Modulation Of Cyclic Nucleotides Research Articles

Metabolic co-dependence of the oocyte and cumulus cells: essential role in determining oocyte developmental competence.

Within the antral follicle, the oocyte is reliant on metabolic support from its surrounding somatic cells. Metabolism plays a critical role in oocyte developmental competence (oocyte quality). In the last decade, there has been significant progress in understanding the metabolism of the cumulus-oocyte complex (COC) during its final stages of growth and maturation in the follicle. Certain metabolic conditions (e.g. obesity) or ART (e.g. IVM) perturb COC metabolism, providing insights into metabolic regulation of oocyte quality. This review provides an update on the progress made in our understanding of COC metabolism, and the metabolic conditions that influence both meiotic and developmental competence of the oocyte. The PubMed database was used to search for peer-reviewed original and review articles. Searches were performed adopting the main terms 'oocyte metabolism', 'cumulus cell metabolism', 'oocyte maturation', 'oocyte mitochondria', 'oocyte metabolism', 'oocyte developmental competence' and 'oocyte IVM'. Metabolism is a major determinant of oocyte quality. Glucose is an essential requirement for both meiotic and cytoplasmic maturation of the COC. Glucose is the driver of cumulus cell metabolism and is essential for energy production, extracellular matrix formation and supply of pyruvate to the oocyte for ATP production. Mitochondria are the primary source of ATP production within the oocyte. Recent advances in real-time live cell imaging reveal dynamic fluctuations in ATP demand throughout oocyte maturation. Cumulus cells have been shown to play a central role in maintaining adequate oocyte ATP levels by providing metabolic support through gap junctional communication. New insights have highlighted the importance of oocyte lipid metabolism for oocyte oxidative phosphorylation for ATP production, meiotic progression and developmental competence. Within the last decade, several new strategies for improving the developmental competence of oocytes undergoing IVM have emerged, including modulation of cyclic nucleotides, the addition of precursors for the antioxidant glutathione or endogenous maturation mediators such as epidermal growth factor-like peptides and growth differentiation factor 9/bone morphogenetic protein 15. These IVM additives positively alter COC metabolic endpoints commonly associated with oocyte competence. There remain significant challenges in the study of COC metabolism. Owing to the paucity in non-invasive or in situ techniques to assess metabolism, most work to date has used in vitro or ex vivo models. Additionally, the difficulty of measuring oocyte and cumulus cell metabolism separately while still in a complex has led to the frequent use of denuded oocytes, the results from which should be interpreted with caution since the oocyte and cumulus cell compartments are metabolically interdependent, and oocytes do not naturally exist in a naked state until after fertilization. There are emerging tools, including live fluorescence imaging and photonics probes, which may provide ways to measure the dynamic nature of metabolism in a single oocyte, potentially while in situ. There is an association between oocyte metabolism and oocyte developmental competence. Advancing our understanding of basic cellular and biochemical mechanisms regulating oocyte metabolism may identify new avenues to augment oocyte quality and assess developmental potential in assisted reproduction.

Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti‐platelet therapy

BackgroundEndothelium‐derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO‐sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. ObjectiveWe investigated using GC‐activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti‐thrombotic effect of both drugs. MethodsIn vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride‐induced arterial thrombosis model were also performed. ResultsThe GC‐activator BAY‐70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra‐platelet cyclic nucleotide levels. Furthermore, mice administered sub‐maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. ConclusionsUsing in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti‐platelet effects without altering blood flow. Therefore, modulation of intra‐platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti‐thrombotic regimen while minimizing vasodilator side effects.

Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos.

Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.

Structure of a eukaryotic cyclic-nucleotide-gated channel

Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-Å-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.

Structure of the SthK carboxy-terminal region reveals a gating mechanism for cyclic nucleotide-modulated ion channels.

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.

Prematuration with cyclic adenosine monophosphate modulators alters cumulus cell and oocyte metabolism and enhances developmental competence of in vitro-matured mouse oocytes.

