Systematic Quantification of GPCR/cAMP-Controlled Protein Kinase A Interactions.

The diffusible second messenger cyclic AMP (cAMP) originates from multiple G protein-coupled receptor (GPCR) cascades activating the intracellular key effector protein kinase A (PKA). Spatially and temporally restricted cAMP-fluxes are directly sensed by macromolecular PKA complexes. The consequences are alterations of molecular interactions, which lead to activation of compartmentalized PKA phosphotransferase activities, regulating a vast array of cellular functions. To decode cell-type and cell-compartment specific PKA functions, the spatio-temporal dynamics of small molecule:protein interactions, protein:protein interactions (PPIs), cAMP-mobilization, and phosphotransferase activities need to be determined directly in the appropriate cellular context. A collection of cell-based reporters has been developed to either visualize or quantitatively measure kinase activities or PKA complex formation/dissociation. In this review, we list a collection of unimolecular and bimolecular PKA biosensors, followed by the specification of the modular design of a Renilla luciferase based protein-fragment complementation assay (PCA) platform for measuring PKA network interactions. We discuss the application spectrum of the PCA reporter to identify, quantify, and dissect dynamic and transient PKA complexes downstream of specific GPCR activities. We specify the implementation of a PCA PKA platform to systematically quantify the concurrent involvement of receptor-cAMP signaling, post-translational modifications, and kinase subunit mutations/perturbations in PKA activation. The systematic quantification of transient PKA network interactions will contribute to a better understanding how GPCR-recognized input signals are streamlined through the compartmentalized and cAMP-interacting PKA signalosome.

Transgenic mouse model reveals an unsuspected role of the acetylcholine receptor in statin-induced neuromuscular adverse drug reactions.

High cholesterol levels are an established risk factor for cardiovascular disease (CVD), the world's leading cause of death. Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (statins) are prescribed to lower serum cholesterol levels and reduce the risk of CVD. Despite the success of statins, many patients abandon treatment owing to neuromuscular adverse drug reactions (ADRs). Genome-wide association studies have identified the single-nucleotide polymorphism (SNP) rs4149056 in the SLCO1B1 gene as being associated with an increased risk for statin-induced ADRs. By studying slow-channel syndrome transgenic mouse models, we determined that statins trigger ADRs in mice expressing the mutant allele of the rs137852808 SNP in the nicotinic acetylcholine receptor (nAChR) α-subunit gene CHRNA1. Mice expressing this allele show a remarkable contamination of end-plates with caveolin-1 and develop early signs of neuromuscular degeneration upon statin treatment. This study demonstrates that genes coding for nAChR subunits may contain variants associated with statin-induced ADRs.

Medical and surgical management of a dystocia because of foetopelvic disproportion in an African lioness (Panthera leo).

Medical and surgical management of a foetopelvic dystocia in an African lioness (Panthera leo) and the post-operative complications are reported. A caesarean section was performed to extract an oversize foetus blocked at the cervical canal; the lioness died 36 h after surgery. At necropsy, an abdominal effusion with sero-haemorrhagic fluid was observed, along with a fibrinopurulent exudate adhered to the serosal surfaces of the pelvic and abdominal cavities. In addition, the pelvic symphysis was not cartilaginous, but formed a firm and rigid joint between the pubis and ischium of each hip. The macroscopic and microscopic findings confirmed the presence of metritis, vaginitis and peritonitis. Dystocia may be caused by the premature ossification of the pelvic symphysis, reducing the dimensions of the pelvic cavity.

Mucor ramosissimus associated with feather loss in canaries (Serinus canarius).

Three canaries showing feather loss on legs, dorsum, neck, and head, and hyperkeratosis on the feet were sacrificed because of their poor corporal condition and submitted to the Unit of Histology and Anatomic Pathology at the Veterinary School of Las Palmas de Gran Canaria. Histologically, skin revealed pronounced epidermal and follicular infundibular hyperplasia associated with orthokeratotic hyperkeratosis. Numerous fungal spores were observed on the stratum corneum of the epidermis and within feather follicles, associated with destruction of the feathers. This fungus was identified as Mucor ramosissimus. To the best of authors' knowledge, this is the first report of dermatitis and feather loss associated with Mucor ramosissimus, not only in canaries but also in birds.

