG > PMEG > ( R)-HPMPG > ( S)-PMPG > PMEDAP = ( R)-PMPDAP = ( S)-PMPDAP; each compound exerted a different type of inhibition.

Yeast GMP Kinase Mutants Constitutively Express AMP Biosynthesis Genes by Phenocopying a Hypoxanthine-Guanine Phosphoribosyltransferase Defect

We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.

Cellular mechanisms involved in carotid body inhibition produced by atrial natriuretic peptide.

Atrial natriuretic peptide (ANP) and its analog, atriopeptin III (APIII), inhibit carotid body chemoreceptor nerve activity evoked by hypoxia. In the present study, we have examined the hypothesis that the inhibitory effects of ANP and APIII are mediated by cyclic GMP and protein kinase G (PKG) via the phosphorylation and/or dephosphorylation of K(+) and Ca(2+) channel proteins that are involved in regulating the response of carotid body chemosensory type I cells to low-O(2) stimuli. In freshly dissociated rabbit type I cells, we examined the effects of a PKG inhibitor, KT-5823, and an inhibitor of protein phosphatase 2A (PP2A), okadaic acid (OA), on K(+) and Ca(2+) currents. We also investigated the effects of these specific inhibitors on intracellular Ca(2+) concentration and carotid sinus nerve (CSN) activity under normoxic and hypoxic conditions. Voltage-dependent K(+) currents were depressed by hypoxia, and this effect was significantly reduced by 100 nM APIII. The effect of APIII on this current was reversed in the presence of either 1 microM KT-5823 or 100 nM OA. Likewise, these drugs retarded the depression of voltage-gated Ca(2+) currents induced by APIII. Furthermore, APIII depressed hypoxia-evoked elevations of intracellular Ca(2+), an effect that was also reversed by OA and KT-5823. Finally, CSN activity evoked by hypoxia was decreased in the presence of 100 nM APIII, and was partially restored when APIII was presented along with 100 nM OA. These results suggest that ANP initiates a cascade of events involving PKG and PP2A, which culminates in the dephosphorylation of K(+) and Ca(2+) channel proteins in the chemosensory type I cells.

Cysteine-rich Protein 2, a Novel Substrate for cGMP Kinase I in Enteric Neurons and Intestinal Smooth Muscle

Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system. The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T. (1993) FEBS Lett. 333, 51-55). Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation. Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus. The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine.

Phosphorylation of Ganciclovir Phosphonate by Cellular GMP Kinase Determines the Stereoselectivity of Anti-Human Cytomegalovirus Activity

A racemic mixture of ganciclovir phosphonate was resolved by stereoselective phosphorylation using GMP kinase. The R-enantiomer of ganciclovir phosphonate was active against human cytomegalovirus but the S-enantiomer was less active. We show that enantiomeric selectivity of antiviral activity for ganciclovir phosphonate was conferred by stereoselective phosphorylations by mammalian enzymes, not by stereoslective inhibition of DNA polymerase from human cytomegalovirus.

Activation of Mitogen-activated Protein Kinase Pathways by Cyclic GMP and Cyclic GMP-dependent Protein Kinase in Contractile Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.

Nitric oxide modulates Na +, K +-ATPase activity through cyclic GMP pathway in proximal rat trachea

The present work demonstrated that nitric oxide (NO) modulates Na +, K +-ATPase activity in the proximal rat trachea. Sodium nitroprusside induced concentration-dependent (10–100 μM) stimulation in proximal trachea Na +, K +-ATPase activity. The effect was specific for Na +, K +-ATPase since Mg-ATPase activity was unaffected. This NO-donor changed neither Na +, K +-ATPase nor Mg-ATPase activity in the distal segment. The modulatory action on Na +, K +-ATPase induced by sodium nitroprusside was linked to an increase in nitrates/nitrites and cyclic GMP levels in proximal segments. Modulation of proximal Na +, K +-ATPase activity by sodium nitroprusside was mimicked by S-nitroso- N-acetylpenicillamine (100 μM) and 8-bromo-cyclic GMP (100 μM). Both sodium nitroprusside and 8-bromo-cyclic GMP effects on Na +, K +-ATPase activity of proximal segments of trachea were blocked by 2 μM of KT 5823 (a cyclic GMP-dependent protein kinase inhibitor), but not by 0.5 μM of KT 5720 (a cyclic AMP-dependent protein kinase inhibitor). Both kinase inhibitors decreased proximal Na +, K +-ATPase activity, but did not change Mg-ATPase activity. Okadaic acid (1 μM), a phosphatase-1 inhibitor, increased proximal Na +, K +-ATPase but not Mg-ATPase activity. The effect of okadaic acid was non-additive with that of 8-bromo-cGMP on Na +, K +-ATPase activity. Our results suggest that NO modulates proximal rat trachea Na +, K +-ATPase activity through cyclic GMP and cyclic GMP-dependent protein kinase.