Oocyte in vitro maturation (IVM) is an important assisted reproductive technology and research tool. The adoption of IVM into routine clinical practice has been hindered by its significantly lower success rates compared to conventional in vitro fertilization. Cyclic AMP (cAMP) modulation and follicle-stimulating hormone (FSH), independently, have long been known to improve IVM oocyte developmental competence. This study comprehensively examined the effects of FSH and cAMP/cGMP modulation, alone and in combination, on IVM oocyte metabolism and developmental outcomes. Mouse cumulus-oocyte complexes (COCs) were subjected to a 1 h prematuration phase ± the cAMP modulator forskolin and cAMP/cGMP modulator 3-isobutyl-1-methylxanthine followed by IVM ± FSH. Prematuration with these cyclic nucleotide modulators or IVM with FSH significantly improved oocyte developmental competence and reduced spindle abnormalities compared to spontaneous IVM (no treatment); however, these two treatments in combination endowed even greater developmental competence (improved subsequent blastocyst rates and quality; P < 0.05), albeit blastocyst yield and quality remained significantly lower than that of oocytes matured in vivo. A significant additive effect of combined IVM treatments was evident as increased COC lactate production and oxygen consumption and enhanced oocyte oxidative metabolism, ATP production, ATP:ADP ratio, and glutathione levels (P < 0.05). Nevertheless, IVM increased reactive oxygen species production, particularly as a consequence of FSH addition, relative to in vivo matured oocytes. In conclusion, improvements in the embryo yield following IVM is associated with increased COC oxygen consumption and oocyte oxidative metabolism, but these remain metabolically and developmentally less competent relative to in vivo derived oocytes.

Absence of Direct Cyclic Nucleotide Modulation of mEAG1 and hERG1 Channels Revealed with Fluorescence and Electrophysiological Methods

Similar to CNG and HCN channels, EAG and ERG channels contain a cyclic nucleotide binding domain (CNBD) in their C terminus. While cyclic nucleotides have been shown to facilitate opening of CNG and HCN channels, their effect on EAG and ERG channels is less clear. Here we explored cyclic nucleotide binding and modulation of mEAG1 and hERG1 channels with fluorescence and electrophysiology. Binding of cyclic nucleotides to the isolated CNBD of mEAG1 and hERG1 channels was examined with two independent fluorescence-based methods: changes in tryptophan fluorescence and fluorescence of an analog of cAMP, 8-NBD-cAMP. As a positive control for cyclic nucleotide binding we used changes in the fluorescence of the isolated CNBD of mHCN2 channels. Our results indicated that cyclic nucleotides do not bind to the isolated CNBD domain of mEAG1 channels and bind with low affinity (K(d) > or = 51 microm) to the isolated CNBD of hERG1 channels. Consistent with the results on the isolated CNBD, application of cyclic nucleotides to inside-out patches did not affect currents recorded from mEAG1 channels. Surprisingly, despite its low affinity binding to the isolated CNBD, cAMP also had no effect on currents from hERG1 channels even at high concentrations. Our results indicate that cyclic nucleotides do not directly modulate mEAG1 and hERG1 channels. Further studies are necessary to determine if the CNBD in the EAG family of K(+) channels might harbor a binding site for a ligand yet to be uncovered.

Distinct spatio-temporal Ca2+ signaling elicited by integrin α2β1 and glycoprotein VI under flow

AbstractWe studied how integrin α2β1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca2+ concentration ([Ca2+]i) of FLUO 3-AM–labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca2+]i oscillations. Rapid α-like peaks were unaffected by the membrane-impermeable Ca2+ chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting γ-like peaks were always preceded by at least one α-like peak and abolished by intracellular or extracellular Ca2+ chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca2+ responses. Human or mouse platelets lacking GPVI function exhibited α-like but not γ-like Ca2+ peaks, whereas those lacking α2β1 showed markedly reduced to absent α-like and no γ-like Ca2+ peaks. Specific α2β1 ligation induced α-like but not γ-like peaks. Thus, α2β1 may generate Ca2+ signals that are reinforced by GPVI and required for subsequent longer-lasting Ca2+ oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of α2β1 in response to collagen stimulation.

