Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen.

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.

5-HT and GABA modulate intrinsic excitability of type I interneurons in Hermissenda.

The sensory neurons (photoreceptors) in the visual system of Hermissenda are one site of plasticity produced by Pavlovian conditioning. A second site of plasticity produced by conditioning is the type I interneurons in the cerebropleural ganglia. Both photoreceptors and statocyst hair cells of the graviceptive system form monosynaptic connections with identified type I interneurons. Two proposed neurotransmitters in the graviceptive system, serotonin (5-HT) and gamma-aminobutyric acid (GABA), have been shown to modify synaptic strength and intrinsic neuronal excitability in identified photoreceptors. However, the potential role of 5-HT and GABA in plasticity of type I interneurons has not been investigated. Here we show that 5-HT increased the peak amplitude of light-evoked complex excitatory postsynaptic potentials (EPSPs), enhanced intrinsic excitability, and increased spike activity of identified type I(e(A)) interneurons. In contrast, 5-HT decreased spike activity and intrinsic excitability of type I(e(B)) interneurons. The classification of two categories of type I(e) interneurons was also supported by the observation that 5-HT produced opposite effects on whole cell steady-state outward currents in type I(e) interneurons. Serotonin produced a reduction in the amplitude of light-evoked complex inhibitory PSPs (IPSPs), increased spontaneous spike activity, decreased intrinsic excitability, and depolarized the resting membrane potential of identified type I(i) interneurons. In contrast to the effects of 5-HT, GABA produced inhibition in both types of I(e) interneurons and type I(i) interneurons. These results show that 5-HT and GABA can modulate the intrinsic excitability of type I interneurons independent of the presynaptic effects of the same transmitters on excitability and synaptic efficacy of photoreceptors.

UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^

Stimulation of renin secretion by nitric oxide is mediated by phosphodiesterase 3

This study aimed to characterize the cellular pathways along which nitric oxide (NO) stimulates renin secretion from the kidney. Using the isolated perfused rat kidney model we found that renin secretion stimulated 4- to 8-fold by low perfusion pressure (40 mmHg), by macula densa inhibition (100 μmol/liter of bumetanide), and by adenylate cyclase activation (3 nmol/liter of isoproterenol) was markedly attenuated by the NO synthase inhibitor nitro-l-arginine methyl ester (l-Name) (1 mM) and that the inhibition by l-Name was compensated by the NO-donor sodium nitroprusside (SNP) (10 μmol/liter). Similarly, inhibition of cAMP degradation by blockade of phosphodiesterase 1 (PDE-1) (20 μmol/liter of 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine) or of PDE-4 (20 μmol/liter of rolipram) caused a 3- to 4-fold stimulation of renin secretion that was attenuated by l-Name and that was even overcompensated by sodium nitroprusside. Inhibition of PDE-3 by 20 μmol/liter of milrinone or by 200 nmol/liter of trequinsin caused a 5- to 6-fold stimulation of renin secretion that was slightly enhanced by NO synthase inhibition and moderately attenuated by NO donation. Because PDE-3 is a cGMP-inhibited cAMP-PDE the role of endogenous cGMP for the effects of NO was examined by the use of the specific guanylate cyclase inhibitor 1-H-(1,2,4)oxodiazolo(4,3a)quinoxalin-1-one (20 μmol). In the presence of 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one the effect of NO on renin secretion was abolished, whereas PDE-3 inhibitors exerted their normal effects. These findings suggest that PDE-3 plays a major role for the cAMP control of renin secretion. Our findings are compatible with the idea that the stimulatory effects of endogenous and exogenous NO on renin secretion are mediated by a cGMP-induced inhibition of cAMP degradation.

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-d-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3α-ol-5β-pregnan-20-one hemisuccinate (3α5βHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3α5βHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3α5βHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3α5βHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.

Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection

A genetic defect in a CC-chemokine receptor (CCR)-5, the principal coreceptor for the macrophage-tropic HIV type 1 (HIV-1), recently was found to naturally protect CCR-5-defective, but healthy, individuals from HIV-1 infection. In this study, we mimic the natural resistance of the CCR-5-defective individuals by designing a strategy to phenotypically knock out CCR-5. The inactivation of the CCR-5 coreceptor is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5. The lymphocytes transduced to express the intracellular chemokine, termed “intrakine,” were found to be viable and resistant to macrophage-tropic HIV-1 infection. Thus, this gene-based intrakine strategy targeted at the conserved cellular receptor for the prevention of HIV-1 entry should have significant advantages over currently described approaches for HIV-1 therapy.