Isoenzymes of GMP Kinase from L1210 Cells: Isolation and Characterization

Guanylate kinase from a mouse leukemic L1210 cells has been purified nearly to homogeneity for the first time. It consists of five isoenzymes with pI 5.95, 5.50, 5.08, 4.83 and 4.51, respectively. The native enzyme is a monomer with a relative molecular mass 25 000. The kinetic constants Km, Vmax and kcat/Km of isoenzymes were estimated. The purified GMP kinase is absolutely specific to GMP and/or dGMP as phosphate acceptor but has a broad specificity to nucleoside 5'-triphosphates as phosphate donors.

CAMP regulates nitric oxide production and ouabain sensitive Na+, K+-ATPase activity in SH-SY5Y human neuroblastoma cells.

We investigated the relation between cyclic AMP (cAMP) and nitric oxide (NO) production, as well as the effect of NO on Na , K+-ATPase activity in the human neuroblastoma cell line SH-SY5Y. Two cAMP agonists, dibutyryl cAMP (DBC) and beraprost sodium (BPS), increased cAMP accumulation and NO production in a time and dose dependent manner at 50 mmol/l glucose. On the other hand, cellular sorbitol and myo-inositol contents and protein kinase C activity were not altered by DBC or BPS. A specific protein kinase A inhibitor, H-89, suppressed increases in nitrite/nitrate and cyclic GMP (cGMP) and protein kinase A activity stimulated by DBC or BPS. This finding suggests that cAMP stimulates NO production by activating protein kinase A via a pathway different from the sorbitol-myo-inositol-protein kinase C pathway. We observed that an NO donor, sodium nitroprusside, and an NO agonist, L-arginine, enhanced ouabain sensitive Na+, K+-ATPase activity at 50 mmol/l glucose. We also found that a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), inhibited Na+, K+-ATPase activity at 5 mmol/l glucose, and partially suppressed the enzyme activity stimulated by DBC or BPS. The results of this study suggest that cAMP regulates protein kinase A activity, NO production and ouabain sensitive Na+, K+-ATPase activity in a cascade fashion. The results also suggest that protein kinase A at least partially regulates Na+, K+-ATPase activity without mediation by NO in SH-SY5Y cells. We speculate that cAMP and NO are two important regulatory factors in the pathogenesis of diabetic neuropathy.

Activation of a nonspecific cation current in rat cultured retinal pigment epithelial cells: involvement of a G(alpha i) subunit protein and the mitogen-activated protein kinase signalling pathway.

1. Whole-cell patch-clamp recording techniques were used to investigate the G protein subtype and related signalling molecules involved in activation of a nonspecific cation (NSC) current in rat cultured retinal pigment epithelial (RPE) cells. 2. Under control conditions, in 130 mM NaCl with K+ aspartate in the pipette, cytosolic dialysis with guanosine-5'-O-(3-triphosphate) (GTPgammaS, 0.1 mM) activated a large non-inactivating NSC current in 80% of the cells recorded from. 3. Loading RPE cells with antibodies (10 microg-ml(-1)) against the alpha subunit of all PTX-sensitive G proteins (G(alpha i/o/t/z)) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed specifically against the alpha subunits of the Gi subclass (G(alpha i-3)) completely abolished current activation. In RPE cells loaded with anti-G(alpha s) activation of the NSC current was unaffected. 4. Investigation of the potential downstream mediators in the G(alpha i) NSC channel pathway revealed that activation of the cation conductance was unaffected by treatment of RPE cells with the selective protein kinase C inhibitor GF 109203X (3 microM) or the selective CaM kinase II inhibitor KN-93 (50 microM). However, NSC current activation was delayed and the current amplitude reduced in the presence of the nonselective kinase inhibitor H-7 (100 microM) or the selective inhibitor of MAPKK (MEK) activation, PD 98059 (50 microM). 5. In the absence of GTPgammaS, the NSC current was not activated by superfusion of the cells with the cyclic GMP kinase activator dibutyryl-cyclic GMP or with the adenylate cyclase activator forskolin. 6. These results support the involvement of a G protein of the G(alpha i) subclass in the activation of a NSC current in rat RPE cells, and suggest a potential modulatory role for MAP kinase-dependent phosphorylation in current regulation.