Differential effects of rolipram on chronic subcutaneous inflammatory angiogenesis and on peritoneal adhesion in mice

The specific PDE4 inhibitor (rolipram) has been shown to attenuate excessive accumulation/activation of inflammatory cells and fibroblasts and cytokine production in several pathological conditions through cyclic nucleotide modulation. Here, using the murine sponge model to induce chronic subcutaneous inflammatory response and to elicit the formation of intraperitoneal adhesions we explored the hypothesis that rolipram would exert beneficial effects on decreasing key components of both processes (inflammatory cell recruitment, angiogenesis, and deposition of extracellular matrix component). Two doses of rolipram (0.2 or 2 mg/kg/day) were administered orally for 7 days in groups of mice bearing either subcutaneous or intraperitoneal polyether–polyurethane implants. Rolipram was effective in inhibiting angiogenesis as assessed by hemoglobin content and VEGF levels in subcutaneous implants (about 40% with both doses) but failed to exert this activity in intraperitoneal implants. Conversely, accumulation of neutrophils and macrophages determined by measuring myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities intraimplant, respectively, was attenuated only in intraperitoneal implants by the treatment. Levels of TNF-α and MCP-1 were also determined and rolipram at both doses decreased the production of both cytokines in intraperitoneal implants. The levels of MCP-1 in the subcutaneous implants were not affected by the treatment. Fibrosis was evaluated by determining the amount of collagen and production of TGF-β1 intraimplant. Both parameters were attenuated by rolipram. These results have shown differential sensitivity of proliferating tissues to PDE4 inhibitor indicating that this agent may be used to target inflammatory angiogenesis selectively.

Cilostazol and pentoxifylline decrease angiogenesis, inflammation, and fibrosis in sponge-induced intraperitoneal adhesion in mice

Aims Adhesion formation following abdominal intervention is an abnormal peritoneal healing process. Our aim was to investigate the effects of controlling adhesion development by inhibiting its key components (angiogenesis, inflammation and fibrosis) using phosphodiesterase (PDE) inhibitors. Main methods Two PDE inhibitors including cilostazol a PDE3 inhibitor (40 and 400 mg/kg), and pentoxifylline (PTX), a PDE 1–5 inhibitor (50 and 500 mg/kg) were used for a period of 7 days to inhibit angiogenesis, inflammation, and fibrosis in a murine model of sponge-induced peritoneal adhesion. Angiogenesis was assessed by hemoglobin content, vascular endothelial growth factor (VEGF) levels, and morphometric analysis. Accumulation of neutrophils and macrophages was determined by measuring myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities, respectively. Levels of TNF-α were also determined. Fibrosis was assessed by determining the amount of collagen in the implant; TGF-β1 levels in the implant were also measured. Key findings Our results show that the treatments attenuated the main components of the adhesion tissue by reducing the amount of fibrovascular tissue that infiltrated the sponge matrix (wet weight). Hemoglobin content and VEGF levels were also decreased by approximately 40%. Neutrophil accumulation was unaffected by the compounds. However, NAG activity was reduced by pentoxifylline, but not by cilostazol. These compounds also decreased the levels of the pro-inflammatory and pro-fibrogenic cytokines TNF-α and TGF-β1, respectively, and collagen synthesis. Significance Our results suggest that cilostazol and PTX decreased the development of peritoneal adhesions in the model, which might be associated with cyclic nucleotide modulation. Therapies to intervene in these pathways may be beneficial for the prevention of these lesions.

Modulation of PMN-endothelial cells interactions by cyclic nucleotides.