Immunohistochemical detection of interleukin-12 and interferon-gamma in pigs experimentally infected with Mycoplasma hyopneumoniae.

The expression of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) was examined immunohistochemically in the lungs of pigs aged 21 days infected experimentally with Mycoplasma hyopneumoniae (Mh). Ten pigs were inoculated intranasally with Mh and killed in pairs weekly from 7 to 35 days post-infection (dpi). Immunolabelling for IL-12 and IFN-gamma was usually associated with inflammation, particularly in macrophages and lymphocytes in the thickened alveolar septa and in the hyperplastic bronchus-associated lymphoid tissue (BALT). Cells positive for both cytokines were detected at 7 dpi, their numbers increasing at 14 and 21 dpi, and slightly decreasing thereafter. The results suggest that IL-12 and IFN-gamma play a role in pulmonary defence mechanisms against Mh infection.

Higher susceptibility of taurine-deficient rats to seizures induced by 4-aminopyridine.

The susceptibility of rats made deficient of taurine by treatment with guanidinoethane sulfonate (GES), to seizures induced by 4-aminopyridine was examined. Guanidinoethane sulfonate, at a concentration of 1% was administered to pregnant rats, in the drinking water 2-3 days prior to delivery and the treatment was continued during nursing. Pups were weaned to the same treatment until 6 weeks of age. This treatment decreased levels of taurine in the cerebral cortex by 70%. 4-Aminopyridine was injected intraperitoneally at doses ranging from 4-7 mg/kg. Taurine-deficient rats showed a greater susceptibility to seizures, as demonstrated by a lowered latency for clonic seizures, an increased incidence of tonic seizures and a higher postseizure mortality. These results suggest an involvement of endogenous taurine in nervous excitability.

Redox sites of NMDA receptors can modulate epileptiform activity in hippocampal slices from kainic acid-treated rats.

Using an animal model of temporal lobe epilepsy, the kainic acid lesioned rat hippocampus, we have evaluated the possibility of modulating glutamate N-methyl-D-aspartate (NMDA) receptor-dependent evoked epileptiform activity through the manipulation of NMDA receptor redox sites. Epileptiform activity was recorded extracellularly from hippocampal slices, in the stratum pyramidale of the CA1 area, and the effects of the oxidizing reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and the reducing agent Tris(2-carboxy ethyl)phosphine (TCEP) on these responses were quantified. Epileptiform activity was substantially reduced in the presence of DTNB but was fully reinstated with the application of TCEP. The effects of both drugs persisted even after wash. Epileptiform activity was totally abolished in the presence of the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid. These results suggest that epileptiform activity can be controlled by manipulation of the redox sites of NMDA receptors and raise the possibility of developing new anticonvulsant drugs which do not fully block NMDA receptor-mediated synaptic transmission.

Epileptiform activity but not synaptic plasticity is blocked by oxidation of NMDA receptors in a chronic model of temporal lobe epilepsy.

Simultaneous extracellular recordings were performed in stratum radiatum and stratum pyramidale of hippocampal slices 7 days following unilateral intracerebroventricular injections of kainic acid. In this ex vivo experimental model of human temporal lobe epilepsy, stimulation of the surviving commissural fibres in stratum radiatum produced graded epileptiform activity in the CA1 area. The oxidizing reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) acting at NMDA receptors redox sites decreases NMDA receptor-mediated responses by half and suppresses evoked epileptiform discharges. We have examined the effect of DTNB on NMDA-dependent bidirectional synaptic plasticity and EPSP/spike coupling. DTNB treatment did not prevent either long-term potentiation induced by tetanic stimulation or long-term depression induced by low frequency stimulation of field EPSPs. Application of DTNB alone did not induce EPSP/spike dissociation. However, both high and low frequency stimulations induced EPSP/spike potentiation indicating that neurons had a high probability to discharge in synchrony. These results suggest that oxidizing reagents may provide novel antiepileptic treatments since they decrease NMDA-dependent evoked epileptiform activity but do not interfere with either NMDA-dependent synaptic plasticity or the probability of synchronous discharge.