A defect in GTP synthesis affects mannose outer chain elongation in Saccharomyces cerevisiae.

We have found that yeast mutants that are defective in mannose outer chain elongation of N-linked glycoproteins show higher cell wall porosity than normal cells, and are hypersensitive to antibiotics with a large molecular weight; such as neomycin and geneticin. Wild-type yeast cells also showed enhanced sensitivity to neomycin in the presence of tunicamycin, an inhibitor of N-glycosylation, suggesting that the extent of N-glycosylation may affect the sensitivity of yeast cells to drugs and that sensitivity to neomycin may be an effective method for screening for yeast mutants defective in N-glycosylation. Pursuing this logic, we isolated neomycin-sensitive yeast mutants and screened them for defects in N-glycosylation. The neomycin-sensitive, N-glycosylation-defective mutants fell into 15 complementation groups including alleles of the previously isolated temperature-sensitive nes mutants nes10, nes17, and nes25. Gene cloning revealed that NES10 was identical to SEC20, which is involved in ER-Golgi protein transport, NES17 was identical to ALG1, which encodes a beta-1,4-mannosyltransferase present in the ER, MSN17, a multicopy suppressor of nes17/alg1, was also isolated and found to be an allele of PSA1, which is involved in GDP-mannose synthesis, NES25 was identical to GUK1, which encodes a GMP kinase. Overexpression of MSN17 increased the GDP-mannose level in a wild-type strain by about threefold, and guk1 decreased the GDP-mannose level to one-fourth, suggesting a close relationship between GTP metabolism and mannose outer chain elongation; the link is presumably provided by the process of GDP-mannose transport in the Golgi membranes.

Involvement of intracellular cyclic GMP and cyclic GMP-dependent protein kinase in alpha-elastin-induced macrophage chemotaxis.

alpha-Elastin with an average molecular mass of 70 kDa, an oxalic acid fragmentation product of highly purified insoluble elastin, induced the migration of macrophages, with maximum activity at 10(-1) microg/ml. Relative to the positive control of 10(-8) M N-formylmethionyl-leucyl-phenylalanine (fMLP), the responsiveness of macrophages to alpha-elastin was nearly the same. Checkerboard analysis demonstrated that the cell movement is chemotaxis and not chemokinesis. A homologous deactivation test showed the possibility of the existence of alpha-elastin-recognizing sites on macrophages. In connection with macrophage chemotaxis in response to alpha-elastin, the intracellular signaling pathway was examined. The guanosine 3', 5'-cyclic monophosphate (cGMP) level was enhanced in macrophages stimulated by alpha-elastin, whereas the adenosine 3',5'-cyclic monophosphate (cAMP) level was not. Chemotaxis assaying of macrophages treated with 8-Br cGMP- and dibutyryl cAMP-loaded macrophages indicated that cGMP promotes cell movement and cAMP suppresses cell locomotion. The possible involvement of protein kinases in the alpha-elastin signaling pathway was explored by use of inhibitors specific for cGMP-dependent protein kinase (PKG), cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and tyrosine kinase. The macrophage chemotactic response to alpha-elastin was inhibited by the PKG inhibitor, but not by the PKA, PKC, or tyrosine kinase inhibitor. These results suggested that the increase in the cGMP level and the activation of PKG in macrophages are involved in alpha-elastin induced macrophage chemotaxis.

