Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs.

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was -18.2 ± 1.8 mV. After treatment with 200 µmol/l SPM, it was -14.7 ± 2.0 mV (P < 0.04). In the presence of claudin-19 antibody, the dilution potential was -12.7 ± 2.1 mV. After SPM, it was -12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from -9.7 ± 1.0 to -6.3 ± 1.1 mV (P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from -19.6 ± 2.6 to -17.2 ± 2.3 mV (P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total (P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

cGMP induces degradation of NKCC2 in the thick ascending limb via the ubiquitin-proteasomal system.

The thick ascending limb of Henle's loop (TAL) reabsorbs NaCl via the apical Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is regulated by surface NKCC2 levels. The second messenger cGMP decreases NKCC2 activity by decreasing surface NKCC2 levels. We found that surface NKCC2 undergoes constitutive degradation. Therefore, we hypothesized that cGMP decreases NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. We measured surface NKCC2 levels by biotinylation of surface proteins, immunoprecipitation of NKCC2, and ubiquitin in TALs. First, we found that inhibition of proteasomal degradation blunts the cGMP-dependent decrease in surface NKCC2 levels [vehicle: 100%, db-cGMP (500 µM): 70.3 ± 9.8%, MG132 (20 µM): 97.7 ± 5.0%, and db-cGMP + MG132: 103.3 ± 3.4%, n = 5, P < 0.05]. We then found that cGMP decreased the internalized NKCC2 pool and that this effect was prevented by inhibition of the proteasome but not the lysosome. Finally, we found that NKCC2 is constitutively ubiquitinated in TALs and that cGMP enhances the rate of NKCC2 ubiquitination [vehicle: 59 ± 14% and db-cGMP (500 µM): 111 ± 25%, n = 5, P < 0.05]. We conclude that NKCC2 is constitutively ubiquitinated and that cGMP stimulates NKCC2 ubiquitination and proteasomal degradation. Our data suggest that the cGMP-induced NKCC2 ubiquitination and degradation may contribute to the cGMP-induced decrease of the NKCC2-dependent NaCl reabsorption in TALs.

The effects of over expressing aquaporins on the cryopreservation of hepatocytes.

During cryopreservation, aquaporins are critical in regulating water transport across cellular membranes and preventing osmotic damages. Hepatocytes express aquaporin (AQP) 0, 8, 9, 11, and 12; this study investigates whether increasing the localization of AQP8 on the cellular membrane would improve cell viability by increasing water transport during cryopreservation. Primary rat hepatocytes were cultured and treated with dibutyryl cAMP (Bt(2)cAMP) or glucagon to increase the expression of AQP8 at the cellular membrane via translocation. This phenomenon is verified through two experiments - confocal immunofluorescence microscopy and cell shrinkage analysis. The immunofluorescence results showed increase in AQP8 on the cellular membrane of treated cells, and cell shrinkage analysis showed an increase in water transport of treated cells compared to controls. Primary rat hepatocytes were treated with Bt(2)cAMP or glucagon and cryopreserved using standard protocols in a controlled rate freezer. This resulted in a significant increase in the cell viability on warming. These results indicate that Bt(2)cAMP or glucagon treated hepatocytes had increased expression of aquaporin in the cellular membrane, increased water transport during cryopreservation, and increased post-thaw viability.

Origin and effect of phototransduction noise in primate cone photoreceptors.

Noise in the responses of cone photoreceptors sets a fundamental limit on visual sensitivity, yet the origin of noise in mammalian cones and its relation to behavioral sensitivity are poorly understood. Our work here on primate cones improves understanding of these issues in three ways. First, we found that cone noise was not dominated by spontaneous photopigment activation or by quantal fluctuations in photon absorption, but was instead dominated by other sources, namely channel noise and fluctuations in cyclic GMP. Second, adaptation in cones, unlike that in rods, affected signal and noise differently. This difference helps to explain why thresholds for rod- and cone-mediated signals have different dependencies on background light level. Third, past estimates of noise in mammalian cones are too high to explain behavioral sensitivity. Our measurements indicate a lower level of cone noise and therefore help to reconcile physiological and behavioral estimates of cone noise and sensitivity.

