Cyclic nucleotides

Carbon dioxide (CO 2 ) is produced by living organisms as a byproduct of metabolism. In physiological systems, CO 2 is unequivocally linked with bicarbonate (HCO 3 -) and pH via a ubiquitous family of carbonic anhydrases, and numerous biological processes are dependent upon a mechanism for sensing the level of CO 2 , HCO 3 , and/or pH. The discovery that soluble adenylyl cyclase (sAC) is directly regulated by bicarbonate provided a link between CO 2 /HCO 3 /pH chemosensing and signaling via the widely used second messenger cyclic AMP. This review summarizes the evidence that bicarbonate-regulated sAC, and additional, subsequently identified bicarbonate-regulate nucleotidyl cyclases, function as evolutionarily conserved CO 2 /HCO 3 /pH chemosensors in a wide variety of physiological systems.

Anterior pituitary cells fire action potentials and release cyclic nucleotides both spontaneously and in response to agonist stimulation, but the relationship between electrical activity and cyclic nucleotide efflux has not been studied. In these cells, a tetrodotoxin-resistant background Na ϩ conductance is critical for firing of action potentials, and multidrug resistance proteins (MRPs) MRP4 and MRP5 contribute to cyclic nucleotide efflux. Here, we show that abolition of the background Na ϩ conductance in rat pituitary cells by complete or partial replacement of extracellular Na ϩ with organic cations or sucrose induced a rapid and reversible hyperpolarization of cell membranes and inhibition of action potential firing, accompanied by a rapid inhibition of cyclic nucleotide efflux. Valinomycin-induced hyperpolarization of plasma membranes also inhibited cyclic nucleotide efflux, whereas depolarization of cell membranes induced by the inhibition of Ca 2ϩ influx or stimulation of Na ϩ

Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity and calcium influx in endocrine pituitary cells, and a role for an unidentified sodium-conducting channel in this process has been proposed. Here we show that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (cAMP-dependent protein kinase) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conduct...

The cellular localization of carnosine-like immunoreactivity was investigated in the adult rat forebrain and in glial cell cultures obtained from newborn rat brain. Using double staining methods, we showed that in vivo carnosine-like immunoreactivity was occurring in a large number of both glial fibrillary acidic protein (GFAP)-positive astrocytes and 2¢3¢-cyclic nucleotide 3¢phosphodiesterase (CNP)-positive oligodendrocytes. In vitro, the carnosineimmunoreactive staining was restricted to a subpopulation of completely differentiated oligodendrocytes, whereas no reaction was detected in immature oligodendrocytes and in astrocytes. These observations could have profound physiopathological implications considering the role suggested for carnosine and related peptides as endogenous antioxidants, free radical scavengers and anti-glycating agents of the central nervous system (CNS).

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

This study explores the protein interactions and evolutionary conservation of phosphodiesterase type 7A (PDE7A) across different species, to identify the species with the highest similarity to humans, which has important applications in many fields such as pharmacology, comparative biology, biochemistry, oncology, endocrinology, and reproductive sciences. Computational analyses of the amino acid sequence of this enzyme revealed a common evolutionary relationship between the selected species and humans, but camels show the most variable evolutionary lineage in their PDE7A protein over time. In searches for homologous proteins, identity analysis results for four returned amino acid sequences were as follows: 91.81 ± 4.25% (mean ± SD), while the similarity between the sequences was 94.66% ± 4.55, and analysis of BLOSUM62 (Blocks Substitution Matrix 62) yielded a minimum value of 0.901 ± 0.055. All species have closely related physicochemical properties. The computational analyses revealed conserved amino acid residues of the gene across the studied species, which likely contribute to the similar expression patterns of the PDE7A gene in various animals. The findings also suggest that rats are a suitable model for gaining deeper insights into human biology.

Background and Purpose: Asthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high-throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca 2+ ] i ), promote human sperm cell motility, and systematically determine the mechanism of action. Experimental Approach: A validated HTS fluorometric [Ca 2+ ] i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. Key Results: Fluorometric assays identified trequinsin as an efficacious agonist of [Ca 2+ ] i , although less potent than progesterone. Functionally, trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. Trequinsin-induced [Ca 2+ ] i responses were cross-desensitised consistently by PGE 1 but not progesterone. Whole-cell patch clamp electrophysiology confirmed that trequinsin activated CatSper and partly inhibited potassium channel activity. Trequinsin also increased intracellular cGMP. Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new agents to improve patient sperm function and fertilisation potential.

Background and PurposeAsthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high‐throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca2+]i), promote human sperm cell motility, and systematically determine the mechanism of action.Experimental ApproachA validated HTS fluorometric [Ca2+]i assay was used to screen an in‐house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer...

Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1 H -indeno[2′,1′:5,6]pyrido[2,3- d ]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis . The e...

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The DedA protein family is a highly conserved and ancient family of membrane proteins with representatives in most sequenced genomes, including those of bacteria, archaea, and eukarya. The functions of the DedA family proteins remain obscure. However, recent genetic approaches have revealed important roles for certain bacterial DedA family members in membrane homeostasis. Bacterial DedA family mutants display such intriguing phenotypes as cell division defects, temperature sensitivity, altered membrane lipid composition, elevated envelope-related stress responses, and loss of proton motive force. The DedA family is also essential in at least two species of bacteria: Borrelia burgdorferi and Escherichia coli . Here, we describe the phylogenetic distribution of the family and summarize recent progress toward understanding the functions of the DedA membrane protein family.

In a great variety of organisms throughout all kingdoms of life, the heme-based-sensor proteins are the key regulators of adaptive responses to fluctuating oxygen, carbon monoxide, and nitric oxide levels. These signal transducers achieve their responses by coupling a regulatory heme-binding domain to a neighboring transmitter. The past decade has witnessed an explosion in the numbers of these modular sensory proteins known, from just two recognized members, FixL and soluble guanylyl cyclase (sGC), to four broad families comprising more than 50 sensors. Heme-based sensors so far feature four different types of heme-binding modules: the heme-binding PAS domain, globin-coupled sensor (GCS), CooA, and heme-NO-binding (HNOB). The transmitters for coupling to such heme-binding domains include histidine protein kinases, cyclic nucleotide phosphodiesterases, chemotaxis methyl-carrier protein receptors, and transcription factors of the basic helix-loop-helix and helix-turn-helix classes. Some well-studied sensors are the FixL, EcDos, AxPDEA1, NPAS2, HemAT-Bs, HemAT-Hs, CooA, and sGC proteins. This review elaborates the defining characteristics of heme-based sensors, examines recent developments on those proteins, and discusses the regulatory hypotheses proposed for those sensors. A general, ''helix-swap'', model is also proposed here for signal transduction by PAS domains.

Robust immune responses are essential for eliminating pathogens but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) produces the second messenger cGAMP to initiate the stimulator of interferon genes (STING) pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double-stranded DNA (dsDNA) stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNA to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNA. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced antimicrobial immune responses.

Enhancing cyclic nucleotides signaling by inhibition of phosphodiesterases (PDEs) is known to be beneficial in disorders associated with cognitive decline. The present study was designed to investigate the effect of vinpocetine (PDE1 inhibitor) on intracerebroventricular (i.c.v.) streptozotocin induced experimental sporadic dementia of Alzheimer's type. Infusion of streptozotocin impaired learning and memory, increased oxidativenitritive stress and induced cholinergic hypofunction in rats. Chronic treatment with vinpocetine (5, 10 and 20 mg/kg i.p.) for 21 days following first i.c.v. streptozotocin infusion significantly improved learning and memory in Morris water maze and passive avoidance paradigms. Further, vinpocetine significantly reduced the oxidative-nitritive stress, as evidenced by decrease in malondialdehyde (MDA) and nitrite levels, and restored the reduced glutathione (GSH) levels. Significant increase in acetylcholinesterase activity and lactate dehydrogenase levels was observed in the present model indicating cholinergic hypofunction and increase in neuronal cell damage. Chronic treatment with vinpocetine also reduced significantly the increase in acetylcholinesterase activity and lactate dehydrogenase levels indicating restorative capacity of vinpocetine with respect to cholinergic functions and preventing the neuronal damage. The observed beneficial effects of vinpocetine on spatial memory may be due to its ability to favorably modulate cholinergic functions, prevent neuronal cell damage and possibly through its antioxidant mechanism also.

The AphA enzyme of Escherichia coli, a molecular class B periplasmic phosphatase that belongs to the DDDD superfamily of phosphohydrolases, was purified and subjected to biochemical characterization. Kinetic analysis with several substrates revealed that the enzyme essentially behaves as a broad-spectrum nucleotidase highly active on 3V-and 5V-mononucleotides and monodeoxynucleotides, but not active on cyclic nucleotides, or nucleotides di-and triphosphate. Mononucleotides are degraded to nucleosides, and AphA apparently does not exhibit any nucleotide phosphomutase activity. However, it can transphosphorylate nucleosides in the presence of phosphate donors. Kinetic properties of AphA are consistent with structural data, and suggest a role for the hydrophobic pocket present in the active site crevice, made by residues Phe 56, Leu71, Trp77 and Tyr193, in conferring preferential substrate specificity by accommodating compounds with aromatic rings. AphA was inhibited by several chelating agents, including EDTA, EGTA, 1,10-phenanthroline and dipicolinic acid, with EDTA being apparently the most powerful inhibitor.