Polymorphonuclear leukocytes (PMN) interact with endothelial cells (EC) under normal and diseased conditions. Common Mechanisms exist regulating PMN-EC interactions in the systemic and pulmonary circulations and adhesion molecules play significant roles in both circulations, however there are important differences. Alterations in PMN deformability appear to be important in the pulmonary circulation because of the unique geometric and hydrodynamic conditions that exist in the pulmonary microvasculature. PMN work as the host's first line of defence against invading pathogens. Under certain circumstances, however, dysregulation of PMN-EC interactions may contribute to local or global tissue injury in diseases such as acute respiratory syndrome and multiple organ failure syndrome. Therefore, a thorough understanding of the regulation of PMN-EC interactions is important to understand the pathogenesis of this type of tissue injury, and modulation of PMN-EC interactions could be applicable to prevent or treat injury. cGMP and cAMP are cyclic nucleotides that work as second messengers and control numerous functions in PMN. This review covers the modulation of PMN-EC interactions with cGMP and cAMP. Recent studies have shown that both cGMP and cAMP have inhibitory effects on events such as rolling, adhesion, migration and deformability change of PMN that are essential to PMN-EC interactions. Therefore, it is expected that the modulation of cyclic nucleotides is applicable for the treatment not only of local inflammatory diseases such as asthma but also of global tissue injury such as acute respiratory distress syndrome.

Regional modulation of cyclic nucleotides by endothelin-1 in rat pulmonary arteries: direct activation of G(i)2-protein in the main pulmonary artery.

The ability of endothelin-1 (ET-1) to modulate the cyclic nucleotides, guanosine 3' 5' cyclic monophosphate (cyclic GMP) and adenosine 3' 5' cyclic monophosphate (cyclic AMP) was assessed in the main elastic pulmonary elastic artery (4 - 5 mm i.d.) and the small muscular pulmonary arteries (150 - 200 micrometer i.d.) of the rat. ET-1 caused an increase in cyclic GMP in the larger vessels but had no effect in the smaller arteries. The increase in cyclic GMP was not dependent on an intact endothelium and was inhibited by the ET(A)-receptor antagonist FR139137 (1 microM). ET-1 caused a decrease in cyclic AMP in the main pulmonary arteries, an effect that was partially blocked by FR139317 but not influenced by the ET(B)-receptor antagonist BQ-788 (1 microM) or removal of the vascular endothelium. In contrast, ET-1 caused an increase in cyclic AMP in the small vessels, an effect that was blocked by BQ-788 but unaffected by FR139317. In the main pulmonary arteries, ET-1 caused enhanced incorporation of radiolabelled ADP-ribose by cholera toxin into G(i)2 in the main pulmonary artery, an indicator of its receptor-mediated activation. In summary, we have shown that in the small muscular pulmonary artery of the rat, (where ET(B) mediated vasoconstriction prevails), there is an ET(B)-mediated increase in cyclic AMP with no net effect on cyclic GMP levels. In the large arteries, (where vasoconstriction is mediated via the ET(A) receptor), there is an ET(A)-mediated increase in cyclic GMP (endothelium independent) and an ET(A)-mediated (endothelium independent) decrease in cyclic AMP.

Involvement of cAMP and protein kinase C in cytomegalovirus enhancement of human immunodeficiency virus replication.

Cytomegalovirus (CMV) infection constitutes a serious threat to patients with acquired immune deficiency syndrome. Recently we reported that human immunodeficiency virus (HIV) infection of CD4+ cells was associated with sustained elevation of cellular levels of cAMP. Moreover, cyclic nucleotide modulators enhanced HIV replication by increasing intracellular levels of cAMP. In this study, the effect of CMV on HIV replication in CMV/HIV mixed infection and its relationship to cAMP were examined. MT-4 cells, CMV strain AD169, and HIV strain IIIB were used. Optimal enhancement (4.4-fold increase) of HIV replication was observed when MT-4 cells were infected with CMV at Day 0 followed by HIV on Day 4 after infection, as determined by reverse transcriptase activity on Day 11 after infection. cAMP (measured by radioimmunoassay) levels in cells infected with CMV alone, HIV alone, or CMV/HIV together were 2-, 3-, and 5-fold above untreated cells, respectively. CMV also enhanced the replication of UV-irradiated HIV 4-fold and this was associated with a 2-fold increase in cAMP as well. Moreover, UV-irradiated CMV enhanced HIV replication 8.8-fold. The same dose of viable and UV-irradiated CMV used in the above experiments increased protein kinase C activity in these cells 3.0- and 8.0-fold, respectively. These findings might suggest that cAMP and protein kinase C are involved in CMV enhancement of HIV replication. These findings may have relevance to the identification of novel target sites for development of antiviral therapeutics.

