The regulation of AMP-activated protein kinase by upstream kinases.

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a major role in maintaining energy homoeostasis. Within individual cells, AMPK is activated by a rise in the AMP/ATP ratio that occurs following a fall in ATP levels. AMPK is also regulated by the adipokines, adiponectin and leptin, hormones that are secreted from adipocytes. AMPK regulates a wide range of metabolic pathways, including fatty acid oxidation, fatty acid synthesis, glycolysis and gluconeogenesis. In peripheral tissues, activation of AMPK leads to responses that are beneficial in counteracting the deleterious effects that arise in the metabolic syndrome. Recent studies have demonstrated that modulation of AMPK activity in the hypothalamus plays a role in feeding. A decrease in hypothalamic AMPK activity is associated with decreased feeding, whereas activation of AMPK leads to increased food intake. Furthermore, signalling pathways occurring in the hypothalamus lead to changes in AMPK activity in peripheral tissues, such as skeletal muscle, via the sympathetic nervous system. AMPK, therefore, provides a mechanism for monitoring changes in energy metabolism within individual cells and at the level of the whole body. Activation of AMPK requires phosphorylation of threonine 172 (Thr-172) within the catalytic subunit. Recent studies have shown that both LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) play important roles in phosphorylating and activating AMPK. In addition, there is evidence that AMPK can be activated by other upstream kinases, although the physiological significance of this is not clear at present. This review focuses on the role of LKB1 and CaMKKbeta in the regulation of AMPK.

Prostaglandin E2 production by dispersed canine fundic mucosal cells. Contribution of macrophages and endothelial cells as major sources.

Endogenous prostaglandins (PGs) influence resistance of the gastric mucosa to injury, but the source of PGs is unknown. Using radioimmunoassay, we studied PG production by dispersed canine fundic mucosal cells. PGE2 production, stimulated by bradykinin, epidermal growth factor, zymosan, and calcium ionophore, was greater in the small-cell elutriator fraction (SCEF) than in the medium and large cell fractions, which contained mucous, chief, and parietal cells. Linear density gradients of SCEF cells revealed maximal PGE2 production in cells of light density. Mast, endocrine, and endothelial cells did not account for this PGE2 production. Macrophages, identified by uptake of acetylated-LDL, immunoreactivity with antibodies to the human Ia antigen, and phagocytosis of fluorescent latex particles, were enriched in the SCEF and correlated with PGE2 production in the density gradient. Magnetic separation of cells in the SCEF-ingesting iron particles enriched PGE2 production. Fractions enriched in endothelial cells present in intact capillary fragments, but depleted of macrophages, also produced PGE2. Regulation of PGE2 production differed among cell types. Fibroblasts were easily cultured from submucosa, but were not detected in the SCEF. We conclude that macrophages and capillary endothelial cells are major producers of PGE2 in the canine fundic mucosa.

Ouabain-sensitive 86Rb(K) influx is linked to transepithelial Na transport in pig kidney cell line.

The pig kidney cell line, LLC-PK1, exhibits rheogenic D-glucose coupled transepithelial Na+ transport that is inhibited by phlorizin. By measuring the difference in initial rates of influx of 86Rb+ with and without coupled Na+ transport, we can demonstrate an 86Rb+ uptake linked to Na+ transport, The simultaneous determination of phlorizin-inhibited Na coupled D-[3H] glucose uptake and 86Rb+ influx allows calculation of an Na+/Rb+ stoichiometry that is consistent with an electrogenic Na+ for Rb+ exchange.

Cognitive function following traumatic brain injury: effects of injury severity and recovery period in a parasagittal fluid-percussive injury model.