The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences. The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5, A. lipoferum 4B, Azospirillum sp. B510, A. thiophilu...

Immunohistochemical techniques were utilized to localize the putative myelin enzyme 2' :3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in the central nervous system (CNS) of normal (+/+ ) and Shiverer (Shi/Shi) mice (Mus musculus). CNP appeared to be only associated with myelinated nerve fibers in the CNS (corpus callosum, subcortical white matter, caudate nucleus, cerebellum and medulla oblongata) of + / + mice. However, little or no immunostaining was observed in the same regions of the CNS of Shi/Shi mice, although these mice have normal levels of a CNS CNP activity. Unexpectedly, oligodendrocytes (cell periphery) were not stained for CNP in either + / + or Shi/Shi mice, although erythrocytes were immunostained.

The olfactory bulb (OB) presents a unique pattern of permanent acquisition of primary afferents and interneurons, but not much detail is known about the differentiation of its oligodendroglial cells. We studied the expression of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase), a protein related to axonal ensheathment by myelinating cells. Expression of CNPase in OB follows a general caudorostral gradient, with the exception of the glomerular layer (GL). At postnatal day 5–6 (P5–P6), the first CNPase+ profiles appeared in the dorsal lateral olfactory tract adjacent to the accessory OB (AOB), followed by rare cell bodies and processes in AOB internal plexiform layer at P7. At P9, the main OB (MOB) granular cell layer (GrCL) already showed intensely stained CNPase+ processes. From P5 to P12, small numbers of CNPase+ cells were found in the subventricular zone (SVZ), throughout its rostral extension (SVZ‐RE), and in the intrabulbar subependymal layer. The appearance of CNPase+ pro...

The differentiation of oligodendrocytes in the forebrain of the opossum (Didelphis marsupialis) has been studied by the immunohistochemical identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and by the autoradiographic detection of the uptake of 3H-thymidine. CNPase is expressed early in oligodendroglia somata and fibre sheaths (myelin) in the forebrain and its persistence in the cell bodies is regionally heterogeneous, being ephemeral

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. The crystal structure of the catalytic domain of PDE9A2, a member of a PDE family specifically hydrolyzing cGMP, has been determined at 2.23-Å resolution. The PDE9A2 catalytic domain closely resembles the cAMP-specific PDE4D2 but is significantly different from the cGMP-specific PDE5A1, implying that each individual PDE family has its own characteristic substrate recognition mechanism. The different conformations of the H and M loops between PDE9A2 and PDE5A1 imply their less critical roles in nucleotide recognition. The nonselective inhibitor 3-isobutyl-1methylxanthine (IBMX) binds to a similar subpocket in the active sites of PDE4, PDE5, and PDE9 and has a common pattern of the binding. However, significantly different orientations and interactions of IBMXs are observed among the three PDE families and also between two monomers of the PDE9A2 dimer. The kinetic properties of the PDE9A2 catalytic domain similar to those of full-length PDE9A imply that the N-terminal regulatory domain does not significantly alter the catalytic activity and the IBMX inhibition.

Monoclonal antibodies against human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.

Background: Berberis orthobotrys Bien ex Aitch. (Berberidaceae) is a plant indigenous of Pakistan that is locally used for the treatment of hypertension. Hypothesis: This study evaluated the vasoactive properties of a Berberis orthobotrys root extract and its fractions, and investigated the role of the endothelium and the underlying mechanism. Study design: An aqueous methanolic extract of Berberis orthobotrys roots was prepared and submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity. Vascular reactivity of the different fractions was assessed using porcine coronary artery rings either with or without endothelium, and in the presence or absence of specific pharmacological tools. The ability of Berberis orthobotrys extracts to affect phosphodiesterase (PDE) activity was evaluated using a radioenzymatic method and purified phosphodiesterases. Results: The aqueous methanol extract induced similar relaxations in coronary artery rings with and without endothelium, and, amongst the three derived preparations, the butanol fraction (BFBO) was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue in rings without endothelium. Analysis of the butanol fraction (BFBO) by LC-ELSD-MS indicated the presence of four major isoquinoline alkaloids including berberine. BFBO significantly potentiated the relaxations induced by cyclic GMP-and cyclic AMP-dependent relaxing agonists, and inhibited contractions to KCl, CaCl 2 , and U46619 in endothelium denuded rings. In contrast, BFBO did not affect relaxations to endothelium-dependent vasodilators. BFBO concentration-dependently inhibited the cyclic GMPhydrolyzing activity of basal PDE1, calmodulin-activated PDE1 and PDE5, and of cyclic AMP-hydrolyzing activity of PDE3 and PDE4 with IC 50 values ranging from 40 to 130 μg/ml. The butanol fraction of the aqueous methanol extract of Berberis orthobotrys roots induced pronounced endothelium-independent relaxations and inhibited contractile responses by acting directly at the vascular smooth muscle in the coronary artery. Moreover, BFBO potentiated relaxations induced by both cyclic GMP-and cyclic AMP-dependent vasodilators most likely due to its ability to inhibit several vascular PDEs, and in particular PDE4 and PDE5.