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension.

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x(L) and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x(L) over-expressing subline (hVMbcl-x(L)) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x(L) cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 +/- 0.8% to 17.2 +/- 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x(L)-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x(L) cell cultures compared with control. We conclude that Bcl-x(L) and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.

Salt-inducible kinase 1 in the rat pinealocyte: adrenergic regulation and role in arylalkylamine N-acetyltransferase gene transcription.

The recognition of the basic leucine zipper domain in the regulation of transcriptional activity of cAMP response element-binding protein by salt-inducible kinase (SIK) prompted our investigation of the regulatory role of this kinase in the induction of Aa-nat and other cAMP-regulated genes in the rat pineal gland. Here we report Sik1 expression was induced by norepinephrine (NE) in rat pinealocytes primarily through activation of beta-adrenergic receptors, with a minor contribution from activation of alpha-adrenergic receptors. Treatments with dibutyryl cAMP, and to a lesser extent, agents that elevate intracellular Ca(2+) mimicked the effect of NE on Sik1 expression. In parallel to the results of the pineal cell culture studies, a marked nocturnal induction of Sik1 transcription was found in whole-animal studies. Knockdown of Sik1 by short hairpin RNA amplified the NE-stimulated Aa-nat transcription and other adrenergic-regulated genes, including Mapk phosphatase 1, inducible cAMP repressor, and type 2 iodothyronine deiodinase in a time-dependent manner. In contrast, overexpressing Sik1 had an inhibitory effect on the NE induction of Aa-nat and other adrenergic-regulated genes. Together, our results indicate that the adrenergic induction of Sik1 in the rat pineal gland is primarily through the beta-adrenergic receptor --> protein kinase A pathway. SIK1 appears to function as part of an endogenous repressive mechanism that regulates the peak and indirectly the duration of expression of Aa-nat and other cAMP-regulated genes. These findings support a role for SIK1 in framing the temporal expression profile of Aa-nat and other adrenergic-regulated genes in the rat pineal gland.

Altered nitric oxide calcium responsiveness of aortic smooth muscle cells in spontaneously hypertensive rats depends on low expression of cyclic guanosine monophosphate-dependent protein kinase type I.

OBJECTIVES: The nitric oxide/cyclic guanosine monophosphate (GMP)/cyclic GMP-dependent protein kinase type I (cGKI) pathway has been extensively investigated in the spontaneously hypertensive rat (SHR) as a possible pathogenetic factor. Therefore, we investigated the role of nitric oxide/cGKI on intracellular calcium dynamics ([Ca2+]i) of aortic smooth muscle cells isolated from control normotensive Wistar Kyoto rats (WKY) and SHR. METHODS: Rat aortic smooth muscle cells (RASMCs) were obtained from 12 to 16-week-old WKY and SHR. [Ca2+]i dynamics were monitored by imaging analysis of fura-2-loaded RASMCs. cGKI mRNA and cGKI protein expression were evaluated by reverse transcription-PCR and western blot. Plasmids codifying for enhanced green fluorescent protein (EGFP) or cGKIalpha-EGFP were transfected on SHR RASMCs. RESULTS: Angiotensin II similarly increased [Ca2+]i in WKY and SHR RASMCs. In WKY RASMCs, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1-100 micromol/l) reduced the decay time of angiotensin II-induced [Ca2+]i transient. On the contrary, in SHR cells, SNAP was ineffective. Dibutyryl cyclic GMP (1-100 nmol/l), a membrane-permeable cyclic GMP analogue, behaved similarly to SNAP. In naive SHR RASMCs, cGKI mRNA and cGKI protein were low or absent. After transfection of a plasmid encoding for cGKIalpha-EGFP, the [Ca2+]i dynamic of SHR-transfected cells regained sensitivity to the nitric oxide/cyclic GMP pathway. CONCLUSION: The low expression of cGKI determines the lack of nitric oxide/cyclic GMP-dependent regulation on [Ca2+]i transient in SHR RASMCs. This alteration may contribute to the development of hypertension and explain suboptimal responses to nitroglycerin and other nitric oxide-releasing molecules in patients.

cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2).

NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal=100%; db-cGMP 100 microM=77+/-7%; 500 microM=54+/-10% and 1,000 microM=61+/-8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal=100%; 8-Br-cGMP=75+/-5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaCl absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48+/-4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL.

The ANP-cGMP-protein kinase G pathway induces a phagocytic phenotype but decreases inflammatory gene expression in microglial cells.

Reactive gliosis is a prominent feature of CNS injury that involves dramatic changes in glial cell morphology together with increased motility, phagocytic activity, and release of inflammatory mediators. We have recently demonstrated that stimulation of the cGMP-protein kinase G (PKG) pathway by NO or atrial natriuretic peptide (ANP) regulates cytoskeleton dynamics and motility in rat astrocytes in culture. In this work, we show that the cGMP-PKG pathway stimulated by ANP, but not by NO, regulates microglial cell morphology by inducing a dramatic reorganization in the actin cytoskeleton. Both ANP (0.01-1.0 microM) and the permeable cGMP analog, dibutyryl-cGMP (1-100 microM), promote a rapid (maximal at 30 min) and concentration-dependent increase in size, rounding, and lamellipodia and filopodia formation in rat brain cultured microglia. These morphological changes involve an augment and redistribution of F-actin and result in increased phagocytic activity. ANP-induced rearrangements in actin cytoskeleton and inert particle phagocytosis are prevented by the PKG inhibitor, Rp-8-Br-PET-cGMPS (0.5 microM), and involve inhibition of RhoA GTPase and activation of Rac1 and Cdc42. However, ANP does not induce NO synthase Type 2 (NOS-2) or tumor necrosis factor-alpha expression and is able to decrease lipopolysaccharide (LPS)-elicited induction of these inflammatory genes. The morphological changes and the decrease of LPS-induced NOS-2 expression produced by ANP in cultured microglia are also observed by immunostaining in organotypic cultures from rat hippocampus. These results suggest that stimulation of the ANP-cGMP-PKG pathway in microglia could play a beneficial role in the resolution of neuroinflammation by removing dead cells and decreasing levels of proinflammatory mediators.

Nitric oxide and cGMP signaling in calcium-dependent development of cell polarity in Ceratopteris richardii.

Single-celled spores of the fern Ceratopteris richardii undergo gravity-directed cell polarity development that is driven by polar calcium currents. Here we present results that establish a role for nitric oxide (NO)/cGMP signaling in transducing the stimulus of gravity to directed polarization of the spores. Application of specific NO donors and scavengers inhibited the calcium-dependent gravity response in a dose-dependent manner. The effects of NO donor exposure were antagonized by application of NO scavenger compounds. Similarly, the guanylate cyclase inhibitors 6-anilino-5,8-quinolinedione and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin, and the phosphodiesterase inhibitor Viagra, which modulate NO-dependent cGMP levels in the cells, disrupted gravity-directed cell polarity in a dose-dependent manner. Viagra effects were antagonized by application of NO scavengers, consistent with the postulate that NO and cGMP are linked in the signaling pathway. To identify other components of the signaling system we analyzed gene expression changes induced by Viagra treatment using microarrays and quantitative real-time reverse transcription-polymerase chain reaction. Preliminary microarray analysis revealed several genes whose expression was significantly altered by Viagra treatment. Three of these genes had strong sequence similarity to key signal transduction or stress response genes and quantitative real-time reverse transcription-polymerase chain reaction was used to more rigorously quantify the effects of Viagra on their expression in spores and to test how closely these effects could be mimicked by treatment with dibutyryl cGMP. Taken together our results implicate NO and cGMP as downstream effectors that help link the gravity stimulus to polarized growth in C. richardii spores. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers BE 640669 to BE 643506, BQ 086920 to BQ 087668, and CV 734654 to CV 736151.

Activators of the PKA and PKG pathways attenuate RhoA-mediated suppression of the KDR current in cerebral arteries.

