Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.     

A comparison of the effects of cyclooxygenase prostanoids on progesterone production by small and large bovine luteal cells

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.     

Methylation in bovine luteal cells as a regulator of luteinizing hormone action

Experiments were conducted to determine if methylation is a part of the mechanism by which luteinizing hormone (LH) and epinephrine stimulate progesterone production by dispersed bovine luteal cells. Corpora lutea (CL) were collected from 24 Holstein heifers on Day 10 of the estrous cycle and dispersed with collagenase. Net progesterone accumulation, representing total progesterone synthesized by 10(6) cells during a 2-h incubation was determined. Cells from 7 CL were treated with 0 and 5 ng LH, in the presence and absence of methylation inhibitor, S-adenosyl-homocysteine (SAH, 1 mM). LH-stimulated progesterone production was inhibited (P less than 0.05) in the presence of SAH(209 +/- 19 vs. 119 +/- 7 ng/10(6) cells). In the absence of LH, progesterone production was unaffected (87 +/- 22 vs. 68 +/- 28) by SAH. Cells from 4 CL were treated with 10 micrograms epinephrine or 10 micrograms isoproterenol with and without SAH. Both epinephrine and isoproterenol-stimulated progesterone production was inhibited (P less than 0.05) by the presence of SAH (204 +/- 24 vs. 125 +/- 18 and 198 +/- 15 vs. 130 +/- 8). Progesterone production by cells from 4 CL was unaffected by the presence of SAH when treated with Medium 199 (M199) (75 +/- 32), 10 micrograms cholera toxin, which directly stimulates adenylate cyclase on the cytoplasmic side of plasma membranes (168 +/- 19), or 3 mM dibutyryl cAMP (210 +/- 40).(ABSTRACT TRUNCATED AT 250 WORDS)     

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca²⁺]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca²⁺]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca²⁺ probe. Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca²⁺]i in both small and large luteal cells, having an effect at concentrations as low as 5μM; and was maximally effective at 50μM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca²⁺]i, with NDGA exerting a greater stimulation of [Ca²⁺i than indomethacin. Prostaglandin F_2α (PGF_2α) as well as prostaglandin E₂ (PGE₂) increased [Ca²⁺ in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca²⁺]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF_2α and PGE₂) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca²⁺]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.     

Traveling Waves and the Processing of Weakly Tuned Inputs in a Cortical Network Module

Recent studies have shown that local cortical feedback can havean important effect on the response of neurons in primary visualcortex to the orientation of visual stimuli. In this work, westudy the role of the cortical feedback in shaping thespatiotemporal patterns of activity in cortex. Two questionsare addressed: one, what are the limitations on the ability ofcortical neurons to lock their activity to rotatingoriented stimuli within a single receptive field? Two, can thelocal architecture of visual cortex lead to the generation ofspontaneous traveling pulses of activity? We study theseissues analytically by a population-dynamic model of ahypercolumn in visual cortex. The order parameter thatdescribes the macroscopic behavior of the network is thetime-dependent population vector of the network. We firststudy the network dynamics under the influence of a weakly tunedinput that slowly rotates within the receptive field. We showthat if the cortical interactions have strong spatialmodulation, the network generates a sharply tuned activityprofile that propagates across the hypercolumn in a path thatis completely locked to the stimulus rotation. The resultantrotating population vector maintains a constant angular lagrelative to the stimulus, the magnitude of which grows with thestimulus rotation frequency. Beyond a critical frequency thepopulation vector does not lock to the stimulus but executes aquasi-periodic motion with an average frequency that is smallerthan that of the stimulus. In the second part we consider thestable intrinsic state of the cortex under the influence of isotropic stimulation. We show that if the local inhibitoryfeedback is sufficiently strong, the network does not settleinto a stationary state but develops spontaneous travelingpulses of activity. Unlike recent models of wave propagation incortical networks, the connectivity pattern in our model isspatially symmetric, hence the direction of propagation ofthese waves is arbitrary. The interaction of these waves withan external-oriented stimulus is studied. It is shown that thesystem can lock to a weakly tuned rotating stimulus if thestimulus frequency is close to the frequency of the intrinsic wave.     

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Lipolytic activity measured at pH 8.6 in bovine corpora lutea exhibited classical properties of lipoprotein lipase (LPL) in terms of serum and heparin stimulation and NaCl inhibition. LPL activity was measured in 23 corpora lutea collected at different stages of the estrous cycle and early pregnancy. The LPL activity in cyclic corpora lutea (mumole FA released/hr/100 mg acetone powder) was low at Days 4-8 of the estrous cycle (3.1 +/- 1.5: mean +/- SE) and at Days 19-20 (1.6 +/- 0.6). However, high activity of the enzyme was found at Days 12-15 of the cycle (11.8 +/- 1.8); these concentrations were significantly (P less than 0.01) elevated over those found at Days 4-8 and 19-20. The enzyme activity began to decline at Days 16-18 of the estrous cycle (5.1 +/- 1.7). Low enzyme activity was found in the corpora lutea removed from two cows at Day 22 of pregnancy. Progesterone concentrations were measured in 16 of the 23 corpora lutea and a good correlation (r = 0.75, P less than 0.01) was found between lipoprotein lipase and progesterone concentrations of the tissue. The data suggest that LPL may be involved in controlling the transfer of fatty acids, including arachidonic, from plasma lipoproteins to luteal tissue.     