The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG) in the eects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca 2+ ] i ) was studied in rat aorta. Cyclic AMP-elevating agents used were a b-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram). 2 In rat intact aorta, the relaxant eect induced by isoprenaline (0.01 ± 0.3 mM) was decreased by a speci®c inhibitor of PKA, H-89, whereas a speci®c inhibitor of PKG, Rp-8-Br-cyclic GMPS, was without eect. No signi®cant dierence in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 mM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca 2+ ] i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPS modi®ed this response. These ®ndings suggest that PKA is only involved in the relaxant eect induced by low concentrations of isoprenaline (0.01 ± 0.3 mM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved. 3 The relaxant eects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no signi®cant eect of H-89. Neither SK&F 94120, nor rolipram at 30 mM signi®cantly modi®ed the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca 2+ ] i . This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no signi®cant eect. These results suggest that PKG is involved in the vascular eects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta. 4 The combination of 5 mM SK&F 94120 with rolipram markedly potentiated the relaxant eect of rolipram. This relaxation was decreased by H-89 and not signi®cantly modi®ed by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors signi®cantly increased the activity ratio of PKA without changing the PKG ratio. The present ®ndings show that PKA rather than PKG is involved in this type of vasorelaxation. The dierences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to dierences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are dierently localized in the cell. 5 These ®ndings support a role for both PKA and PKG in cyclic AMP-mediated relaxation in rat aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.

Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFa secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/ J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtype (IC 50 = 1.4 nM) than the other subtypes (PDE4A, IC 50 = 44 nM; PDE4B, IC 50 = 48 nM; and PDE4D, IC 50 = 14 nM). Interestingly, its affinity for the High Affinity Rolipram Binding Site is relatively low (K i = 148 nM) in comparison to rolipram (K i = 3 nM). Finally, as also observed using MRL/lpr peripheral blood lymphocytes (PBLs), NCS 613 inhibits basal and LPS-induced TNFa secretion from PBLs of lupus patients, suggesting a therapeutic potential of NCS 613 in systemic lupus. This study reveals that PDE4 represent a potential therapeutic target in lupus disease.

Hearts from AC8TG mice develop a higher contractility (LVSP) and larger Ca 2؉ transients than NTG mice, with (surprisingly) no modification in L-type Ca 2؉ channel current (I Ca,L ) (1). In this study, we examined the cardiac response of AC8TG mice to ␤-adrenergic and muscarinic agonists and IBMX, a cyclic nucleotide phosphodiesterase (PDE) inhibitor. Stimulation of LVSP and I Ca,L by isoprenaline (ISO, 100 nM) was twofold smaller in AC8TG vs. NTG mice. In contrast, IBMX (100 M) produced a twofold higher stimulation of I Ca,L in AC8TG vs. NTG mice. IBMX (10 M) increased LVSP by 40% in both types of mice, but contraction and relaxation were hastened in AC8TG mice only. Carbachol (10 M) had no effect on basal contractility in NTG hearts but decreased LVSP by 50% in AC8TG mice. PDE assays demonstrated an increase in cAMP-PDE activity in AC8TG hearts, mainly due to an increase in the hydrolytic activity of PDE4 and PDE1 toward cAMP and a decrease in the activity of PDE1 and PDE2 toward cGMP. We conclude that cardiac expression of AC8 is accompanied by a rearrangement of PDE isoforms, leading to a strong compartmentation of the cAMP signal that shields L-type Ca 2؉ channels and protects the cardiomyocytes from Ca 2؉ overload.

Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored. Rats developed arterial hypertension associated with a slight cardiac hypertrophy (+24%). cAMP-PDE4 activity was specifically increased while cGMP-PDE activities were broadly increased (+130% for PDE1; +76% for PDE2; +113% for PDE5) and associated with increased expressions for PDE1A, PDE1C and PDE5A. The cGMP-PDE1 activation by Ca 2+ /CaM was reduced. BNP expression was increased by 3.5-fold, while NOX2 expression was reduced by 66% and AMP kinase activation was increased by 64%. In early cardiac hypertrophy induced by angiotensin II, all specific PDE activities in left cardiac ventricles were increased, favoring an increase in cGMP hydrolysis by PDE1, PDE2 and PDE5. Increased cAMP hydrolysis was related to PDE4. We observed the establishment of two cardioprotective mechanisms and we suggest that these mechanisms could lead to increase intracellular cGMP: i) increased expression of BNP could increase ''particulate'' cGMP pool; ii) increased activation of AMPK, subsequent to increase in PDE4 activity and 59AMP generation, could elevate ''soluble'' cGMP pool by enhancing NO bioavailability through NOX2 downregulation. More studies are needed to support these assumptions. Nevertheless, our results suggest a potential link between PDE4 and AMPK/NOX2 and they point out that cGMP-PDEs, especially PDE1 and PDE2, may be interesting therapeutic targets in preventing cardiac hypertrophy.

Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFa secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/ J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtype (IC 50 = 1.4 nM) than the other subtypes (PDE4A, IC 50 = 44 nM; PDE4B, IC 50 = 48 nM; and PDE4D, IC 50 = 14 nM). Interestingly, its affinity for the High Affinity Rolipram Binding Site is relatively low (K i = 148 nM) in comparison to rolipram (K i = 3 nM). Finally, as also observed using MRL/lpr peripheral blood lymphocytes (PBLs), NCS 613 inhibits basal and LPS-induced TNFa secretion from PBLs of lupus patients, suggesting a therapeutic potential of NCS 613 in systemic lupus. This study reveals that PDE4 represent a potential therapeutic target in lupus disease.

The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG) in the eects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca 2+ ] i ) was studied in rat aorta. Cyclic AMP-elevating agents used were a b-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram). 2 In rat intact aorta, the relaxant eect induced by isoprenaline (0.01 ± 0.3 mM) was decreased by a speci®c inhibitor of PKA, H-89, whereas a speci®c inhibitor of PKG, Rp-8-Br-cyclic GMPS, was without eect. No signi®cant dierence in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 mM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca 2+ ] i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPS modi®ed this response. These ®ndings suggest that PKA is only involved in the relaxant eect induced by low concentrations of isoprenaline (0.01 ± 0.3 mM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved. 3 The relaxant eects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no signi®cant eect of H-89. Neither SK&F 94120, nor rolipram at 30 mM signi®cantly modi®ed the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca 2+ ] i . This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no signi®cant eect. These results suggest that PKG is involved in the vascular eects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta. 4 The combination of 5 mM SK&F 94120 with rolipram markedly potentiated the relaxant eect of rolipram. This relaxation was decreased by H-89 and not signi®cantly modi®ed by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors signi®cantly increased the activity ratio of PKA without changing the PKG ratio. The present ®ndings show that PKA rather than PKG is involved in this type of vasorelaxation. The dierences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to dierences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are dierently localized in the cell. 5 These ®ndings support a role for both PKA and PKG in cyclic AMP-mediated relaxation in rat aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.

is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). 2 The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 mM) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 mM), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 mM) also inhibited significantly (22%) the cAMP-PDE activity. 3 Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC 50 ¼ 3.4 nM) the activity of MPA PDE5 partially purified by HPLC. 4 Sildenafil (0.1 nM-50 mM) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 mM). The potency of sildenafil (IC 50 ¼ 11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC 50 ¼ 600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 mM) and H89 (1 mM), potent inhibitors of PKG and PKA, respectively. 5 In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10 -100 nM) antagonized ATP-and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca 2+ ] i response. 6 This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC 50 ϭ25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (K i ϭ12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca 2ϩ ] i ) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca 2ϩ ] i levels and agonist-induced rises in [Ca 2ϩ ] i in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 mol/L), and db-cAMP (100 mol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca 2ϩ ] i increases in HUVECs without modifying resting [Ca 2ϩ ] i levels. Similar results were obtained by using a Ca 2ϩ -free bath solution. EGb 761 (100 mg/L), but not rolipram (50 mol/L) or db-cAMP (100 mol/L), also inhibited Ca 2ϩ influx into cells having thapsigargin-depleted internal Ca 2ϩ stores and bathed in a Ca 2ϩ -free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca 2ϩ ] i in HUVECs, mainly by inhibition of Ca 2ϩ mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca 2ϩ signaling in endothelial cells. (Arterioscler Thromb Vasc Biol. 2000;20:e34-e40.

Angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family, is induced by hypoxia in both tumor and endothelial cells. It is highly expressed in tumor cells from conventional renal carcinoma and in hypoxic perinecrotic areas of numerous types of human tumors (1).

