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PubMed1984

Developmental regulation of the cyclic-nucleotide-phosphodiesterase mRNA of Dictyostelium discoideum. Analysis by cell-free translation and immunoprecipitation.

Extracellular cyclic-nucleotide phosphodiesterase of Dictyostelium discoideum has previously been purified and characterized [Orlow et al. (1981) J. Biol. Chem. 256, 7620-7627]. Antisera have been raised against the purified enzyme. Following cell-free translation of RNA extracted from cells at various stages of development and immunoprecipitation with anti-phosphodiesterase serum, cAMP phosphodiesterase synthesized in vitro and labeled with L-[35S]methionine can be detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. The cell-free translation product is an Mr-48 000 polypeptide and can be immunoprecipitated with antiserum raised against active Mr-50 000 cAMP phosphodiesterase or antiserum raised against heat-denatured cAMP phosphodiesterase. Purified native cAMP phosphodiesterase blocks immunoprecipitation of the cAMP-phosphodiesterase polypeptide synthesized in vitro. A detectable level of cAMP-phosphodiesterase mRNA is present in axenically grown cells. After starvation of the cells in phosphate buffer for 1 h an increase of translatable cAMP-phosphodiesterase mRNA occurs, followed by a decrease and another increase. When cells are starved in the presence of the slowly hydrolyzed cAMP analogue, adenosine 3',5'-thiophosphate, the level of translatable cAMP-phosphodiesterase mRNA increases about tenfold and does not show a temporary decline. A maximum of 0.015% of the total acid-insoluble radioactivity is incorporated into the Mr-48 000 cAMP-phosphodiesterase polypeptide.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Growth factor-like effects mediated by muscarinic receptors in PC12M1 cells.

Rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors (PC12M1) undergo morphological changes when stimulated by muscarinic agonists. These changes, which include the outgrowth of neurite-like processes, are blocked by the muscarinic antagonist atropine and are not observed in PC12 cells. The observed morphological changes, which are independent of RNA and protein synthesis, are blocked by the methylation inhibitor 5'-deoxy-5'-methylthioadenosine, suggesting that methylation plays a role in this process. Analysis of cyclic AMP accumulation and phosphoinositide turnover reveals that both processes are enhanced on activation by muscarinic agonist. Our data suggest, however, that the muscarinic-dependent neurite-like outgrowth processes are not mediated by cyclic AMP, Ca2+, or protein kinase C pathways. The muscarinic-dependent neurite outgrowth effect is enhanced by nerve growth factor, with a resulting increase in both the number of neurite-extending cells and the length of the neurite. In addition, activation of muscarinic receptors in PC12M1 cells stimulates the induction of marker genes for neuronal differentiation. Muscarinic receptors may therefore mediate growth factor-like effects in these cells.

Adenylyl Cyclases↗
PubMed2006

Expressing and responding to pain and stoicism in home-care nurse-patient interactions.

Addressing pain and suffering are critical issues for home-care nurses. Pain is frequently experienced by people living at home with chronic illnesses, as well as acutely ill people discharged early from hospitals. The purpose of this qualitative ethology study was to explore and describe the interactions and experiences expressing and responding to pain in home-care nurse-patient interactions. A qualitative video ethology design was appropriate to inductively describe micropatterns of communicative behaviour in natural settings. Ten home-care nurse-patient dyads were videotaped over multiple visits. Data consisted of 31 videotaped visits (over 19 hours) and accompanying participant interviews. Recursive and cyclical qualitative analysis was used to describe the patterns of communication with which stoicism or endurance of pain and suffering was supported or challenged. Dominant patterns of interaction were concerned with: negotiating appropriate forms of stoicism; negotiating ways to express, understand and measure pain and suffering; and dealing with intractable or inflicted pain. The methodology of this study allows an in-depth view of typical nurse-patient interactions. It explores the subtle communicative expertise of nurses by investigating the types of communication used in nursing encounters and by explicating behavioural patterns of expressing and responding to suffering. Observational research of interaction-as-it-occurs must continue to better understand how nurses and patients co-construct personal identities of suffering and stoicism.