Previous work in this laboratory has demonstrated that rats show substantial deficits on the cued and hidden versions of the Morris water maze, as well as an apparent time-dependent recovery over a period of months, following moderate parasagittal fluid-percussion (FP) injury. However, the longitudinal nature of those studies precluded definitive statements regarding recovery because of the possible confound of practice-dependent improvements in performance. The present experiments were undertaken to address this issue and to investigate more closely the relationship between impact severity and posttraumatic learning/memory deficits, which have not been examined thoroughly in this model. Separate groups of rats were subjected to mild (1.1 to 1.4 atm), moderate (1.8 to 2.1 atm), or severe (2.2 to 2.7 atm) FP injury (or sham surgery) and tested on several water maze tasks at either 5 days or 8 weeks after injury. Moderately and severely injured animals showed impairment in acquisition of the hidden platform task at both time points. Cued platform task performance was impaired significantly in severely injured animals 8 weeks after insult. Mildly injured animals exhibited no significant deficits on either task at either time point. The results indicate that deficits on the hidden platform task are more robust than those on the cued platform task, and that performance on both tasks is dependent on injury severity. They also indicate that the learning/memory deficits in this model are relatively enduring, suggesting that the model is a reasonable one for assessing potential treatment regimens.

Sex differences, context preexposure, and the immediate shock deficit in Pavlovian context conditioning with mice.

The acquisition of context fear in rats is affected by variables such as the sex of the animal, the placement to shock interval (PSI), and preexposure to the context. The current experiments assessed the effects of these variables on context conditioning in mice (C57BL/6). In Experiment 1, mice were placed in a chamber and received a single shock 5s, 20 s, 40s, 60s, 180s, or 720s later. Increasing the PSI produced corresponding increases in conditional freezing during the context test. In addition, male mice acquired more context conditioning than female mice did but only at intermediate PSIs. In Experiment 2, preexposure to the context before training alleviated the sex difference found with an intermediate PSI. The results are discussed in terms of configural learning theory and are argued to be contrary to the predictions of scalar expectancy theory.

Physiological evidence for hippocampal disinhibition resulting from activation of the median raphe.

The mechanism underlying median raphe (MR)-induced facilitation of hippocampal synaptic transmission was investigated by recording stimulus-evoked field potentials and unitary responses in urethane-anesthetized rats. Stimulation of the MR 40 ms prior to perforant path (PP) activation significantly increased the magnitude of PP-evoked granule cell population spikes (median increase = 78%) without affecting population EPSP slope. Injection of homocysteic acid into the vicinity of the MR also facilitated PP-evoked granule cell population spikes, in a dose-dependent manner. Nineteen dentate hilar units were characterized as putative interneurons on the basis of their waveform characteristics and their response to PP stimulation. Electrical activation of the MR inhibited spontaneous or PP-evoked activity in the majority (75%) of these cells; the remaining cells were unaffected. MR stimulation also inhibited spontaneous activity in a large proportion (60%) of putative interneurons in CA1. The present results provide evidence that neurons within the raphe modulate hippocampal throughput by altering discharge of non-principal cells. These data, thus, support the idea that disinhibition is a common mechanism by which extrahippocampal structures modulate information flow through the hippocampus.

Behavioral, electrophysiological, and histopathological consequences of mild fluid-percussion injury in the rat.

Metabolic dysfunction in the relay nuclei of the rat vibrissa circuit follows traumatic brain injury (TBI). This study examined the effects of mild (1.4-1.5 atm) parasagittal fluid-percussion injury on the electrophysiology of this circuit. TBI caused significant reductions in slope and increases in latency of vibrissa-evoked field potentials 3 days after injury. Assessment of open-field swimming revealed an increase in thigmotaxis 2 days after injury. TBI caused mild selective cortical damage and limited axonal swelling at the injury site. Thus mild injury disrupts somatosensory electrophysiology and exploratory behavior.

Chronic failure in the maintenance of long-term potentiation following fluid percussion injury in the rat.

Traumatic brain injury (TBI) can produce chronic cognitive learning/memory deficits that are thought to be mediated, in part, by impaired hippocampal function. Experimentally induced TBI is associated with deficits in hippocampal synaptic plasticity (long-term potentiation, or LTP) at acute post-injury intervals but plasticity has not been examined at long-term survival periods. The present study was conducted to assess the temporal profile of LTP after injury and to evaluate the effects of injury severity on plasticity. Separate groups of rats were subjected to mild (1.1-1.4 atm), moderate (1.8-2.1 atm), or severe (2.2-2.7 atm) fluid percussion (FP) injury (or sham surgery) and processed for hippocampal electrophysiology in the first or eighth week after injury. LTP was defined as a lasting increase in field excitatory post-synaptic potential (fEPSP) slope in area CA1 following tetanic stimulation of the Schaffer collaterals. The fEPSP slope was measured for 60 min after tetanus. Assessment of LTP at the acute interval (6 days) revealed modest peak slope potentiation values (129-139%), which declined in each group (including sham) over the hour-long recording session and did not differ between groups. Eight weeks following injury, slices from all groups exhibited robust maximal potentiation (134-147%). Levels of potentiation among groups were similar at the 5-min test interval but differed significantly at the 30- and 60-min test intervals. Whereas sham slices showed stable potentiation for the entire 60-min assessment period, slices in all of the injury groups exhibited a significant decline in potentiation over this period. These experiments reveal a previously unknown effect of TBI whereby experimentally induced injury results in a chronic inability of the CA1 hippocampus to maintain synaptic plasticity. They also provide evidence that sham surgical procedures can significantly influence hippocampal physiology at the acute post-TBI intervals. The results have implications for the mechanisms underlying the impaired synaptic plasticity following TBI.