The patterns of expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE) have been studied in developing and adult rat brain using affinity-purified polyclonal antibodies against CaM-PDE. An immunocytochemical map of adult brain regions expressing CaM-PDE, constructed from serial coronal brain sections, illustrated that CaM-PDE was expressed in specific neuronal subpopulations throughout the adult rat brain. Immunoblot analysis coupled with subcellular fractionation indicated that CaM-PDE was primarily localized to cytoplasmic fractions, with a small amount associated with synaptosomal membranes. Immunoblots from developing brain indicated that CaM-PDE expression increased dramatically during postnatal days 7-20 (PND 7-20); parallel increases in CaM-PDE enzyme activity occurred during this same time. Immunocytochemical studies indicated that several distinct patterns of CaM-PDE expression occurred during development. Neocortex showed low levels of CaM-PDE immunoreactivity in neuronal somata of layers III, V and VI on PND 4 that increased by PND 11; the adult somatodendritic pattern of immunoreactivity was observed by PND 60. Similar patterns were observed in cerebellar Purkinje cells, with somatodendritic staining observed by PND 12. By contrast, caudate-putamen, the inferior olive and the hypoglossal nuclei expressed high levels of CaM-PDE on PND 4, with levels considerably lower in the adult animal. The different patterns of expression suggest that in neocortex and cerebellum, CaM-PDE increases during the period of neuronal differentiation and active synaptogenesis, while in the caudate-putamen, inferior olive and hypoglossal nucleus, high levels may be required early in development.

In a previous study, our laboratory reported that sildenafil citrate, a cyclic nucleotide phosphodiesterase type 5 inhibitor, reversed a learning impairment in rats induced by systemic inhibition of nitric oxide synthase (60 mg/kg, i.p., N ω -nitro-L-arginine methyl ester; L-NAME). To limit the peripheral effects of L-NAME and further localize the site of action of sildenafil, L-NAME (48 μg, i.c.v.) was infused bilaterally into the lateral cerebral ventricles 30 min prior to maze training. Saline or sildenafil citrate (1.5 or 3.0 mg/kg, i.p.) was administered systemically 15 min before training. Drug injections occurred 24 h after pretraining rats to avoid foot shock on a one-way active avoidance straight runway. Following drug treatment, the rats received 15 training trials on a 14-unit T-maze task that requires learning a complex sequence of turns to avoid mild foot shock. This complex maze paradigm is sensitive to aging and blockade of cholinergic, N-methyl-D-aspartate and nitric oxide signaling systems. Behavioral measures of performance included deviations from the correct pathway (errors), runtime from start to goal (latency), shock frequency and shock duration. Statistical analysis revealed that central infusion of L-NAME impaired maze performance and that sildenafil (3.0 mg/kg) significantly attenuated the impairment. These results suggest that sildenafil citrate may serve as a cognitive enhancer by modulating central nitric oxide/cGMP signal transduction following N-methyl-D-aspartate receptor activation. This pathway has been implicated in age-related cognitive decline and may be a useful target for pharmacological intervention of neurodegenerative disease.

We examined whether treatment with sildenafil citrate (the active compound of Viagra), a cyclic nucleotide phosphodiesterase type 5 inhibitor (PDE5), would reverse the learning impairment induced by cholinergic muscarinic (mACh) receptor blockade [0.75 mg/kg scopolamine HCl, intraperitoneal (i.p.) injections]. Rats were pretrained in a one-way active avoidance of foot shock in a straight runway and the next day received 15 training trials in a 14-unit T-maze. Performance in this maze paradigm requires accurate responding to avoid mild foot shock and has been shown to be sensitive to aging and to impairment in central cholinergic systems. Intraperitoneal (i.p.) injections of scopolamine or saline and sildenafil or vehicle were given 30 and 15 min before training, respectively. The combined treatment conditions were as follows: saline+vehicle (control), scopolamine (0.75 mg/kg)+vehicle, and scopolamine (0.75 mg/kg)+sildenafil (1.5, 3.0, or 4.5 mg/ kg). Behavioral measures of performance included deviations from the correct pathway (errors), run time from start to goal, shock frequency, and duration. Statistical analysis revealed that scopolamine impaired maze performance and that sildenafil (3.0 mg/kg) significantly attenuated this impairment in a dose-dependent manner. These results suggest that sildenafil citrate may serve as a cognitive enhancer for therapeutic treatment of cholinergic dysfunction in age-related cognitive decline and Alzheimer's dementia (AD).

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3,5-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4-and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20 -600 M, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A 1 and E 1 , the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterasemediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.