Synthesis and antiviral activity of phosphonate derivatives of enantiomeric dihydro-2 H-pyranyl nucleosides

A synthetic approach to phosphonate derivatives of 2,5- cis-substituted dihydro-2 H-pyranyl nucleosides has been developed and both series of enantiomers (3 and 4) have been prepared. The key step in the synthetic pathway was the introduction of the phosphonomethoxy moiety on pentopyranosyl glycals through a Ferrier-type rearrangement. The heterocyclic base was then incorporated under Mitsunobu conditions. The resulting nucleoside derivatives were more stable towards acidic degradation than their natural isomers. However, they were found to be inactive against the replication of human immunodeficiency virus (HIV), herpes simplex virus (HSV) and other herpes viruses [i.e. varicella-zoster virus (VZV), cytomegalovirus (CMV)] in cell culture, which could at least partially be ascribed to an inefficient phosphorylation by cellular enzymes (i.e. GMP kinase).

The vasodilatory effect of endogenous nitric oxide is a major counter-regulatory mechanism in the spontaneously hypertensive rat

A decreased responsiveness to endothelium-dependent vasodilatory substances is characteristically seen in isolated arteries from spontaneously hypertensive rats (SHR). However, the precise status and role of nitric oxide (NO), which is, at least in part, the endothelium-derived relaxing factor, remains unclear in SHR. The objective of the present study was to evaluate the importance of NO release in vivo. The effect on systolic blood pressure of chronic administration of NG-nitro-L-arginine methyl ester (L-NAME, an NO-synthase inhibitor) was studied. Twenty SHR and 10 Wistar-Kyoto (WKY) rats were given 25 mg/kg per day L-NAME by gavage. Thirteen SHR and 14 WKY rats given water for the same period were used as controls. Rats were killed after 15 days and the aortic wall cyclic GMP (cGMP, the second messenger of NO) and cGMP-dependent kinase (the effector of cGMP) concentrations were assessed. During the trial, 11 of the 20 SHR given L-NAME died. Mean +/- SD systolic blood pressure increased from 131 +/- 8 to 171 +/- 10 mmHg in WKY rats given L-NAME and from 185 +/- 10 to 249 +/- 22 mmHg in SHR given L-NAME. Aortic cGMP content was similar in control WKY rats (2122 +/- 707 fmol/mg protein) and in control SHR (2098 +/- 704 fmol/mg protein), and was decreased in the L-NAME-treated WKY rats and SHR to 308 +/- 87 and 644 +/- 222 fmol/mg protein, respectively. The aortic concentrations of cGMP-dependent kinase were not different in any group. Basal release of NO does not appear to be impaired in SHR, but represents a major counter-regulatory mechanism in this genetic model of arterial hypertension.

Guanylate kinase of Escherichia coli K-12

We have identified the gene gmk, in the same operon as rpoZ, spoT, and recG at about 82 minutes on the Escherichia coli chromosome. The gmk (GMP kinase) gene encodes a peptide of 23,592 Da, possessing extensive similarity to the amino acid sequence of guanylate kinase from yeast. To confirm that gmk truly encodes guanylate kinase and to explore some of its enzymatic features, we have overproduced the product of gmk and purified it to homogeneity. Unlike guanylate kinases purified from eukaryotic sources, E. coli guanylate kinase is multimeric, and ionic conditions dictate its protomeric state; under low ionic conditions it appears to be a tetramer while under high ionic conditions it is a dimer. Kinetic analysis reveals that guanylate kinase, again, unlike eukaryotic guanylate kinases, binds GMP cooperatively and that the observed cooperatively changes with ionic strength. These results indicate that, despite extensive sequence similarity to its eukaryotic counterparts, E. coli guanylate kinase is structurally and enzymatically different.

Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity.

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.