Ergonomic strategies for dental professionals.

This study examined the extent to which dental professionals have made changes within their practice environments to decrease the potential for developing a cumulative trauma disorder. A survey was disseminated to a sample of 95 dental professionals. The survey addressed the presence and location of pain, changes professionals made within their workplaces, and whether or not these changes were perceived as effective. Fifty two surveys were returned for a response rate of 55%. Results indicated that 96% noted pain during or after work; 88% had made changes in their work practices. Common strategies used to promote health were stretching, good posture, personal relaxation, and instrument maintenance. Use of ergonomic instruments, workstations, and new instrumentation strategies were seen as effective, but rated slightly lower than other categories.

Characterization of receptors regulating secretory function in the fundic mucosa.

A model for a present view of the major pathways and receptors mediating function in the canine fundic mucosa is depicted in Figure 1. Gastrin has direct actions on the parietal cell and on the somatostatin cell; action on the parietal cell, but not somatostatin cell, is potentiated by histamine. In contrast, gastrin action on the somatostatin cell is potentiated by beta-adrenergic agonists. The potency of H2 blockers against gastrin may reflect blockage by these inhibitors of the stimulatory (parietal cell), but not the inhibitory (somatostatin cell), component of gastrin action, thus shifting the balance of gastrin effects toward the inhibitory side. The profound effects of H2 antagonists on gastrin action may also reflect an effect mediated by histamine release, but this possibility awaits direct confirmation. Cholinergic pathways also have at least dual sites of action: stimulation of the parietal cell, and blockage of the release of the inhibitory transmitter somatostatin. Anticholinergic agents may therefore have a dual acid inhibitory effect by reducing direct parietal cell stimulation and enhancing somatostatin release. There is little doubt that this model will rapidly evolve, but the concept that the pathways mediating acid secretion both converge in parallel at the parietal cell, and act in series to cause the release of paracrine transmitters, is attractive and likely to persist.

Potassium metabolism in seawater teleosts. I. The use of 86Rb as a tracer for potassium.

Comparison of the movements of rubidium and potassium between blood and tissues, and between blood and external medium, was undertaken in seawater adapted rainbow trout (Salmo gairdneri) and sculpin (Leptocottus armatus). Qualitative observations suggested that the two ions behaved similarly in exchanges within both fish. More than 90% of an injected dose of both ions disappeared from the blood within 1 h. Complete equilibration of both required about 5 h, and the decrease in blood concentration during the last 4 h followed similar kinetics. After injection and equilibration of 86Rb into trout the specific activity of most tissue pools (c.p.m. 86Rb/microeq tissue K) was close to that of blood (c.p.m. 86Rb/microeq plasma K). Skeletal muscle was the sole exception; its specific activity was less than 15% that of plasma even 4-5 h after injection. Injection of ouabain into trout caused cells to lose both 86Rb and K at similar rates. The same thing occurred when [K] in the external medium was raised abruptly. Exchanges of the two ions between blood and the external medium were also similar. Both ions were extruded and taken up rapidly across the gills. Double-labelling (86Rb and 42K fluxes measured simultaneously) showed that JK/JRb = 1.27 for both influx and efflux in trout. The same value was obtained for effluxes in sculpin. Exposing sculpin to seawater lacking Na and K caused both 42K and 86Rb efflux to fall by 75-80%; 86Rb efflux decreased similarly in trout. Alkali metal-free sea water also caused the voltage across the trout gill to change from +10 mV to -20 mV, and in the sculpin from +25 mV to -25 mV. Addition of either Rb or K to the ion-deficient medium partially repolarized the gill in both fish. Repolarization was slightly greater after K repletion than after Rb repletion. These observations suggest that 86Rb is an adequate tracer for potassium movement in seawater-adapted fish. The gills of trout and sculpin are a little more permeable to K than to Rb, and 86Rb fluxes must be multiplied by 1.3 to provide accurate estimates of transbranchial K movement. The quantitative relationship between JK and JRb within the animal was not established, but some data suggest that cell membranes in fish, like the gills, are somewhat more permeable to K than to Rb.