Adaptation, Psychological↗
PubMed1974

Analysis of the role of cyclic adenosine 3',5'-monophosphate in catecholamine release.

1. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels and catecholamine release were measured in cat adrenal glands perfused in situ with Locke solution.2. Cyclic AMP was present in the medulla in an amount which represented approximately one fifth of that present in the cortex.3. Perfusion with acetylcholine (ACh) or nicotine increased cyclic AMP both in the intact adrenal and in the perfusate. The time course of the changes in tissue cyclic AMP during stimulation was out of phase with the time course of catecholamine release. Maximal increases in cyclic AMP were not manifest until after 8 min of exposure to the secretogogue, whereas maximal rates of secretion occurred during the first minute.4. Theophylline (0.5 mM) increased basal and stimulated adrenal cyclic AMP levels, but did not potentiate the secretory response to ACh or nicotine.5. Perfusion with cyclic AMP or its dibutyryl derivative (0.2-4 mM) failed to effect a consistent or significant increase in the rate of catecholamine release and was unable to potentiate the secretory response to a submaximal concentration of ACh or calcium.6. The results suggest that, unlike calcium, cyclic AMP is not a direct mediator of medullary secretion.

Acetylcholine↗
PubMed2003

GAF domains: cyclic nucleotides come full circle.

Cyclic nucleotides are ubiquitous signaling molecules that are present in life forms ranging from bacteria to yeast to humans. In higher eukaryotes, conserved molecular machinery processes signals through both cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP). Hurley describes how one widespread cyclic nucleotide recognition module, the GAF (cyclic GMP, adenylyl cyclase, FhlA) domain, formerly thought to bind only cyclic GMP, has now been found to bind cyclic AMP as well. Recent structural analysis of a cyclic GMP-binding GAF domain shows how conserved elements among the cyclic nucleotide-binding subgroup of GAF domains recognize the common chemical moieties in the two compounds.

Animals↗
PubMed2004

Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets.

Nutrients that induce biphasic insulin release, such as glucose and leucine, provide acetyl-CoA and anaplerotic input in the beta-cell. The first phase of release requires increased ATP production leading to increased intracellular Ca(2+) concentration ([Ca(2+)](i)). The second phase requires increased [Ca(2+)](i) and anaplerosis. There is strong evidence to indicate that the second phase is due to augmentation of Ca(2+)-stimulated release via the K(ATP) channel-independent pathway. To test whether the phenomenon of time-dependent potentiation (TDP) has similar properties to the ATP-sensitive K(+) channel-independent pathway, we monitored the ability of different agents that provide acetyl-CoA and anaplerotic input or both of these inputs to induce TDP. The results show that anaplerotic input is sufficient to induce TDP. Interestingly, among the agents tested, the nonsecretagogue glutamine, the nonhydrolyzable analog of leucine aminobicyclo[2.2.1]heptane-2-carboxylic acid, and succinic acid methyl ester all induced TDP, and all significantly increased alpha-ketoglutarate levels in the islets. In conclusion, anaplerosis that enhances the supply and utilization of alpha-ketoglutarate in the tricarboxylic acid cycle appears to play an essential role in the generation of TDP.

Acetyl Coenzyme A↗
PubMedDate not supplied

Evidence for atrial natriuretic factor induced natriuretic peptide receptor subtype switching in rat proximal tubular cells during culture.

Culture and natriuretic peptide dependent changes in the expression of the natriuretic peptides atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and the natriuretic peptide receptors A, B, and C in primary cultures of rat proximal tubular cells were demonstrated using polymerase chain reaction analysis and cyclic guanosine monophosphate response to ANF and CNP. Freshly isolated cells expressed mRNA coding for the natriuretic peptide receptor C only, with no expression of the natriuretic peptides or the natriuretic peptide receptors A or B. At confluence natriuretic peptide receptor C expression was lost, while mRNA transcripts for both ANF and BNP and the A and B receptors became apparent. The appearance of mRNA transcripts for the natriuretic peptide receptors A and B during cell growth correspond with a significant increase in the cyclic guanosine monophosphate response to both ANF and CNP, confirming the presence of functionally active guanylate cyclase linked A and B natriuretic peptide receptors. The observed changes in peptide receptor expression during culture were preceded by changes in natriuretic peptide mRNA expression, suggesting the possibility that natriuretic peptide receptor subtype switching may be under the control of endogenous peptide release. Incubation of freshly isolated proximal tubular cells with ANF, BNP, or CNP for 3 h induced similar changes in receptor expression. Incubation with ANF induced expression of the natriuretic peptide receptor B and CNP while inhibiting natriuretic peptide receptor C. Incubation with BNP induced expression of the natriuretic peptide receptor B and CNP. Incubation with CNP induced expression of the natriuretic peptide receptors A and B and CNP. These results suggest that primary cultures of rat proximal tubular cells may experience natriuretic peptide and natriuretic peptide receptor subtype switching as they approach confluence under the control of endogenously expressed natriuretic peptides.