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIα subunit would reveal those tissues and signaling events that require anchored PKA. RIIα knockout mice appear normal and healthy. In adult skeletal muscle, RIα protein levels increased to partially compensate for the loss of RIIα. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIα knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA–AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIα subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIα subunit. The potentiation of the L-type Ca2+ channel in RIIα knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIα is capable of physiologically relevant anchoring interactions.

Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes

The neurosteroid 3α-hydroxysteroid-5α-pregnan-20-one (allopregnanolone) acts as a positive allosteric modulator of γ-aminobutyric acid at γ-aminobutyric acid type A receptors and hence is a powerful anxiolytic, anticonvulsant, and anesthetic agent. Allopregnanolone is synthesized from progesterone by reduction to 5α-dihydroprogesterone, mediated by 5α-reductase, and by reduction to allopregnanolone, mediated by 3α-hydroxysteroid dehydrogenase (3α-HSD). Previous reports suggested that some selective serotonin reuptake inhibitors (SSRIs) could alter concentrations of allopregnanolone in human cerebral spinal fluid and in rat brain sections. We determined whether SSRIs directly altered the activities of either 5α-reductase or 3α-HSD, using an in vitro system containing purified recombinant proteins. Although rats appear to express a single 3α-HSD isoform, the human brain contains several isoforms of this enzyme, including a new isoform we cloned from human fetal brains. Our results indicate that the SSRIs fluoxetine, sertraline, and paroxetine decrease the Km of the conversion of 5α-dihydroprogesterone to allopregnanolone by human 3α-HSD type III 10- to 30-fold. Only sertraline inhibited the reverse oxidative reaction. SSRIs also affected conversions of androgens to 3α- and 3α, 17β-reduced or -oxidized androgens mediated by 3α-HSD type IIBrain. Another antidepressant, imipramine, was without any effect on allopregnanolone or androstanediol production. The region-specific expression of 3α-HSD type IIBrain and 3α-HSD type III mRNAs suggest that SSRIs will affect neurosteroid production in a region-specific manner. Our results may thus help explain the rapid alleviation of the anxiety and dysphoria associated with late luteal phase dysphoria disorder and major unipolar depression by these SSRIs.

Involvement of Type 4 cAMP-Phosphodiesterase in the Myogenic Differentiation of L6 Cells

Myogenic cell differentiation is induced by Arg8-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

Cloning and characterization of PDE7B, a cAMP-specific phosphodiesterase

A member of the phosphodiesterase (PDE)7 family with high affinity and specificity for cAMP has been identified. Based on sequence homologies, we designate this PDE as PDE7B. The full-length cDNA of PDE7B is 2399 bp, and its ORF sequence predicts a protein of 446 amino acids with a molecular mass of 50.1 kDa. Comparison of the predicted protein sequences of PDE7A and PDE7B reveals an identity of 70% in the catalytic domain. Northern blotting indicates that the mRNA of PDE7B is 5.6 kb. It is most highly expressed in pancreas followed by brain, heart, thyroid, skeletal muscle, eye, ovary, submaxillary gland, epididymus, and liver. Recombinant PDE7B protein expressed in a Baculovirus expression system is specific for cAMP with a Km of 0.03 μM. Within a series of common PDE inhibitors, it is most potently inhibited by 3-isobutyl-1-methylxanthine with an IC50 of 2.1 μM. It is also inhibited by papaverine, dipyridamole, and SCH51866 at higher doses. PDE7A and PDE7B exhibit the same general pattern of inhibitor specificity among the several drugs tested. However, differences in IC50 for some of the drugs suggest that isozyme selective inhibitors can be developed.

Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis

A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated Keq′ (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25°C is 110, and its standard-state free-energy change value (ΔG′) is −2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (Km = 0.5 mM). Pi and phosphorylated compounds such as 3-phosphoglycerate, PPi, ATP, ADP, NADP+, and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-α-d-glucan 4-α-d-glucosyltransferase; EC 2.4.1.21) for ADPG, thus markedly blocking the starch biosynthesis.