Effects of pharmacologic modulation of cyclic nucleotides on the respiratory burst of human neutrophils

Effects of pharmacologic modulation of cyclic nucleotides on the respiratory burst of human neutrophils

Differential Effect of Cyclic Nucleotide Modulators on HIV Replication

Differential Effect of Cyclic Nucleotide Modulators on HIV Replication

Lithium modulation of cortical cyclic nucleotides: evidence for the Yin-Yang hypothesis

Rats were subjected to chronic treatment with lithium chloride (0.2–0.3%) over a period of 3 weeks. The activity of cortical phosphodiesterase (EC 3.1.4.17) was determined simultaneously with cyclic AMP and cyclic GMP content and compared to control, untreated animals. Lithium, at therapeutic serum concentrations was found to suppress cyclic AMP levels with a concomitant increase in cyclic AMP-phosphodiesterase activity. A simultaneous two-fold increase in cyclic GMP was observed. Through the alteration of cortical cholinergic activity with physostigamine and the use of cyclic GMP as a cholinergic marker, we were able to demonstrate a novel cholinotropic property of lithium to stimulate synthesis of cyclic GMP. This effect appears to be linked, in a Yin-Yang mechanism, to the observed suppression of cyclic AMP induced by lithium through activation of cyclic AMP-phosphodiesterase.

Synergistic disaggregation of platelets by tissue-type plasminogen activator, prostaglandin E1, and nitroglycerin.

Endothelial cells produce at least three substances that can attenuate the platelet aggregation response: tissue-type plasminogen activator; the platelet inhibitory prostaglandins I2 and E1; and endothelium-derived relaxing factor, one form of which exhibits properties of nitric oxide. Since platelet aggregates formed in vivo are involved in the initiation of many clinically important occlusive vascular syndromes, we tested the hypothesis that these endothelial products act synergistically to disperse platelet aggregates. Our data reveal that tissue-type plasminogen activator, prostaglandin E1, and nitroglycerin (an organic nitrate activator of guanylate cyclase analogous to endothelium-derived relaxing factor) act synergistically to disaggregate platelets and do so in part by modulation of platelet cyclic nucleotides. These data suggest a potential mechanism by which the endothelium protects against the formation of platelet aggregates in vivo and offer a potential strategy for improving the efficacy of thrombolytic therapy.

Egg-laying hormone, serotonin, and cyclic nucleotide modulation of ionic currents in the identified motoneuron B16 of Aplysia

We have used the 2-electrode voltage-clamp technique to analyze the effects of the neuropeptide, egg-laying hormone (ELH), and the biogenic amine 5-HT on ionic currents in the buccal motoneuron B16 of Aplysia. When B16 is voltage-clamped near resting membrane potential, bath-applied ELH induces a prolonged inward shift in holding current. The ELH-induced inward current is not due to a decrease in the transient, the delayed, or the calcium-activated potassium currents. Current-voltage measurements, along with ion substitution and channel-blocking experiments, indicate that ELH primarily induces or increases a voltage-dependent slow inward current carried by sodium. Serotonin also causes a prolonged inward shift in holding current in B16. Like ELH, 5-HT induces or enhances the voltage-dependent inward current carried by sodium. In addition, 5-HT increases an inwardly rectifying potassium current, and, in some preparations, decreases an outward current that is activated when the cell is depolarized to -40 mV and above. None of the currents modulated by ELH or by 5-HT are affected by 200 microM ouabain or by reducing extracellular chloride concentration. Extracellular application of isobutylmethylxanthine (IBMX), forskolin, 8-bromo-cAMP and 8-bromo-cGMP, and intracellular injection of cAMP elicits the slow inward current carried by sodium. The inward current response to ELH is blocked by prior application of 8-bromo-cAMP, while, under these conditions, 5-HT continues to elicit the increase in inward-rectifier potassium current, but decreases the slow inward sodium current. Serotonin also reduces the slow inward sodium current when applied after ELH. These results suggest that the modulation of B16 by ELH may be mediated entirely by either cAMP or cGMP, while at least a portion of the response to 5-HT may involve an unidentified second messenger in addition to cyclic nucleotides.