This study tested whether activation of protein kinase A (PKA) and G (PKG) pathways would attenuate the ability of RhoA to suppress the delayed rectifier K(+) (K(DR)) current and limit agonist-induced depolarization and constriction. Smooth muscle cells from rat cerebral arteries were enzymatically isolated, and whole cell K(DR) currents were monitored with conventional patch-clamp electrophysiology. The K(DR) current averaged 21.2 +/- 2.3 pA/pF (mean +/- SE) at +40 mV and was potently inhibited by UTP. Current suppression was eliminated in the presence of C3 exoenzyme, confirming that this modulation is dependent on RhoA. Activation of PKA (dibutyryl-cAMP, forskolin) or PKG (dibutyryl-cGMP, sodium nitroprusside, nitric oxide) similarly abolished the ability of UTP to suppress K(DR) and did so without effect on baseline current. Using pressure myography techniques, we stripped cerebral arteries of endothelium and preconstricted them with UTP; these were subsequently shown to hyperpolarize and dilate to both forskolin and sodium nitroprusside. An increase in K(V) channel activity was found to partly underlie these associated changes, as constriction to 4-aminopyridine (K(DR) channel blocker) was greater after PKA or PKG activation. We conclude from our electrophysiological and functional observations that the PKA and PKG pathways attenuate the ability of UTP to depolarize and constrict cerebral arteries in part by minimizing the RhoA-mediated suppression of the K(DR) current.

Nitric oxide inhibits gastric acid secretion by increasing intraparietal cell levels of cGMP in isolated human gastric glands.

We have previously identified cells containing the enzyme nitric oxide (NO) synthase (NOS) in the human gastric mucosa. Moreover, we have demonstrated that endogenous and exogenous NO has been shown to decrease histamine-stimulated acid secretion in isolated human gastric glands. The present investigation aimed to further determine whether this action of NO was mediated by the activation of guanylyl cyclase (GC) and subsequent production of cGMP. Isolated gastric glands were obtained after enzymatic digestion of biopsies taken from the oxyntic mucosa of healthy volunteers. Acid secretion was assessed by measuring [(14)C]aminopyrine accumulation, and the concentration of cGMP was determined by radioimmunoassay. In addition, immunohistochemistry was used to examine the localization of cGMP in mucosal preparations after stimulation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). SNAP (0.1 mM) was shown to decrease acid secretion stimulated by histamine (50 microM); this effect was accompanied by an increase in cGMP production, which was histologically localized to parietal cells. The membrane-permeable cGMP analog dibuturyl-cGMP (db-cGMP; 0.1-1 mM) dose dependently inhibited acid secretion. Additionally, the effect of SNAP was prevented by preincubating the glands with the GC inhibitor 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (10 microM). We therefore suggest that NO in the human gastric mucosa is of physiological importance in regulating acid secretion. Furthermore, the results show that NO-induced inhibition of gastric acid secretion is a cGMP-dependent mechanism in the parietal cell involving the activation of GC.

Protein kinase A activity and protein phosphorylation during the mouse sperm acrosomal reaction.

Using two-dimensional gel electrophoresis, changes in protein phosphorylation caused by cyclic nucleotide-dependent protein kinases were analyzed with or without exposure to a protein kinase inhibitor, H-8, during the mouse sperm acrosomal reaction. The acrosomal reaction, induced by the treatment of sperm with dibutyryl cyclic AMP or dibutyryl cyclic GMP, was inhibited by H-8. The activities of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase induced by the sperm extract were also inhibited by H-8. When endogenous PKA in sperm was activated by the addition of cyclic AMP, a 45-kDa protein spot identified by electrophoresis indicated the occurrence of phosphorylation in vivo. Furthermore, the enhanced phosphorylation of the 45-kDa protein spot was inhibited by H-8. These results suggest that the PKA-catalyzed phosphorylation of the 45-kDa protein may be involved in the regulation of the mouse sperm acrosomal reaction.

Effects of cyclic nucleotides on the function of prestin.

