Angiotensin II Receptor Type I-Regulated Anion Secretion in Cystic Fibrosis Pancreatic Duct Cells

The β-adrenergic (cAMP-dependent) regulation of Cl⁻ conductance is defective in cystic fibrosis (CF). The present study explored alternative regulation of anion secretion in CF pancreatic ductal cells (CFPAC-1) by angiotensin II (AII) using the short-circuit current (I _SC ) technique. An increase in I _SC could be induced in CFPAC-1 cells by basolateral or apical application of AII in a concentration-dependent manner (EC₅₀ at 3 μm and 100 nm, respectively). Angiotensin receptor subtypes were identified using specific antagonists, losartan and PD123177, for AT₁ and AT₂ receptors, respectively. It was found that losartan (1 μm) could completely inhibit the AII-induced I _SC , whereas, PD123177 exerted insignificant effect on the I _SC , indicating predominant involvement of AT₁ receptors. The presence of AT₁ receptors in CFPAC-1 cells was also demonstrated by immunohistochemical studies using specific antibodies against AT₁ receptors. Confocal microscopic study demonstrated a rise in intracellular Ca²⁺ upon stimulation by AII indicating a role of intracellular Ca²⁺ in mediating the AII response. Depletion of intracellular but not extracellular pool of Ca²⁺ diminished the AII-induced I _SC . Treatment of the monolayers with a Cl⁻ channel blocker, DIDS, markedly reduced the I _SC , indicating that a large portion of the AII-activated I _SC was Cl⁻-dependent. AII-induced I _SC was also observed in monolayers whose basolateral membranes had been permeabilized by nystatin, suggesting that the I _SC was mediated by apical Cl⁻ channels. Our study indicates an AT₁-mediated Ca²⁺-dependent regulatory mechanism for anion secretion in CF pancreatic duct cells which may be important for the physiology and pathophysiology of the pancreas. Received: 17 June 1996/Revised: 14 November 1996     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: I. Selectivity of Protection Against Oxidative Stress

Abstract: Previous research has suggested that the initial effects of cellular free radical neurotoxic insult involve large increases in intracellular Ca²⁺. However, the exact role of oxidative stress on the various parameters involved in these increases has not been specified. The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 µM H₂O₂ for 30 min in growth medium alone or containing either nifedipine (L-type Ca²⁺ antagonist), conotoxin (N-type antagonist), Trolox (vitamin E analogue), or α-phenyl-n-tert-butylnitrone (nitrone trapping agent; PBN). The concentrations of H₂O₂ were chosen by examining the degree of cell killing induced by exposure to graded concentrations of H₂O₂. The 5 and 25 µM concentrations of H₂O₂ produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 µM concentration produced a moderate degree of cell killing that did not increase between the two times. Fluorescent imaging was used to visualize intracellular Ca²⁺ changes in fura-2-loaded cells. Baseline (pre-30 mM KCI) Ca²⁺ levels were increased significantly by H₂O₂ treatment (e.g., 300 µM, 200%), but the rise in the level of free intracellular Ca²⁺ after KCI stimulation (i.e., peak) was decreased (e.g., 300 µM, 50%) and the cell's ability to sequester or extrude the excess Ca²⁺ (i.e., Ca²⁺ recovery time) after depolarization was decreased significantly. All compounds prevented baseline Ca²⁺ increases and, with the exception of conotoxin, antagonized the peak decreases in Ca²⁺. It is interesting that after 300 µM H₂O₂ exposure, only Trolox was partially effective in preventing these deficits in recovery. Conotoxin increased the decrement recovery in the absence of H₂O₂. However, in cells exposed to 5 or 25 µM H₂O₂, conotoxin as well as the other agents were effective in preventing the deficits in recovery.     

Ethanol Promotes Apoptosis in Cerebellar Granule Cells by Inhibiting the Trophic Effect of NMDA

Abstract: When primary cultures of cerebellar granule neurons are grown in a physiological concentration of KCl (5 m M ) they undergo apoptosis, which can be prevented by growing the cells in the presence of N -methyl- d -aspartate (NMDA). We now show that ethanol inhibits this trophic effect of NMDA, i.e., promotes apoptosis, and also inhibits the NMDA-induced increase in intracellular Ca²⁺ concentration in cells grown in 5 m M KCl. Both effects of ethanol show a similar concentration dependence and are reversed by a high concentration of glycine, the co-agonist at the NMDA receptor. The data suggest that the effect of ethanol on apoptosis is mediated, at least in part, by inhibition of NMDA receptor function. This effect of ethanol to increase apoptosis could contribute to the previously described in vivo sensitivity of the developing cerebellum to ethanol-induced damage.     

Mast Cells Contain Large Quantities of Secretagogue-Sensitive N-Acetylaspartate

Abstract: Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N -acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.     

Hypoxia Dramatically Increases the Nonspecific Transport of Blood-Borne Proteins to the Brain

Abstract: Increased cerebrovascular permeability is an important factor for the development of cerebral edema. To investigate the effect of hypoxia on the transport of blood-borne proteins to the brain, we used a cell culture model of the blood-brain barrier (BBB) consisting of a coculture of brain capillary endothelial cells and astrocytes that closely mimics the in vivo situation. The permeability of albumin, a marker of the nonspecific transcellular route, is extremely low in this in vitro model of the BBB. After hypoxia, a huge increase in the permeability of albumin is detected. Despite the opening of the tight junctions already demonstrated after hypoxia, the increase in the permeability of albumin is mainly attributed to an increase of the nonspecific vesicular transport in the cell, attested by the temperature dependence of the phenomenon and the visualization of labeled apotransferrin in the cytoplasm. The increase of this pathway could participate in the development of brain edema during hypoxia.     

N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABAAα6 Subunit in Cerebellar Granule Cells

Abstract: We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 µM) results in the transient increase in content of GABA_Aα6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABA_B receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in α6 levels induced by 30 µM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional α6-containing GABA_A receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA_Aα6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA_A receptor subunit composition.     

Ethanol Specifically Inhibits NMDA Receptors with Affinity for Ifenprodil in the Low Micromolar Range in Cultured Cerebellar Granule Cells

Abstract: The effect of ethanol on the intracellular Ca²⁺ concentration response to NMDA in rat cerebellar granule cells grown in low or high KCI concentrations has been studied using image analysis. The cells grown in low KCI displayed high sensitivity for glycine. The subtype-selective antagonist ifenprodil inhibited the response with high (in the low micromolar range) and low (in the high micromolar range) potency. Ethanol affected the high-potency component in these cultures. In cells grown in high KCI the glycine sensitivity was lower, and a low potency for ifenprodil (high micromolar) dominated. These cells were not significantly sensitive to ethanol. The results indicate that the component displaying potency for ifenprodil in the low micromolar range with properties of the NR2B subunit is the target for ethanol action on the NMDA receptor.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

Oleic Acid Inhibits Gap Junction Permeability and Increases Glucose Uptake in Cultured Rat Astrocytes

Abstract: The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape-loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose-dependent inhibition of gap junction permeability by 79.5% at 50 µ M , and no further inhibition was observed by increasing the oleic acid concentration to 100 µ M . The oleic acid-mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid-related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.