Segmental synthesis and actions of prostaglandins along the nephron.

The sites of synthesis and action of prostaglandins (PGs) along the renal tubule are examined. We focused our attention on experiments performed on well-defined nephron segments, using direct quantitative measurements of prostaglandin synthesis by radio- or enzyme-immunoassay. On the other hand, we selected, among the described effects of PGs, those obtained on precisely defined tubular segments. Among PGs, PGE2 synthesis is largely predominant all along the tubule. Its main sites of synthesis are the medullary collecting tubule and, to a lesser extent, the cortical collecting tubule and the thin limb of Henle's loop. Synthesis of PGE2 is amplified approximately tenfold in the presence of an excess exogenous substrate, arachidonic acid, compared with values measured without addition of substrate. Other eicosanoids have roughly the same distribution along the tubule as PGE2. Their rate of synthesis is, however, much less than that of PGE2, approximately 20-fold lower for PGF2 alpha and 6-keto-PGF1 alpha, and 100-fold lower for thromboxane B2 (TxB2). This contrasts with glomerular PG synthesis, where the difference between the production of PGE2 and other eicosanoids is much less marked. Most studies agree that antidiuretic hormone (ADH) and kinins augment PGE2 synthesis, whereas corticosteroids decrease it, at least in the collecting tubule. Direct effects of PGE2 have been described mainly in the medullary thick ascending limb and collecting tubule. They generally consist of a decrease in transepithelial potential difference and reabsorptive rates of water and solutes, in particular sodium and chloride. However, whatever the solute or tubular segment concerned, some studies failed to find such effects. The bulk of evidence suggests that ADH and PGs interact in kidney tubular cells. It is generally accepted that PGs antagonize the hydrosmotic effects of ADH in the collecting tubule. The mechanisms underlying these complex interactions are still under discussion: they probably involve several types of receptors and pathways for ADH action, which intervene in the modulation of both PG synthesis and cyclic nucleotides, and several types of PG receptors, either stimulatory or inhibitory to adenylate cyclase.

Ca 2+ responses in cytomegalovirus-infected fibroblasts of human origin

Cytomegalovirus (CMV) infection of fibroblasts of human origin is associated with a cascade of cellular responses (rounding, “contraction,” ”relaxation,” and enlargement). Since in other systems these cellular responses are regulated by intracellular free Ca 2+ activity ([Ca 2+] i), we measured intracellular Ca 2+ responses to CMV infection. At relatively high multiplicities of infection (m.o.i), an influx of Ca 2+ was observed within the first hour after CMV infection (p.i.) at which time it was at its maximum rate. Both the time of occurrence and the magnitude of this Ca 2+ influx were dependent on the calculated input m.o.i. In CMV-infected cells, [Ca 2+] i rose gradually from 80 n M at 0 hr to 174 n M at 48 hr p.i. (about 2.7 times the [Ca 2+] i found in mock-infected cells at this time). At 8 and 12 hr p.i. CMV-infected cells consistently contained a somewhat greater level of 45Ca 2+ than mock-infected cells, despite the fact that there was only a small increase in [Ca 2+] i between CMV and mock-infected cells in the same period. This observation suggests that there may be significant amounts of Ca 2+ taken up into intracellular stores. This Ca 2+ in intracellular stores may, at later times after infection, contribute to the increase in [Ca 2+] i observed from 12 to 48 hr p.i. Ca 2+ influx blockers, such as nifedipine and verapamil, inhibited the rise in [Ca 2+] i. The increase in [Ca 2+] i in response to CMV infection was shown to (1) be dependent on the m.o.i., (2) require infectious virus, and (3) occur under conditions consistent with the expression of immediate-early CMV genes. The capability of inducing such Ca 2+ responses was conserved among three laboratory strains of CMV. The CMV-induced Ca 2+ responses may be related not only to the development of CMV cytopathology, but also to the replication of CMV, since in other studies cyclic nucleotide modulators and Ca 2+ influx blockers were found to inhibit the replication of CMV.