Potassium metabolism in seawater teleosts: II. Evidence for active potassium extrusion across the gill.

Unidirectional K-fluxes were estimated in unanaesthetized trout and sculpin and in anaesthetized sculpin from observed 86Rb movement (JK = 1.3 JRb). In all three groups efflux exceeded influx; Jo/Jin was 2-3. The values predicted by the flux ratio equation were 0.5 for trout and 1.0 for sculpin, so active K-extrusion is indicated. The results also show that more than one half of the total influx must be ingested with food rather than passing across the gills. Flux data show that the gills are more permeable to K+ than to Na+, PK/PNa was 5.4 in trout and 2.6 in sculpin. Changes in K-concentration in the external medium did not appear to affect efflux; there was no exchange component in the total fluxes. When both Na+ and K+ were omitted from the bathing solution, efflux decreased to about 15% of the normal seawater value. This is more than would be expected if the flux were purely diffusive and supports the conclusion that extrusion contains an active component. Repletion of the ion-deficient medium with K+ (alone) increased K-efflux. However, it also repolarized the gill and increased plasma [K+], and the flux change could be accounted for by the augmented driving force; i.e. it was diffusive. The additional plasma K came from the intracellular compartment, rather than an augmented influx from the medium.

Transepithelial glucose transport in cell culture.

Pig kidney cell line LLC-PK1 cultured on a collagen-coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by circumferential occluding junctions. In the presence of 5.5 mM D-glucose, a potential difference (PD) of 2.8 mV, apical bath negative, short-circuit current Isc of 13.2 microA . cm-2, and transepithelial resistance of 211 omega . cm2 were recorded. Isc and PD were reduced by phlorizin added to the apical bath but were unaffected when phlorizin was placed in the basolateral bath. Ouabain or the replacement of Na by tris-(hydroxymethyl)aminomethane or choline abolished the Isc. The sugar concentrations required to produce the half-maximal Isc were 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methylglucose. When [Na] was reduced, the D-glucose required for half-maximal SCC increased. Isotopically 3H- and 14C-labeled D-glucose were used to determine simultaneous bidirectional fluxes; a resultant net apical-to-basolateral flux was present and could be abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism. The cell culture model provides advantages for investigation of mechanisms of transepithelial glucose transport.

Transepithelial transport in cell culture: D-glucose transport by a pig kidney cell line (LLC-PK1).

The pig kidney cell line LLC-PK1 cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5 mM D-glucose, a PD of 2.8 mV. apical surface negative a SCC of 13 microA cm-2 and transepithelial resistance of 211 ohm.cm2 were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methyl-glucose. When [Na] was reduced, the concentration of D-glucose required for half-maximal SCC increase. Isotopically labeled 3H and 14C D-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK1, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.

Transepithelial transport in cell culture: stoichiometry of Na/phlorizin binding and Na/D-glucose cotransport. A two-step, two sodium model of binding and translocation.

The renal cell line LLC-PK1 cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependent D-glucose coupled transport. The short-circuit current (Isc) was accounted for by net apical-to-basolateral D-glucose coupled Na flux, which was 0.53 +/- 0.09(8) mueq cm-2hr-1, and Isc, 0.50 +/- 0.50(8) mueq cm-2hr-1. A linear plot of concurrent net Na vs. net D-glucose apical-to-basolateral fluxes a gave a regression coefficient of 2.08. As support for a 2:1 transepithelial stoichiometry, sodium was added in the presence of D-glucose and the response of Isc analyzed by a Hill plot. A slope of 2.08 +/- 0.06(5) was obtained confirming a requirement of 2 Na for 1 D-glucose coupled transport. A Hill plot of Isc increase to added D-glucose in the presence of Na gave a slope of 1.02 +/- 0.02(5). A direct determination of the initial rates of Na and D-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2. A coupling ratio of 2 for Na, D-glucose uptake, doubles the potential energy available for Na-gradient coupled D-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependent-phlorizin binding was 1.1 +/- 0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na].(ABSTRACT TRUNCATED AT 250 WORDS)

Transepithelial transport in cell culture: bioenergetics of Na-, D-glucose-coupled transport.