Animals↗
PubMedDate not supplied

Comparison of initial fixation strength between biodegradable and metallic interference screws and a press-fit fixation technique in a porcine model.

The objective of this study was to evaluate the initial fixation strength of a biodegradable interference screw compared with press-fit fixation and a titanium interference screw in anterior cruciate ligament reconstruction using a bone-patellar tendon-bone graft. Porcine lower limbs were used. The specimens underwent 500 loading cycles between 60 and 250 N. This corresponds to loads in the graft during aggressive rehabilitation. Thereafter, intact specimens were loaded to failure. Failure mode was defined by visual analysis. Under cyclic loads none of the interference screw fixations failed. In the press-fit group (angle between load axis and tunnel axis 80 degrees), five specimens failed. The mean maximal load to failure was 945 N (+/- 87) for the titanium screw, 797 N (+/- 60) for the biodegradable screw, and 708 N (+/- 211) for the five press-fit specimens that did not fail during cyclic loading. With respect to primary fixation strength, biodegradable screws are a reasonable alternative to titanium interference screws. The press-fit fixation did not provide a secure fixation in all cases. Five press-fit specimens failed under cyclic loads comparable with those seen under conditions of accelerated rehabilitation.

Absorbable Implants↗
PubMed1998

Effects of nebulized nitroprusside on pulmonary and systemic hemodynamics during pulmonary hypertension in piglets.

We tested the effects of nebulized nitroprusside (Neb-NP) on pulmonary and systemic hemodynamics during pulmonary hypertension induced by hypoxia or group B streptococci infusion in piglets. Twenty-three anesthetized and mechanically ventilated piglets received Neb-NP under four experimental conditions: 1) normoxia; 2) 15 and 60 min of pulmonary hypertension induced by hypoxia; 3) after pretreatment with dipyridamole; 4) pulmonary hypertension induced by infusion of group B streptococci. In addition, Neb-NP was contrasted to nebulization of tolazoline. During hypoxia-induced pulmonary hypertension, Neb-NP significantly reduced pulmonary artery pressure [PAP; -8.4+/-0.9 (SEM) mm Hg] and pulmonary vascular resistance (-25+/-2.1%) (both p < 0.001), whereas neither systemic arterial pressure nor cardiac output changed significantly. Selective pulmonary vasodilation began within 2 min of the onset of Neb-NP, and did not wane over 1 h. In contrast, within 5 min after Neb-NP was discontinued while hypoxia persisted, PAP rose significantly. Pretreatment with dipyridamole did not enhance the pulmonary vasodilation induced by Neb-NP, but did reduce systemic arterial pressure. Nebulized tolazoline did not reduce PAP significantly, but did lower systemic arterial pressure. Selective pulmonary vasodilation induced by Neb-NP was significantly smaller during group B streptococci-induced versus hypoxia-induced pulmonary hypertension. In sum, Neb-NP produced prompt, significant, selective reduction of PAP in piglets with pulmonary hypertension. Cautious extrapolation of these findings to selected clinical conditions in human infants may be warranted.

Administration, Inhalation↗
PubMed2004

Ras and Gpa2 mediate one branch of a redundant glucose signaling pathway in yeast.

