Phosphoinositides as regulators in membrane traffic

Phosphorylated products of phosphatidylinositol play critical roles in the regulation of membrane traffic, in addition to their classical roles as second messengers in signal transduction at the cell surface. Growing evidence suggests that phosphorylation-dephosphorylation of the polar heads of phosphoinositides (polyphosphorylated inositol lipids) in specific intracellular locations signals either the recruitment or the activation of proteins essential for vesicular transport. Cross talk between phosphatidylinositol metabolites and guanosine triphosphatases is an important feature of these regulatory mechanisms.     

Mechanical and morphometrical changes in progressive bilateral pneumothorax and pleural effusion in normal rats

A variety of neurotransmitter receptor changes occur after a course of electroconvulsive seizures (ECS) in rats, including an increased density of adenosine A1 sites. Adenosine antagonism has been related to the proconvulsant action of methylxanthines such as caffeine. We determined tonic-clonic seizure duration in rats given ECS with caffeine (0-175 mg/kg, IP) after a course of one or six daily ECS. A single day of ECS did not affect the dose-dependent proconvulsant action of caffeine. After six daily ECS, the proconvulsant action of caffeine was reduced. After nine daily ECS, an A1 antagonist (8-cyclopentyl-1,3-dipropylxanthine) and an A2A antagonist (1-allyl-3,7-dimethyl-8-p-sulfophenylxanthine) showed reduced proconvulsant activity. The results suggest that the reduced proconvulsant action of caffeine after chronic ECS depends on adenosine antagonism.     

Regulation of phosphatidic acid phosphohydrolase by epidermal growth factor. Reduced association with the EGF receptor followed by increased association with protein kinase Cepsilon

An important component of receptor-mediated intracellular signal transduction is the generation of lipid second messengers. Lipid second messenger production is a complex process involving a variety of regulatory enzymes that control the intracellular response to the extracellular signal. Phosphatidic acid (PA) is generated in response to phospholipase D and can be converted to other lipid second messengers including diacylglycerol (DG) and lysophosphatidic acid. PA is converted to DG by PA phosphohydrolase (PAP). We report here that PAP activity can be detected in epidermal growth factor (EGF) receptor immunoprecipitates. Following treatment with EGF, there is a substantial reduction in the PAP activity that co-precipitates with the EGF receptor. The loss of EGF receptor-associated PAP activity occurs with a concomitant increase in PAP activity associated with the epsilon isoform of protein kinase C (PKC). The PAP activity associated with PKCepsilon was dependent upon the PKC co-factors phosphatidylserine and DG but was independent of the kinase activity of PKCepsilon. These data suggest a novel signaling mechanism for the regulation of lipid second messenger production and implicate PAP as an important regulatory component for lipid second messenger production in receptor-mediated intracellular signaling.     

Passive avoidance behavior in rats after electroconvulsive shock: Facilitative effect of response retardation.

In 3 experiments, a total of 178 male Wistar rats were trained in a one-trial passive avoidance task and then were submitted to electroconvulsive shock (ECS) or to sham ECS. 24 hrs later they were tested for retention, with the door opened either immediately or 30 sec after the beginning of the test. Ss initially forced to avoid for 30 sec continued to avoid for the entire test, but the others had the usual low step-through latencies seen with ECS-treated Ss. Activity measures for those Ss stepping through differentiated groups having received footshock from those not having footshock and ECS. A retest 5-10 min later showed "recovery" in the amnestic Ss and continued avoidance behavior for those that avoided on the first test. Results are taken as evidence that ECS effects are not on memory storage but on the capacity of the animal to organize information effectively and quickly in order to produce an adaptive response. (20 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Infectious gastroenterocolitides in children: an update on emerging pathogens

The recognition that bacterial infections induce signal transduction responses in infected epithelial cells also provides new avenues to consider as novel forms of therapy. For example, the chemokine interleukin-8, which attracts neutrophils to sites of mucosal infection, is produced by epithelial cells of gastric and intestinal origin in response to bacterial infection. Inhibitors of chemokine production or inhibition of the biologic effects of neutrophil chemoattractants have the potential to reduce both mucosal inflammatory responses and the attendant clinical sequelae. Eukaryotic cells also respond to infection with elevations in cytosolic second messengers, including inositol triphosphate (IP3) and calcium ([Ca2+]i). In intestinal epithelium, these second messengers can mediate the diarrheal response to infection. Calcium/calmodulin inhibitors may have a beneficial effect in treating those gastrointestinal infections mediated through changes in the level of cytosolic free calcium. DuPont and colleagues showed, for example, that oral therapy with zaldaride maleate relieves symptoms of disease and shortens the duration of diarrhea in travelers with ETEC-induced diarrhea. Evaluation of additional signal transduction responses to microbial infections should provide both new insights into the pathogenesis of gastrointestinal infectious diseases and novel approaches to consider for the prevention and therapy for these human illnesses.     

