SUMMARY Background The angiotensin (Ang) converting enzyme 2 (ACE2)/Ang-(1-7)/Mas receptor pathway is an important component of the renin–angiotensin system and has been suggested to exert beneficial effects in ischemic stroke. Aims This study explored whether the ACE2/Ang-(1-7)/Mas pathway has a protective effect on cerebral ischemic injury and whether this effect is affected by age. Methods We used three-month and eight-month transgenic mice with neural over-expression of ACE2 (SA) and their age-matched non-transgenic (NT) controls. Neurological deficits and ischemic stroke volume were determined following middle cerebral artery occlusion (MCAO). In oxygen and glucose deprivation (OGD) experiments on brain slices, the effects of the Mas receptor agonist (Ang1-7) or antagonist (A779) on tissue swelling, Nox2/Nox4 expression reactive oxygen species (ROS) production and cell death were measured. Results (1) Middle cerebral artery occlusion -induced ischemic injury and neurological deficit were reduced in SA mice, especially in eight-month animals; (2) OGD-induced tissue swelling and cell death were decreased in SA mice with a greater reduction seen in eight-month mice; (3) Ang-(1–7) and A779 had opposite effects on OGD-induced responses, which correlated with changes in Nox2/Nox4 expression and ROS production. Conclusions Angiotensin converting enzyme 2/Ang-(1-7)/Mas axis protects brain from ischemic injury via the Nox/ROS signaling pathway, with a greater effect in older animals.

Membrane conductances during hypoosmotic swelling were characterized in rat astrocytes in primary tissue culture. Using whole cell patch clamp techniques, mean ± SEM cell conductance in isoosmotic phosphate‐buffered saline (PBS) was 55.6 ± 5.8 pS/pF. Cell conductance (mean ± SEM) increased from this initial value to 187 ± 46%, 561 ± 188%, and 1216 ± 376% within 9 min of exposure to 220 mOsm, 190 mOsm, and 145 mOsm PBS, respectively. With each of these hypoosmotic exposures, no change occurred in membrane capacitance. When CsCl replaced KCl in the microelectrode solution, a similar conductance increase was obtained at each osmolality. However, when gluconate salts were used in place of chloride salts in the electrode solution, no significant conductance increase was observed with 190 mOsm PBS. With a KCl microelectrode solution, all conductance increase which occurred in 190 mOsm PBS was inhibited by 200 μM niflumic acid, but not by 5 mM BaCl2. Both niflumic acid and BaCl2 inhibited 60–80% of the conductance increase of cells in 145 mOsm PBS. Using a microelectrode solution containing taurine as the major anion, membrane conductance increased 5‐fold when cells were placed in 250 mOsm medium. This conductance increase was completely inhibited by 200 μM niflumic acid. Thus, independent chloride and potassium conductances are activated by hypoosmotic swelling of cultured astrocytes while plasma membrane area is unaltered. The chloride conductance pathway is activated at a significantly lower degree of hypoosmotic exposure than that which activates the potassium pathway and may be permeable to anionic taurine. These conductance pathways may mediate diffusive loss of potassium, chloride, and taurine from these cells during volume regulation following hypoosmotic swelling. GLIA 20:254–261, 1997. © 1997 Wiley‐Liss, Inc.

Taurine transport is important for volume regulation of cultured neurons and astroglial cells. Both cell types utilize similar mechanisms for taurine accumulation and efflux. However, taurine lost from cerebellar Purkinje cells in vivo is accumulated by adjacent astrocytes during hypoosmotic hyponatremia. To examine mechanisms for transfer of taurine between cell types, we measured relative sensitivities of taurine loss from cultured neurons and astrocytes. Primary cultures of hippocampal neurons and astrocytes were grown from embryonic and neonatal rat brain, respectively. Neurons were used after 10-14 days in culture. Astrocytes were used after 14 days in culture and were grown in the same culture medium used for neurons for 3 days prior to experimentation. Cells were incubated at 37 degrees C for 30 min in isoosmotic (290 mOsm) phosphate-buffered saline (PBS). The PBS was then changed to fresh isoosmotic or to hypoosmotic PBS (270 mOsm or 250 mOsm), made by reducing the NaCl concentration. Cell volume and taurine content were determined immediately before hypoosmotic exposure or 3, 15, or 30 min later. In isoosmotic PBS, astrocytes contained 162 +/- 18 nmol taurine/mg protein (mean +/- SEM), equivalent to an intracellular concentration of 30.2 +/- 2.1 mM. No taurine loss was detectable after 3 or 15 min in either hypoosmotic PBS, but after 30 min in 270 or 250 mOsm PBS, astrocyte taurine was reduced by 8.0% or 22.2%, respectively. Neurons initially contained 114 +/- 13 nmol taurine/mg protein, equivalent to an intracellular taurine concentration of 22.2 +/- 2.5 mM. After 3 min of exposure to 270 or 250 mOsm PBS, the cells had lost 17 +/- 5% or 25 +/- 4% of their taurine content, respectively. Cell volumes of each cell type were similarly affected by hypoosmotic exposure. We conclude that taurine loss from cultured hippocampal neurons is more sensitive to osmotic swelling than taurine loss from cultured hippocampal astrocytes. This characteristic, if present in cells of the hippocampus in vivo, could lead to net transfer of taurine from neurons to glial cells during pathological conditions which cause cell swelling.

No abstract

We examined the calmodulin dependence of anion channel activation during hypo-osmotic swelling in rat cerebral astrocytes. Control cells bathed in iso-osmotic (290 mOsm) phosphate-buffered saline (PBS) and recorded using a patch electrode containing 140 mM KCl increased membrane conductance threefold over basal levels after 12 min in hypo-osmotic (200 mOsm) PBS. Cells injected with monoclonal anticalmodulin antibody demonstrated no increase in membrane conductance during a subsequent exposure to hypo-osmotic PBS. In contrast, cells iontophoretically injected with monoclonal antiglial fibrillary acidic protein antibody or with anticalmodulin antibody absorbed with an excess of free calmodulin demonstrated an increase in conductance during hypo-osmotic exposure similar to that of control cells. Conductance in iso-osmotic conditions was unchanged by antibody injection. Similar results were obtained when using patch electrode and bath solutions containing chloride as the only cell permeant ion, indicating a calmodulin-dependent anion current is activated with this degree of hypo-osmotic treatment. Western blots confirmed the specificity of the anticalmodulin and antiglial fibrillary acidic protein antibodies used in this study for proteins of 17 and 51 kD, respectively. In addition, in vitro studies demonstrated inhibition of the calmodulin-dependent activation of phosphodiesterase by the anticalmodulin antibody. Thus, binding of this antibody to calmodulin causes functional inhibition of calmodulin activity. No change in the intensity or cellular distribution of calmodulin immunostaining was observed during 30 min of hypo-osmotic exposure. However, increased immunostaining for activated calmodulin kinase IIalpha was observed after 10 min of hypo-osmotic exposure, suggesting initiation of calmodulin-dependent processes by cell swelling. The data indicate calmodulin activity is critical for activation of volume-regulated anion channels in rat cerebral astrocytes.