Effects of Cyclic AMP and Andre-gens on in vitro Brain Aromatase Enzyme Activity During Prenatal Development in the Rat

In the rat, there is a marked but transient increase in hypothalamic aromatase activity during the last week of fetal life. The present study was undertaken to gain insight into the regulation of this developmental pattern. Hypothalamic fragments comprising the medial basal hypothalamus and the suprachiasmatic region (henceforth referred to as preoptic area) were explanted and cultured in serum-free medium for 2 to 5 days. Aromatase activity was measured by the formation of (3) H(2) O, utilizing either [1ß-(3) H]androstene-dione or [1ß-(3) H]testosterone as substrate. Maximal rates of activity were obtained at a saturating concentration of 0.3 μM [1ß-(3) H]testosterone. Confirmation of the identity of the [(3) H]estradiol formed was demonstrated by recrystallization of the derivatized estradiol to constant specific activity following incubation with [1,2,6,7-(3) H]testosterone. In agreement with previous reports, in vivo hypothalamic aromatase activity was negligible before gestational day (GD) 16, increased strikingly by GD19 (>5.0 pmol/h/mg protein) and decreased, thereafter, to low levels at GD22 (∼1.0 pmol/h/mg protein). Medial basal hypothalamus-preoptic area fragments explanted before GD17 failed to develop aromatase activity in vitro. If the tissue was explanted on GD17 or 18 (i.e. when the in vivo rate of activity was increasing), the enzyme activity did not continue to increase, but it was rather maintained for 2 days before decreasing in a manner that closely mimicked the decline observed in vivo. A similar, butimmediate decline was observed when the tissue was explanted on GD19 (i.e. at the time when theactivity peaks in vivo). Exposure of explants to either growth factors (insulin-like growth factor II, epidermal growth factor, and basic or acidic fibroblast growth factor), or steroids (estradiol-17ß, progesterone, testosterone, dihydrotestosterone and corticosterone) failed to either increase aromatase activity before the peak at GD19 or ameliorate its perinatal decline. Increase of Ca(2+) fluxes with the ionophore A23187 or activation of the cyclic AMP, cyclic GMP, or protein kinase C pathways were similarly ineffective, as was angiotensin II, a recently proposed stimulator of neural aromatase. In contrast, aromatase activity was suppressed 2- to 4-fold by activation of the cyclic AMP pathway (with either forskolin or 8-bromo-cyclic AMP) or by the androgens, testosterone and dihydrotestosterone. These results suggest that: 1) the appearance of aromatase activity in the rat hypothalamus before GD17 requires the unfolding of extrahypothalamic events, 2) the increase in aromatase activity that occurs before GD19 also requires extrahypothalamic inputs and does not involve any of the known intracellular signal transduction pathways, and 3) the decline in activity observed after GD19 is regulated within the hypothalamus, and appears to be determined, at least in part, by the activation of cyclic AMP formation. A potential role for androgens is discussed.

Regulation of GTP biosynthesis

In the regulation of GTP biosynthesis, complex interactions are observed. A major factor is the behavior of the activity of IMPDH, the rate-limiting enzyme of de novo GTP biosynthesis, and the activity of GPRT, the salvage enzyme of guanylate production. The activities of GMP synthase, GMP kinase and nucleoside-diphosphate kinase are also relevant. In neoplastic transformation, the activities and amounts of all these biosynthetic enzymes are elevated as shown by kinetic assays and by immunotitration for IMPDH. In cancer cells, the up-regulation of guanylate biosynthesis is amplified by the concurrent decrease in activities of the catabolic enzymes, nucleotidase, nucleoside phosphorylase, and the rate-limiting purine catabolic enzyme, xanthine oxidase. The up-regulation of the capacity for GTP biosynthesis is also manifested in the stepped-up capacity of the overall pathways of de novo and salvage guanylate production. The linking with neoplasia is also seen in the elevation of the activities of IMPDH and GMP synthase and de novo and salvage pathways as the proliferative program is expressed as cancer cells enter log phase in tissue culture. The activity of GMP reductase showed no linkage with neoplastic or normal cell proliferation; however, in induced differentiation in HL-60 cells the activity increased concurrently with the decline in the activity of IMPDH. This reciprocal regulation of the two enzymes is observed in differentiation induced by retinoic acid, DMSO or TPA in HL-60 cells. In support of enzyme-pattern-targeted chemotherapy, evidence was provided for synergistic chemotherapy with tiazofurin (inhibitor of IMPDH) and hypoxanthine (competitive inhibitor of GPRT and guanine salvage activity) in patients and in tissue culture cell lines. These investigations should contribute to the clarification of the controlling factors of GMP biosynthesis, the role of the various enzymes, the behavior of GMP reductase in mammalian cells and the application of the approaches of enzyme-pattern-targeted chemotherapy in patients.