Hypothalamic regulation of pituitary FSH secretion

Temporal changes in plasma LH and FSH concentrations were monitored during the afternoon of proestrus in controls and in rats in which the spontaneous LH/FSH surges were blocked with Nembutal. These values were compared with those obtained following electrochemical stimulation (ECS) of either the medial preoptic area (MPOA) or the dorsal anterior hypothalamic area (DAHA) in similar Nembutal-blocked animals. Whereas MPOA-ECS (60 muA/60 sec) elicited a release of both FSH and LH, similar unilateral stimulation of the DAHA resulted in a pronounced increase in plasma FSH and only a slight elevation in plasma LH. Increasing the amount of DAHA tissue stimulated (100 muA/60 sec) caused a significantly greater release of FSH but not LH. Bilateral DAHA-ECS (60 muA/60 sec) failed to produce a greater release of FSH than that observed after unilateral 100 muA/60 sec ECS but resulted in increased concentrations of LH in plasma. Surgical separation of the MPOA from the DAHA, leaving the preopticotuberal fibers intact, did not alter the spontaneous temporal patterns of discharge of FSH or LH 19-21 days post-operatively, although peak LH concentrations were reduced. Further, unilateral ECS (60 muA/60 sec) of the MPOA in such preparations elicited a release of FSH and LH similar to that observed in intact MPOA-ECS rats. In contrast, unilateral DAHA ECS (60 muA/60 sec) in rats with transected hypothalami, caused no release of LH and an attenuated FSH discharge when compared with intact DAHA-ECS rats (peak valued 189 +/- 8 ng/ml vs 274 +/- 11 ng/ml). These studies suggest the existence of specific cell bodies in the DAHA which can cause selective release of FSH when activated. Coexisting with this system is that level of control which is believed to be responsible for the cyclic discharge of both FSH and LH of which the MPOA is a component part.     

Comparison of amnesias induced by electroconvulsive shock administered after training-trial footshock or noncontingent footshock in rats.

Used a 1-trial fear-conditioning paradigm in 2 experiments with 75 and 105 male Long-Evans rats, respectively. The amnesia pattern resulting from ECS delivered .5 sec after the training-trial footshock (FS/ECS) was compared with the amnesia resulting from an ECS delivered .5 sec after a noncontingent footshock (NCFS/ECS) administered 24 hrs after the training-trial footshock. FS/ECS produced a relatively strong and permanent amnesia, whereas NCFS/ECS resulted in a relatively weak and transient amnesia. Several possible explanations of these results are discussed, and it is suggested that both amnesias resulted from a disruption of memory-retrieval processes. (27 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Activation of phosphatidylinositol-specific phospholipase C in response to HDL3 and LDL is markedly reduced in cultured fibroblasts from Tangier patients

We compared HDL3- and LDL-induced signal transduction in normal and Tangier fibroblasts to elucidate whether impaired signal transduction responses to lipoproteins might contribute to disturbed cellular lipid and lipoprotein metabolism in Tangier disease, a rare autosomal disorder of cellular lipid and lipoprotein metabolism. In several cell types HDL and LDL activate a currently unknown isoform of phosphatidylinositol-specific phospholipase C (PI-PLC) that results in the generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Compared with normal fibroblasts, Tangier fibroblasts stimulated with HDL3 or LDL resulted in a significantly reduced accumulation of inositol phosphates and 1,2-diacylglycerol formation. Furthermore, in Tangier fibroblasts both lipoproteins failed to mobilize calcium from internal pools, and the cytosol-to-membrane redistribution of protein kinase C (in both the alpha and epsilon isoforms) was markedly reduced. Thus, the data indicate an impaired PI-PLC activation in response to lipoproteins in Tangier fibroblasts.     