Mammalian Target of Rapamycin Complex 2 (mTORC2) Is a Critical Determinant of Bladder Cancer Invasion

Bladder cancer is the fourth most common cause of cancer in males in the United States. Invasive behavior is a major determinant of prognosis. In this study, we identified mammalian target of rapamycin complex 2 (mTORC2) as a central regulator of bladder cancer cell migration and invasion. mTORC2 activity was assessed by the extent of phosphorylation of Ser473 in AKT and determined to be approximately 5-fold higher in specimens of invasive human bladder cancer as opposed to non-invasive human bladder cancer. The immortalized malignant bladder cell lines, UMUC-3, J82 and T24 demonstrated higher baseline mTORC2 activity relative to the benign bladder papilloma-derived cell line RT4 and the normal urothelial cell line HU1. The malignant bladder cancer cells also demonstrated increased migration in transwell and denudation assays, increased invasion of matrigel, and increased capacity to invade human bladder specimens. Gene silencing of rictor, a critical component of mTORC2, substantially inhibited bladder cancer cell migration and invasion. This was accompanied by a significant decrease in Rac1 activation and paxillin phosphorylation. These studies identify mTORC2 as a major target for neutralizing bladder cancer invasion.     

Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average V_max of 0.86±0.01 μmol min^–1mL^–1 and K_M of 76±6 μM. The enzyme was inhibited by the DPP-IV family inhibitor l-threo-Ile-thiazolidide (K_i=64.0±0.53 nM), competitively inhibited by bacitracin (K_i=0.16±0.01 mM) and bestatin (K_i=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC₅₀ value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn²⁺ and Ca²⁺, for which a mixed inhibition mechanism was observed (K_i values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca²⁺ ions preferentially bound to the β-propeller domain of the rat and human enzymes, while Zn²⁺ ions to the α-β hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.     

Effects of heat and drought stress on post‐illumination bursts of volatile organic compounds in isoprene‐emitting and non‐emitting poplar

Over the last decades, post‐illumination bursts (PIBs) of isoprene, acetaldehyde and green leaf volatiles (GLVs) following rapid light‐to‐dark transitions have been reported for a variety of different plant species. However, the mechanisms triggering their release still remain unclear. Here we measured PIBs of isoprene‐emitting (IE) and isoprene non‐emitting (NE) grey poplar plants grown under different climate scenarios (ambient control and three scenarios with elevated CO₂ concentrations: elevated control, periodic heat and temperature stress, chronic heat and temperature stress, followed by recovery periods). PIBs of isoprene were unaffected by elevated CO₂ and heat and drought stress in IE, while they were absent in NE plants. On the other hand, PIBs of acetaldehyde and also GLVs were strongly reduced in stress‐affected plants of all genotypes. After recovery from stress, distinct differences in PIB emissions in both genotypes confirmed different precursor pools for acetaldehyde and GLV emissions. Changes in PIBs of GLVs, almost absent in stressed plants and enhanced after recovery, could be mainly attributed to changes in lipoxygenase activity. Our results indicate that acetaldehyde PIBs, which recovered only partly, derive from a new mechanism in which acetaldehyde is produced from methylerythritol phosphate pathway intermediates, driven by deoxyxylulose phosphate synthase activity.     

Mechanisms of firing patterns in fast-spiking cortical interneurons

Cortical fast-spiking (FS) interneurons display highly variable electrophysiological properties. Their spike responses to step currents occur almost immediately following the step onset or after a substantial delay, during which subthreshold oscillations are frequently observed. Their firing patterns include high-frequency tonic firing and rhythmic or irregular bursting (stuttering). What is the origin of this variability? In the present paper, we hypothesize that it emerges naturally if one assumes a continuous distribution of properties in a small set of active channels. To test this hypothesis, we construct a minimal, single-compartment conductance-based model of FS cells that includes transient Na(+), delayed-rectifier K(+), and slowly inactivating d-type K(+) conductances. The model is analyzed using nonlinear dynamical system theory. For small Na(+) window current, the neuron exhibits high-frequency tonic firing. At current threshold, the spike response is almost instantaneous for small d-current conductance, gd, and it is delayed for larger gd. As gd further increases, the neuron stutters. Noise substantially reduces the delay duration and induces subthreshold oscillations. In contrast, when the Na(+) window current is large, the neuron always fires tonically. Near threshold, the firing rates are low, and the delay to firing is only weakly sensitive to noise; subthreshold oscillations are not observed. We propose that the variability in the response of cortical FS neurons is a consequence of heterogeneities in their gd and in the strength of their Na(+) window current. We predict the existence of two types of firing patterns in FS neurons, differing in the sensitivity of the delay duration to noise, in the minimal firing rate of the tonic discharge, and in the existence of subthreshold oscillations. We report experimental results from intracellular recordings supporting this prediction.