Outer hair cells (OHCs) in the mammalian organ of Corti display electromotility, which is thought to provide the local active mechanical amplification of the cochlear response. Prestin is the key molecule responsible for OHC electromotility. Several compounds, including cGMP, have been shown to influence OHC electromotility. There are two potential cAMP/cGMP-dependent protein kinase phosphorylation sites on prestin. Whether these sites are involved in cGMP-dependent reactions is as yet unknown. In this study, prestin cDNA was transiently transfected into TSA 201 cells. Cells that expressed prestin were selected to measure non-linear capacitance (NLC), a signature of outer hair cell motility. We applied cGMP and cAMP analogues and a protein kinase G (PKG) antagonist to the cells. Furthermore, nine mutations at putative phosphorylation sites of prestin were produced. The neutral amino acid alanine replaced serine/threonine at phosphorylation sites to change the conserved phosphorylation motif in order to mimic the dephosphorylated state of prestin, whereas replacement with the negatively charged aspartic acid mimicked the phosphorylated state. The properties of such modified prestin-expressing cells were examined, through measurement of NLC and with confocal microscopy. Our data demonstrate that cGMP is significantly more influential than cAMP in modifying the non-linear, voltage-dependent charge displacement in prestin-transfected cells. The electrical properties of the single and double mutations further indicate a possible interaction between the two PKG target sites. One of these sites may influence the membrane targeting process of prestin. Finally, a new topology map of prestin is proposed.

Regulation of interleukin-1beta and interleukin-1beta inhibitor release by human airway epithelial cells.

In asthma, human airway epithelial cells (HAECs) regulate the intensity of mucosal inflammation, in part, by releasing the pro-inflammatory cytokine interleukin (IL)-1beta. However, the IL-1beta inhibitors, IL-1 receptor antagonist (IL-1RA) and soluble IL-1 receptor type II (sIL-1RII), regulate IL-1beta bioactivity. In order to better understand the control of IL-1beta activity in the airway mucosa, the role(s) of tumour necrosis factor (TNF)-alpha, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the release of IL-1beta and its inhibitors by cultured HAECs were examined. HAECs were treated with TNF-alpha (2-200 ng.mL(-1)), dibutyryl cAMP (0.01-1 mM), 8-bromo-cGMP (0.01-1 mM) or vehicle for 24 h, and cytokine levels in the HAEC-conditioned medium were measured by enzyme-linked immunosorbent assay. HAECs produced IL-1beta, IL-1RA and sIL-1RII constitutively, but the inhibitor concentrations greatly exceeded that of IL-1beta (by approximately 100- and approximately 550-fold, respectively). TNF-alpha dose-dependently increased the levels of all IL-1beta cytokine family members. However, over the range of TNF-alpha concentrations studied, IL-1beta concentration increased more than those of its inhibitors. cAMP increased constitutive and TNF-alpha-stimulated IL-1beta release but reduced that of sIL-1RII. In contrast, cGMP had no effect on IL-1beta but reduced IL-1RA and sIL-1RII release. Under basal conditions, the disproportionate release of inhibitors relative to interleukin-1beta by human airway epithelial cells probably prevents interleukin-1beta-mediated biological effects. Tumour necrosis factor-alpha, cyclic adenosine monophosphate and cyclic guanosine monophosphate may potentiate mucosal inflammation by increasing interleukin-1beta levels relative to those of its inhibitors in the airway mucosa.

Aquaporin-1 is recruited to the plasma membrane by hyperosmotic stimuli via a protein kinase A-dependent pathway in rat peritoneal mesothelial cells.