Epithelial and pancreatic choristoma in bovine lymph nodes.

Lymph nodes from 186 cows were evaluated as part of a bovine tuberculosis eradication programme. The mediastinal lymph nodes of 13 animals contained atypical structures. In 12 cases (6.45%) these consisted of multiple epithelial structures and, in one case, of pancreatic-like tissue. Immunohistochemistry (IHC) revealed that the epithelial structures were consistent with respiratory epithelium and with ectopic pancreatic tissue, respectively. To the best of our knowledge these are the first histological and immunohistochemical descriptions of epithelial and pancreatic choristomas in bovine lymph nodes.

Effects of the ionophores X537A and A23187 on GABA, glycine, and taurine release from chick retinal subcellular fractions.

The ionophore X537A at concentrations of 5--20 microM stimulated the release of [3H]GABA and [35S]taurine from retinal subcellular crude nuclear (P1) and crude synaptosomal (P2) fractions. The release of [3H]GABA increased 114% and 136% over control values in P1 and P2 fractions, respectively. The efflux of [35S]taurine from P1 was increased by 45% and that from P2 by 21%. X537A increased 45Ca2+ uptake in the P2 fraction but not in the P1 fraction. The effect of X537A on the amino acid release was not dependent on the presence of exogenous calcium. X537A did not affect [3H]GABA or [35S]taurine uptake by the retinal fractions. A23187 enhanced [3H]GABA release from P1 and P2 by 52% and 105%, respectively. The ionophore also increased [14C]glycine liberation in both P1 (35%) and P2 (50%) but failed to stimulate [35S]taurine release. A23187 produced a transient increase of 45Ca2+ uptake of 38% in P1 and 30% in P2. The effects of A23187 on the release of amino acids were calcium dependent. The amino acid uptake was not affected by the ionophore. These results are consistent with the suggested neurotransmitter role for GABA at the outer synaptic layer and for GABA and glycine at the inner synaptic layer of the retina. A neurotransmitter role for taurine is not supported by the present results.

Efflux of osmolyte amino acids during isovolumic regulation in hippocampal slices.

The efflux of potassium (K(+)) and amino acids from hippocampal slices was measured after sudden exposure to 10% (270 mOsm), 25% (225 mOsm) or 50% (150 mOsm) hyposmotic solutions or after gradual decrease (-2.5 mOsm/min) in external osmolarity. In slices suddenly exposed to 50% hyposmotic solutions, swelling was followed by partial (74%) cell volume recovery, suggesting regulatory volume decrease (RVD). With gradual hyposmotic changes, no increase in cell water content was observed even when the solution at the end of the experiment was 50% hyposmotic, showing the occurrence of isovolumic regulation (IVR). The gradual decrease in osmolarity elicited the efflux of (3)H-taurine with a threshold at -5 mOsm and D-[(3)H]aspartate (as marker for glutamate) and at -20 mOsm for [(3)H]GABA. The efflux rate of [(3)H]taurine was always notably higher than those of [(3)H]GABA and D-[(3)H]aspartate, with a maximal increase over the isosmotic efflux of about 7-fold for [(3)H]taurine and 3- and 2-fold for [(3)H]GABA and D-[(3)H]aspartate, respectively. The amino acid content in slices exposed to 50% hyposmotic solutions (abrupt change) during 20 min decreased by 50. 6% and 62.6% (gradual change). Taurine and glutamate showed the largest decrease. An enhancement in (86)Rb efflux and a corresponding decrease in K(+) tissue content was seen in association with RVD but not with IVR. These results demonstrate the contribution of amino acids to IVR and indicate their involvement in this mechanism of cell volume control.

Taurine distribution in different cat muscles as visualized by immunohistochemistry: changes with stimulus state.