Human CD5 signaling and constitutive phosphorylation of C-terminal serine residues by casein kinase II

CD5 is a lymphocyte surface glycoprotein with a long cytoplasmic domain suitable for phosphorylation and signal transduction, which is involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals. In this study, we use Jurkat T cell transfectants of CD5 cytoplasmic tail mutants to reveal phosphorylation sites relevant to signal transduction. Our results show that casein kinase II (CKII) is responsible for the constitutive phosphorylation of CD5 molecules at a cluster of three serine residues located at the extreme C terminus (S458, S459, and S461). Furthermore, the yeast two-hybrid system demonstrates the specific association between the C-terminal regions of the CD5 cytoplasmic tail and the regulatory beta subunit of CKII. We demonstrate that CKII associates with and phosphorylates the C-terminal region of CD5, a conserved domain known to be relevant for the generation of second lipid messengers, and thereby enables at least one component of its signaling function.     

Lack of correlation between apoptosis and DNA single-strand breaks in X-irradiated human peripheral blood mononuclear cells in the course of ageing

Inositol(1,3,4,5)tetrakisphosphate (InsP4) and phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42IP4 previously purified from pig cerebellum, which binds InsP4 (Kd approximately 2 nM) and PtdInsP3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42IP4 (pig) is highly homologous to centaurin-alpha, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42IP4 is expressed in rat brain and how it might be related to centaurin-alpha. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42IP4, but not to centaurin-alpha. The existence of p42IP4 in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-alpha-like protein in rat brain; and (3) the amino acid sequences deduced from p42IP4 cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42IP4 from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.     

Leukocyte polarization in cell migration and immune interactions

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.     

Refractory hypothalamic adenylate cyclase in anorectic tumor-bearing rats: implications for NPY-induced feeding

Entorhinal-dentate evoked potentials were measured in rats before and after: (1) eight electroconvulsive shock (ECS) seizures, or (2) matched handling. In animals that received ECS, evoked potentials were significantly enhanced, as evidenced by a long-lasting increase in the amplitude of the population spike. This increase in population-spike amplitude lasted for at least 3 months after the last ECS trial. No evoked-potential changes were observed in the subjects that received matched handling. These data suggest that ECS seizures produce long-lasting, perhaps permanent, changes in the brain.     

Long-term enhancement of entorhinal-dentate evoked potentials following 'modified' ECS in the rat

Electroconvulsive therapy (ECT) is widely used as a treatment for drug-resistant depression. The animal analogue of ECT is electroconvulsive shock (ECS) seizures. We have recently shown that repeated ECS seizures cause a long-lasting, perhaps permanent, enhancement in entorhinal-dentate evoked potentials in the rat. Our study, however, involved 'unmodified' ECS, whereas in clinical practice ECT is now usually given in its 'modified' form (with near-threshold currents, a short-acting barbiturate, muscle relaxant and oxygen). We have therefore repeated our experiments using modified ECS. Entorhinal-dentate evoked potentials were measured in Long-Evans rats before and after: (1) eight modified ECS seizures; or (2) eight sham modified ECS trials. Despite the use of the modified procedure, a significant and long-lasting enhancement in population spike amplitude was seen in the ECS group. We conclude that the modified procedure does not protect rats against the long-lasting enhancement of evoked potentials. Similar changes may be occurring in the brains of patients subjected to modified ECT.     

Impairment of signal transduction in T cells from old mice

T cells from old mice showed impaired proliferative response to antigenic stimulation. To understand the mechanism underlying the age-related impairment of T cell functions, the signal transduction pathway was examined and compared between T cells from young and old mice, and between T cell clones established from a young and old mouse. The age-related changes in T cells were as follows: (1) reduction in the expression and the activation of protein tyrosine kinases associated with T cell receptor (TCR) after antigenic stimulation; (2) reduced phosphorylation of phospholipase C gamma 1 (PLC gamma 1); (3) reduced production of second messengers such as inositoltrisphosphate (IP3) and diacylglycerol (DAG); and (4) reduced influx of Ca2+ ion. Thus, a T cell clone established from an old mouse showed impaired proliferation by stimulation with anti-CD3 antibody, but was fully activated to the level of a T cell clone from a young mouse by stimulation with phorbol acetate myristate (PMA) plus ionomycin (INM). However, splenic T cells freshly prepared from old mice did not show full recovery by the same treatment. The results indicate that one major blockade in the signal transduction of T cells from old mice is present in the pathway just after TCR, but besides this, the blockade is also present in multiple sites down-stream, which can not be bypassed by stimulation with PMA plus INM.     