Several ATP-binding cassette (ABC) transporters can transport drugs out of cells against steep concentration gradients resulting in resistance to the drugs transported. Recent work has shown that at least three members of the family of human Multidrug Resistance-associated Proteins (MRPs), MRP4, 5 and 8, are able to transport some nucleoside-monophosphate analogs. This can result in resistance to the base, nucleoside or nucleotide precursors of these results, at least in cell lines with high levels of transporter. The affinity of these transporters for the nucleotide analogs studied thus far is relatively low (millimolar rather than micromolar), and this limits their potential impact on the resistance. We briefly review how ABC transporters in general, and MRPs in particular, could affect the disposition and cellular accumulation of antiviral compounds.

Introduction: Heart failure (HF) is the leading cause of death worldwide. Most large and small animal disease models of HF are based on surgical procedures. A common surgical technique to induce HF is transverse aortic constriction (TAC), which induces pressure overload. The conventional TAC (cTAC) procedure is a highly invasive surgery that is associated with severe inflammation and excessive perioperative deaths. Aim: To establish an improved, minimally invasive TAC (mTAC) procedure that does not require thoracotomy. Methods and results: Following anesthesia, mice were intubated, and a small incision was made at the neck and chest. After cutting the sternum about 4 mm, the aortic arch was approached without opening the pleural cavity. A suture was placed between the brachiocephalic artery and the left common carotid artery. This model was associated with low perioperative mortality and a highly reproducible constriction evidenced by an increased rightto-left carotid blood flow velocity ratio in mTAC mice (5.9 ± 0.2) vs. sham controls (1.2 ± 0.1; P < 0.001). mTAC mice exhibited progressive cardiac remodeling during the 8 weeks post-TAC, resulting in reduced left ventricular (LV) contractility, increased LV end-systolic diameter, left atrial enlargement and diastolic dysfunction, and an increased heart weight to tibia length ratio (mTAC: 15.0 ± 0.8 vs. sham: 10.1 ± 0.6; P < 0.01). Conclusion: Our data show that the mTAC procedure yields a highly reproducible phenotype consisting of LV contractile dysfunction and enlargement, combined with left atrial enlargement and diastolic dysfunction. Potential impact of the findings: This model may be used to test the molecular mechanisms underlying atrial remodeling associated with HF development or to evaluate therapeutic strategies to treat these conditions.

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3,5-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4-and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20 -600 M, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A 1 and E 1 , the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterasemediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.

Several ATP-binding cassette (ABC) transporters can transport drugs out of cells against steep concentration gradients resulting in resistance to the drugs transported. Recent work has shown that at least three members of the family of human Multidrug Resistance-associated Proteins (MRPs), MRP4, 5 and 8, are able to transport some nucleoside-monophosphate analogs. This can result in resistance to the base, nucleoside or nucleotide precursors of these results, at least in cell lines with high levels of transporter. The affinity of these transporters for the nucleotide analogs studied thus far is relatively low (millimolar rather than micromolar), and this limits their potential impact on the resistance. We briefly review how ABC transporters in general, and MRPs in particular, could affect the disposition and cellular accumulation of antiviral compounds.

The effects of hypothyroidism on oligodendroglial differentiation and myelination are for the first time studied by immunohistochemical localization of an early oligodendroglial marker, the 2%3%cyclic nucleotide 3%phosphodiesterase (E.C. 3.1.4.37-CNPase), in developing rats. Two groups received methimazol; one during gestation (H) and another postnatally (PN). One H sub-group received thyroxine after birth (T). We observed a delay in CNPase expression followed by a decrease in the number of CNPase immunoreactive fibers in both H and PN groups. The T sub-group was not different from controls. Furthermore, the immunoreactive fibers, in mature hypothyroid animals, showed a continuous pattern of staining in contrast with a discontinuous one in controls. Myelinogenesis is a highly regulated timed event. CNPase links myelin related proteins to the cytoskeleton also interacting with membrane lipids during extension and wrapping of the oligodendroglial process around the axon (ensheathment phase). In mature myelinated fiber the CNPase is absent from compact myelin sheath, being located only in the inner and outer loops and in paranodal loops. Thus, our data suggest a disorder in myelin compaction and point once more to the post-natal period as critical for the mechanisms that are thyroid hormone regulated in myelinogenesis.