Addition of glucose to starved yeast cells elicits a dramatic restructuring of the transcriptional and metabolic state of the cell. While many components of the signaling network responsible for this response have been identified, a comprehensive view of this network is lacking. We have used global analysis of gene expression to assess the roles of the small GTP-binding proteins, Ras2 and Gpa2, in mediating the transcriptional response to glucose. We find that 90% of the transcriptional changes in the cell attendant on glucose addition are recapitulated by activation of Ras2 or Gpa2. In addition, we find that protein kinase A (PKA) mediates all of the Ras2 and Gpa2 transcriptional effects. However, we also find that most of the transcriptional effects of glucose addition to wild-type cells are retained in strains containing a PKA unresponsive to changes in cAMP levels. Thus, most glucose-responsive genes are regulated redundantly by a Ras/PKA-dependent pathway and by one or more PKA-independent pathways. Computational analysis extracted RRPE/PAC as the major response element for Ras and glucose regulation and revealed additional response elements mediating glucose and Ras regulation. These studies provide a paradigm for extracting the topology of signal transduction pathways from expression data.

Alleles↗
PubMed2005

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA.

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.

Amino Acid Sequence↗
PubMed2001

Characterization of uptake and compartmentalization of 3,5,3'-tri-iodothyronine in cultured neonatal rat cardiomyocytes.

The uptake of tri-iodothyronine (T(3)) in cultured neonatal rat cardiomyocytes was investigated and compared with the uptake of reverse T(3 )(rT(3)) and thyroxine (T(4)). Cellular compartmentalization of T(3) was studied by distinguishing T(3) activity associated with the plasma membrane from that in the cytosol or incorporated in the cell nucleus. T(3) and T(4) uptake displayed similar temperature dependencies which, in magnitude, differed from that of rT(3) uptake. T(3) uptake was Na(+ )independent, and sensitive to oligomycin and monodansylcadaverine (42-49% and 25% inhibition of 15-min cellular uptake respectively). Furthermore, T(3) uptake could be inhibited by tryptophan (20%) and tyrosine (12%), while 2-aminobicyclo[2,2,1]heptane-carboxylic acid had no effect. Co-incubation with tryptophan and oligomycin resulted in an additive inhibition of T(3) uptake (77%). We therefore conclude that (i) T(3) uptake is energy dependent, (ii) receptor-mediated endocytosis may be involved and (iii) the aromatic amino acid transport system T may play a role, while system L is not involved in T(3) transport in cardiomyocytes. Co-incubation with unlabeled iodothyronines showed that 3,3'-di-iodothyronine and T(3) itself were the most effective inhibitors of T(3) uptake (30% and 36% inhibition of 15-min cellular uptake respectively). At 15-min incubation time, 38% of the total cell-associated T(3) was present in the cytosol and nucleus, and 62% remained associated to the plasma membrane. Unidirectional uptake rates did not saturate over a free T(3) concentration range up to 3.9 microM. We have concluded that T(3) uptake in neonatal rat cardiomyocytes occurs by an energy- and temperature-dependent mechanism that may include endocytosis and amino acid transport system T, and is not sensitive to the Na(+) gradient. Elucidation of the molecular basis for the T(3) transporter is the subject of current investigation.

Amino Acids↗
PubMedDate not supplied

Memantine and ACPC affect conditioned place preference induced by cocaine in rats.

The influence of uncompetitive NMDA receptor antagonist, 1-amino-3,5--dimethyl-adamantane (memantine) and partial glycineB site agonist, 1-amino-cyclopropanecarboxylic acid (ACPC) on cocaine-induced conditioned place preference (CPP) were examined in male Wistar rats. After determination of initial preference, animals were conditioned with cocaine (5 mg/kg, ip) for 3 conditioning trials, alone or in combination with memantine (7.5 mg/kg, ip) or ACPC (50.0 mg/kg, ip). Memantine prevented acquisition and expression of the place preference produced by cocaine, while ACPC prevented only acquisition effect. Neither of the NMDA antagonists displayed any reinforcing properties by itself. Our current data suggest that the NMDA receptor complex may be involved in the rewarding effect of cocaine.

Amino Acids, Cyclic↗
PubMedDate not supplied

[Analysis of the level of cyclic adenosine-3',5'-monophosphate in structures of the 'informational' and 'motivational' system of the rat brain during active conditioning].