The renal cell line LLC-PK1 cotransports Na and D-glucose from the apical to the basolateral side of the cell monolayer, and the short-circuit current (Isc) measures the net amount of Na transported. Under conditions of maximal cotransport, the addition of phlorizin or removal of Na reversibly decreased oxygen consumption by one-half. In the absence of glycolytic substrates, alpha-methyl-D-glucoside stimulated Isc and oxygen consumption, although the Isc came to a steady state 50% less than when glycolytic substrates were present. The addition of other aerobic substrates did not increase Isc; however, when non-cotransported glycolytic substrates were introduced the Isc returned to a maximum with an associated fall in oxygen consumption and increased lactate production. Thus, in the absence of glycolytic substrates aerobic ATP formation may be rate-limiting for Na, D-glucose cotransport. For this epithelium glycolysis makes an important contribution to the provision of energy for transport. Oxygen consumption does not correlate well with Isc and is not a good measure of the energy used in transport.

Regulation of pepsinogen release from canine chief cells in primary monolayer culture.

To study the regulation of pepsinogen secretion by chief cells, we have developed techniques for the isolation, enrichment, and short-term culture of chief cells from canine stomach. The fundic mucosa was enzyme dispersed and chief cells were enriched to a content of about 70% using an elutriator rotor. After 36 h in culture confluent monolayers formed that were highly enriched in chief cells. Carbachol induced a time-dependent release of pepsinogen into the medium, with about a threefold increase in pepsinogen secretion over controls found after 60 min of incubation. Carbachol stimulation of pepsinogen secretion was dose dependent, with 5 microM producing 50% of the maximal response found at a carbachol concentration of 100 microM. Atropine (100 microM) produced a rightward shift of the dose-response curve, indicating the presence of a muscarinic receptor. Dibutyryl cAMP, 8-bromo-cAMP, and forskolin also markedly stimulated pepsinogen secretion. Secretin and vasoactive intestinal peptide (VIP) stimulated pepsinogen secretion, but the response were of smaller magnitude than found with carbachol or the cAMP analogues. The phosphodiesterase inhibitor isobutylmethylxanthine also caused a small stimulation of pepsinogen secretion but did not enhance the response to secretin or VIP. These findings indicate that epithelial monolayers can spontaneously form from isolated canine chief cells and retain functional differentiation evident by a response to stimulation. Canine chief cells in culture possess muscarinic and secretin receptors and respond to cAMP.

The apical surface of canine chief cell monolayers resists H+ back-diffusion.

The resistance of the gastric mucosa to acid and peptic injury is reflected by a resistance to the back-diffusion of H+ from gastric lumen to blood. The nature of this 'barrier', however, remains undefined. Using Ussing chambers, we have now studied the acid-barrier function of monolayers prepared from dispersed canine fundic chief cells. These monolayers secrete pepsinogen in response to stimulation. We found that, on acidification of the apical solution to pH 2, transepithelial resistance (R) increased 2.6-fold and the monolayers maintained this 1:100,000 H+ concentration gradient for more than 4 h. The addition of aspirin to the acidified apical solution caused a rapid decay in R, as did acidification of the basolateral solution to a pH less than 5.5. Ouabain-treated monolayers displayed the rise in R expected with apical acidification, while potential difference (V) and short-circuit current (Isc) decreased essentially to zero, indicating impermeability to H+. However, if the integrity of the ouabain-treated monolayers was disrupted by low apical pH, H+ permeation occurred, reflected by an Isc that was dependent on the H+ gradient across monolayers. These data indicate that the apical surface of chief cells is a very tight barrier to H+ diffusion and may be an important element resisting acid-peptic injury.