The distribution of endogenous taurine in the cardiac pectinate muscle, the intestinal wall smooth muscle and in nine striated muscles of the cat was studied by immunohistochemistry. It was found that taurine-like immunoreactivity (TLI) is distributed homogeneously in the cardiac and smooth muscles selected where it is present in every single fibre. In contrast, TLI appeared unevenly distributed in eight out of nine skeletal muscles, the soleus being atypical. Under resting conditions, TLI is present in only a portion of the fibre population, the proportion being specific for each muscle. Under nerve stimulation the TLI negative/positive ratio increased, suggesting release activity, while an opposite trend occurred after denervation. Glutamate-like immunoreactivity (GLI), although present in every fibre, showed a stronger reaction in those fibres positive to taurine, suggesting parallel movements of both amino acids. Results are discussed in terms of two functional possibilities.

Effect of light deprivation on the ERG responses of taurine-deficient rats.

The decrease of taurine levels in the retina of taurine-depleted rats treated with guanidinoethane sulfonate (GES) reduced the amplitude of the a- and b-waves of the electroretinogram, registered as a function of the log relative intensity stimulus. The effect was observed either in rats exposed to a light-darkness cycle or to continuous darkness; this effect was somewhat more pronounced in retinas of rats exposed to light. Values obtained from the Naka-Rushton equation for the V-log I curve for the a-wave showed that the half-saturating intensity was unchanged but Vmax was decreased by GES treatment. Implicit times for the b-wave delayed whereas those for the a-wave were unaffected by the treatment.

Effect of guanidinoethane sulfonate on taurine uptake by rat retina.

Guanidinoethane sulfonate (GES) markedly decreased 3H-taurine accumulated in the retina by the high-affinity uptake process. The effect of GES was dose-dependent. Analysis of the kinetics of GES effect revealed that it is a competitive inhibitor. The uptake of taurine by GES was less affected in rat cerebral cortex slices, where the inhibition by 1 mM GES was only 28%. Taurine accumulation by tissues of rats treated with GES (0.1% and 1% in the drinking water) was found to be particularly decreased in the retina, although accumulation by heart and liver was also affected by the higher dose. Taurine uptake by cerebellum and cerebral cortex slices was unaffected by GES. Treatment of rats with GES is known to produce an alteration in the structure and function of the retina, but apparently not in other organs. We discuss whether the marked effect of GES on taurine transport by the retina is related to the deleterious effect of the inhibitor in this organ.

Minimal nephrotoxicity with cephalosporin-aminoglycoside combinations in patients with neoplastic disease.

Patients with cancer and suspected sepsis were treated in a prospective, randomized trial with one of four cephalosporin-aminoglycoside combinations: cephalothin and tobramycin; cephalothin and gentamicin; cefamandole and tobramycin; or cefamandole and gentamicin. Carbenicillin was added if the absolute granulocyte count was less than 1,000/mm3. Of 199 patients receiving 20 to more doses of an aminoglycoside and having serial determination of serum creatinines, nephrotoxicity developed in seven (3.5%) given any of the four combinations. There were no significant differences between patients receiving either cephalosporin or either aminoglycoside. Nephrotoxicity developed less frequently among children (2 or 125; 1.6%) than adults (5 of 74; 6.8%).

Light-stimulated release of taurine from retinas of kainic acid-treated chicks.

The light-stimulated release of [3H]taurine from chick retina was studied in chicks intraocularly injected with kainic acid (60 nmol). This treatment produced a loss of more than 80% of the inner nuclear and the inner synaptic layers, sparing the outer retinal layers. Concomitantly, the treatment produced a marked decrease of endogenous GABA and glycine but not of taurine. The activity of glutamate decarboxylase was also markedly decreased in the kainic acid-treated retinas. The release of [3H]taurine, either spontaneous or stimulated by light, was unaffected by the treatment. These results suggest that the light-stimulated efflux of taurine occurs from the retinal layers which are not affected by the kainic acid treatment.