Myelination depends on the proper differentiation of oligodendrocytes and several factors may influence this event. For instance, thyroid hormone (T3) affects the timing of differentiation and regulates the expression of several enzymes involved in the synthesis of complex lipids and in the expression of some myelin structural proteins. We investigated the effect of T3 deficiency on oligodendroglial differentiation and in the distribution of oligodendrocyte/myelin proteins 2 0 3 0 -cyclic nucleotide 3 0 -phosphodiesterase (CNPase) and myelin basic protein (MBP). Oligodendroglial-enriched cultures were obtained from cerebra of neonate rats grown in a modified medium. The T3-deficient status was induced by using medium devoid of T3. We observed a delay, in T3-deficient cultures, in oligodendroglial maturation characterized by less extensive processes and membrane vellum than in controls. In control cultures, CNPase immunoreactivity was punctated, showing cell bodies and processes at earlier stages and redistribution to cytoskeleton vein-like structures in later stages. In T3-deficient cultures, CNPase remained in a punctated pattern and only at 10 days in vitro we observed CNPase redistribution to the presumptive cytoskeleton vein-like structures. MBP in control cultures was distributed through the whole cell body and processes whereas in T3-deficient cultures, MBP immunoreactivity was concentrated in the perinuclear region. These results reinforce the hypothesis that T3 is an important factor in oligodendrocyte differentiation, particularly regarding the distribution of myelin proteins.

The steroid hormone, progesterone (P), modulates neuroendocrine functions in the central nervous system resulting in alterations in physiology and reproductive behavior in female mammals. A wide body of evidence indicates that these neural effects of P are predominantly mediated via their intracellular progestin receptors (PRs) functioning as "ligand-dependent" transcription factors in the steroid-sensitive neurons regulating genes and genomic networks. In addition to P, intracellular PRs can be activated by neurotransmitters, growth factors and cyclic nucleotides in a ligand-independent manner via crosstalk and convergence of pathways. Furthermore, recent studies indicate that rapid signaling events associated with membrane PRs and/or extra-nuclear, cytoplasmic PRs converge with classical PR activated pathways in neuroendocrine regulation of female reproductive behavior. The molecular mechanisms, by which multiple signaling pathways converge on PRs to modulate PR-dependent female reproductive behavior, are discussed in this review.

In plants, Ca 2+ has emerged as the predominant second messenger for signal transduction, as cyclic nucleotides are not known to play any significant role in this system. Earlier, we characterized an interesting Ca2+-dependent protein kinase, WbCDPK (winged bean calmodulin-like domain protein kinase), from the soluble fraction of winged bean (Psophocarpus tetrayonolobus) shoot extract. Here an isoform of WbCDPK is purified to apparent homogeneity from the same winged bean shoot extract. It is a single polypeptide chain protein-serine kinase, having an Mr of about 70,000 and like WbCDPK, its preferred substrates are histone HI, syntide 2 and MLC-peptide (a synthetic myosin light chain related peptide) and it is totally dependent on Ca 2÷ for its activity, but exogenous calmodulin (CAM) does not stimulate it. However, it is strongly inhibited by CaM antagonists, indicating the presence of a CaM-like domain, as in WbCDPK. The two enzymes do not cross react immunologically and the isoform differs significantly from WbCDPK in its apparent inability to catalyse the autophosphorylation reaction, which is known to cause down-regulation of substrate phosphorylation in the case of WbCDPK.

Non-restrictive, porous, external stents inhibit neointima formation in porcine vein grafts. Since the mechanisms underlying these effects are unknown we investigated the impact of this external stent on factors known to inhibit vascular smooth muscle cell proliferation: prostacyclin (PGI 2 ), nitric oxide (NO), cAMP and cGMP formation in different regions of stented and unstented porcine vein grafts. Paired stented and unstented saphenous vein -carotid artery interposition grafting was carried out in Landrace pigs. One month after surgery, the vessels were excised and the formation of PGI 2 , cAMP and cGMP determined using radioimmunoassay and nitric oxide synthase (NOS) distribution studied using autoradiography and histochemistry. There were no significant differences between PGI 2 , cAMP and cGMP (nitroprusside-stimulated) formation in the medial/intimal regions of grafts of stented vein graft and ungrafted saphenous vein whereas all were significantly reduced in unstented vein graft. A23187-stimulated cGMP formation (mediated by NO release) and NOS content was significantly greater in the medial/intimal region of stented and unstented vein graft compared to ungrafted saphenous vein, indicating induction of endothelial NOS (eNOS) in both types of graft. This normalisation of the PGI 2 -cAMP axis and guanylyl cyclase activity in the medial/intimal region may contribute to the beneficial impact of the external stent on vein graft thickening. The increase in eNOS in both stented and unstented vein grafts mitigates against this isoform as playing a role in mediating the inhibitory effect of the stent on neointima formation. In the adventitia of both stented and unstented grafts there was an increase in PGI 2 , cAMP and cGMP formation compared to ungrafted saphenous vein, the production being greater in the stented compared to the unstented graft. In the adventitia of stented veini grafts, NOS, detected with NAPDH diaphorase staining, was associated with microvessels as well as with inflammatory cells. Taken together, these data are suggestive of a role for PGI 2 and NO in promoting microangiogenesis in the adventitia of stented vein grafts which may in turn minimize graft hypoxia, an established contributory factor to neointima formation.