Repeated exposure of adult rats to Aroclor 1254 causes brain region-specific changes in intracellular Ca2+ buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic. In vitro studies from this laboratory indicated that noncoplanar PCBs perturbed intracellular signal transduction mechanisms including Ca2+ homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (PKC). In the present study, we examined the effects of PCBs in vivo by dosing adult male Long-Evans rats orally with Aroclor 1254 (0, 10, or 30 mg/kg/day; 5 days/week for 4 weeks) in corn oil. At 24 h after the last dose, rats were tested for motor activity in a photocell device for 30 min. Immediately, the rats were euthanized, blood was collected for thyroid hormone analysis, and brains were removed, dissected into regions (cerebellum, frontal cortex, and striatum), and subcellular fractions were obtained for neurochemical analysis. Following Aroclor 1254 treatment, body weight gain in the high-dose group was significantly lower than the control and low-dose groups. Horizontal motor activity was significantly lower in rats dosed with 30 mg/kg Aroclor 1254. Ca2+ buffering by microsomes was significantly lower in all three brain regions from the 30 mg/kg group. In the same dose group, mitochondrial Ca2+ buffering was affected in cerebellum but not in cortex or striatum. Similarly, total cerebellar PKC activity was decreased significantly while membrane-bound PKC activity was significantly elevated at 10 and 30 mg/kg. PKC activity was not altered either in cortex or the striatum. Neurotransmitter levels in striatum or cortex were slightly altered in PCB-exposed rats compared to controls. Furthermore, repeated oral administration of Aroclor 1254 to rats did not significantly alter forebrain tyrosine hydroxylase immunoreactivity or enzymatic activity. Circulating T4 (total and free) concentrations were severely depressed at both doses in Aroclor 1254-exposed rats compared to control rats, suggesting a severe hypothyroid state. These results indicate that (1) in vivo exposure to a PCB mixture can produce changes in second messenger systems that are similar to those observed after in vitro exposure of neuronal cell cultures; (2) second messenger systems seem to be more sensitive than alterations in neurotransmitter levels or tyrosine hydroxylase involved in dopamine synthesis during repeated exposure to PCBs; and (3) the observed motor activity changes were independent of changes in striatal dopamine levels.     

Adenylyl cyclase signal transduction and alcohol-induced sedation

This study examined adenylyl cyclase (AC) signal transduction in alcohol-sensitive brain regions of rats selectively bred for high (HAD) and low (LAD) alcohol drinking and correlated these findings with differences in sensitivity and tolerance to alcohol-induced sedation found within these lines. LAD rats were more sensitive to the sedative effects of alcohol than were HAD rats as evidenced by a shorter latency to lose the righting response (RR) after a single alcohol challenge. When time to recover the RR was compared after each of two alcohol challenges, HAD rats recovered the RR more rapidly following the second challenge compared to the first, indicating that the HAD rats rapidly developed tolerance to the sedative effects of alcohol. Tolerance did not develop in rats of the LAD line. Two months after completion of behavioral testing, adenylyl cyclase (AC) signal transduction was examined in alcohol-sensitive brain regions of rats from both lines. Immunoblot analyses indicated that LAD rats had greater Gs alpha expression in the frontal cortex (FC) and hippocampus (HIP) compared to HAD rats. Rats with the highest HIP and FC Gs alpha levels were more rapidly affected by the sedative properties of alcohol than were rats with lower Gs alpha levels. G protein expression and AC activity in the FC, HIP, cerebellum (CERE), and nucleus accumbens (ACB) were also correlated with sensitivity to the sedative properties of alcohol and with the rapid development of tolerance to this alcohol effect. The results suggest that sensitivity and tolerance to alcohol-induced sedation may be mediated in part through AC signal transduction.     

Anticipatory cues can interfere with inhibitory operant behavior in the rat

In four separate experiments a total of 24 male rats were trained for 30 min. daily in the same temporal order to inhibit their responses for at least 6 sec. before a response-contingent reward was delivered (DRL-6 sec.). The rats tested as the second group each day displayed about twice the number of errors (effect size = 40%) shown by rats tested in the first or third groups. These results suggest that anticipatory cues, acquired within two sessions, interfere with response inhibition during an appetitive task for a limited time (between a few minutes to about one hour). The results are consistent with the hypothesis that learned anticipation of reward may decrease inhibitory mechanisms by facilitating limbic lability.