The content of cyclic adenosine-3',5'-monophosphate (cAMP) was studied in structures of the "motivational" and "infromational" systems of rat brain after the active avoidance conditioning procedure in rats. Three groups of animals were examined: naive rats, trained (conditioned) rats, and group of the active control presented with uncombined conditioned (light) and unconditioned (electric footshock) stimuli. The content of cAMP was determined in the frontal cortex, hippocampus, amygdala, and hypothalamus of both hemispheres immediately after the retrieval of conditioned reaction one day after conditioning. A significant increase in cAMP level was bilaterally observed in the hypothalamus in the group of active control, and in both hippocampi and the right frontal cortex in the conditioned animals. Positive correlations between the cAMP levels in symmetrical regions of the frontal cortex, amygdala, and hypothalamus were revealed in all the examined groups. Additionally, intra- and interhemispheric correlations were found in the active control and conditioned rats. Patterns of correlation were specific for each of these groups. The observed phenomenon is discussed in term of involvement of "informational" and "motivational" brain structures in the mechanisms of adaptive behavior.

Animals↗
PubMed2003

Atrial natriuretic peptide ameliorates hypoxic pulmonary vasoconstriction without influencing systemic circulation.

UNLABELLED: Hypoxic pulmonary vasoconstriction (HPV) is encountered during ascent to high altitude. Atrial natriuretic peptide (ANP) could be an option to treat HPV because of its natriuretic, diuretic, and vasodilatory properties. Data on effects of ANP on pulmonary and systemic circulation during HVP are conflicting, partly owing to anesthesia, surgical stress or uncontrolled dietary conditions. Therefore, ten conscious, chronically tracheotomized dogs were studied under standardized dietary conditions. The dogs were trained to breathe spontaneously at a ventilator circuit. PROTOCOL: 30min of normoxia [inspiratory oxygen fraction (F(i)O(2))=0.21] were followed by 30min of hypoxia without ANP infusion (Hypoxia I, F(i)O(2)=0.1). While maintaining hypoxia an intravenous infusion of atrial natriuretic peptide was started with 50ng x kg body wt(-1) x min(-1) for 30min (Hypoxia+ANP1=low dose), followed by 1000ng x kg body wt(-1) x min(-1) for 30min (Hypoxia+ANP2=high dose). Thereafter, ANP infusion was stopped and hypoxia maintained for a final 30min (Hypoxia II). Compared to normoxia, mean pulmonary arterial pressure (MPAP) (16+/-0.7 vs. 26+/-1.3mmHg) and pulmonary vascular resistance (PVR) (448+/-28 vs. 764+/-89dyn x s(-1) x cm(-5)) increased during Hypoxia I and decreased during Hypoxia+ANP 1 (MPAP 20+/-1mmHg, PVR 542+/-55dyn x s(-1) x cm(-5)) (P<0.05). The higher dose of ANP did not further decrease MPAP or PVR, but started to have a tendency to decrease mean arterial pressure and cardiac output. We conclude that low dose ANP is able to reduce HPV without affecting systemic circulation during acute hypoxia.

Angiotensin II↗
PubMedDate not supplied

[Electrochemical reduction of cytochrome P450s based on electrodes with immobilized substrates].

A new approach for the electrochemical reduction of cytochromes P450 (P450s, CYPs) with electrodes chemically modified with appropriate substrates of P450s ("reverse" electrodes) has been proposed. The method is based on the analysis of cyclic voltammograms, square wave voltammograms, amperograms and determination of such electrochemical characteristics as catalytic current and redox potential. The sensitivity of the proposed method is 0.2-1 nmol P450/electrode. The differences of maximal current and potentials in square wave voltammograms and catalytic current in amperometric measurements are more sensitive and reliable. Planar regime of screen-printed electrodes permits to use 20-60 microl of electrolyte volume. We investigated P450 2B4--benzphetamine or P450scc--cholesterol enzyme - substrate pairs. Electrochemical parameters of electrodes with nonspecific P450 substrate were differed from electrodes with appropriate substrates.

Aryl Hydrocarbon Hydroxylases↗