Aquaporin-1 (AQP1) has been reported to play an important role in water permeability in peritoneal dialysis. To determine the mechanism involved in this process, we used cultured rat peritoneal mesothelial cells (RPMCs) to examine the glucose-induced translocation of AQP1 to the plasma membrane. Cultured RPMCs obtained from male Sprague-Dawley rats were incubated in a combination of Dulbecco modified Eagle medium (DMEM) and F12 medium at 37 degrees C for 15 minutes. The plasma membrane of the RPMCs was separated by Percoll gradient, and the quantity of AQP1 in the membrane fraction was determined by Western blot analysis. The amount of AQP1 was significantly increased by the addition of 5% glucose (139.5% +/- 38.7% of control, p < 0.05) or of dibutyryl cyclic adenosine monophosphate (db-cAMP), a cAMP analog to the medium (139.5% +/- 21.9% of control, p < 0.05). However glucose-induced enhancement of AQP1 disappeared with the addition of H-89, a protein kinase A (PKA)--specific inhibitor (103% +/- 17.5% of control, p < 0.05 as compared with 5% glucose). We also examined the effect of 5% glucose on PKA activity separately in the cytosol fraction, the crude membrane fraction, and the pure plasma membrane fraction. In the cytosol fraction of 5% glucose-stimulated RPMCs, PKA activity was decreased (70.5% +/- 11.5% of control, p < 0.01), but in the crude membrane fraction, it was significantly increased (143.9% +/- 52.9% of control, p < 0.01). In the pure plasma membrane fraction, PKA activity did not change. From those findings, we hypothesize that 5% glucose augments the PKA-dependent translocation of AQP1 to the plasma membrane, mediated by PKA translocation to the intracellular AQP1 store.

Vasodilator-stimulated phosphoprotein regulates proliferation and growth inhibition by nitric oxide in vascular smooth muscle cells.

OBJECTIVE: Vasodilator-stimulated phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition. METHODS AND RESULTS: Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed. CONCLUSIONS: Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase.

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.

Additive antinociceptive effect of the combination of diazoxide, an activator of ATP-sensitive K+ channels, and sodium nitroprusside and dibutyryl-cGMP.

Using the rat paw pressure test, in which increased sensitivity is induced by intraplantar injection of prostaglandin E2, we assessed the antinociceptive effect of the ATP-sensitive K+ channel opener diazoxide and the large-conductance Ca(2+)-activated K+ channel opener NS-1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one) on the peripheral hyperalgesia induced by prostaglandin E2. Diazoxide, administered locally into the right hindpaw (20, 38, 75, 150, 300 and 600 microg), elicited a dose-dependent antinociceptive effect on prostaglandin E2-induced hyperalgesia (2 microg/paw). The effect of diazoxide at the dose of 300 microg/paw was shown to be local since it did not produce any effect when administered in the contralateral paw. The action of diazoxide (300 microg/paw) as an ATP-sensitive K+ channel opener seems to be specific, since its effect was antagonized in a dose-dependent manner by glibenclamide (40, 80 and 160 microg/paw), a specific blocker of these channels, while tetraethylammonium (7.5, 15 and 30 microg/paw), dequalinium (12.5, 25 and 50 microg/paw) or charybdotoxin (0.5, 1 and 2 microg/paw), blockers of voltage-dependent K+ channels and of small- and large-conductance Ca(2+)-activated K+ channels, respectively, were not able to abolish the antinociception induced by diazoxide. The peripheral antinociceptive effect of diazoxide was not prevented by prior administration of naloxone (12.5, 25 and 50 microg/paw), an opioid receptor antagonist, or methylene blue (75, 125 and 300 microg/paw), an agent that inhibits the activation of guanylate cyclase by nitric oxide. A low dose of diazoxide (20 microg/paw) administered together with a low dose of sodium nitroprusside (125 microg/paw) or dibutyryl cGMP (db-cGMP, 50 microg/paw) induced a marked antinociceptive effect similar to that observed when each drug was administered alone. NS1619 (75, 150 and 300 microg/paw), a specific opener of large-conductance Ca(2+)-activated K+ channels, had no antinociceptive action on prostaglandin E2-induced hyperalgesia. This series of experiments provides evidence for a peripheral antinociceptive action of diazoxide and supports the suggestion that the activation of ATP-sensitive K+ channels could be the mechanism by which sodium nitroprusside and db-cGMP induce peripheral antinociception, excluding the involvement of large-contuctance Ca(2+)-activated K+ channels in the process.