Dose-dependent effects of angiotensin-(1–7) on the NHE3 exchanger and [Ca2+]i in in vivo proximal tubules

The acute direct action of angiotensin-(1–7) [ANG-(1–7)] on bicarbonate reabsorption ( JHCO3−) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO3− is 2.82 ± 0.078 nmol·cm−2·s−1 (50). ANG-(1–7) (10−12 or 10−9 M) in luminally perfused tubules decreases JHCO3− (36 or 60%, respectively), but ANG-(1–7) (10−6 M) increases it (80%). A779 increases JHCO3− (30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1–7) on it. S3226 decreases JHCO3− (45%) and changes the stimulatory effect of ANG-(1–7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1–7). Our results indicate that in the basal condition endogenous ANG-(1–7) inhibits JHCO3− and that the biphasic dose-dependent effect of ANG-(1–7) on JHCO3− is mediated by the Mas receptors via the Na+/H+ exchanger 3 (NHE3). The control value of intracellular Ca2+ concentration ([Ca2+]i), as monitored using fura-2 AM, is 101 ± 2 nM ( 6 ), and ANG-(1–7) (10−12, 10−9, or 10−6M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca2+]i (25%) but impairs the stimulatory effect of all doses of ANG-(1–7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1–7) dose-dependent effects on [Ca2+]i and JHCO3−. Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1–7) on [Ca2+]i and JHCO3− may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca2+]i modification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

The Effects of HCl and CaCl2 Injections on Intracellular Calcium and pH in Voltage-clamped Snail (Helix aspersa) Neurons

To investigate the mechanisms by which low intracellular pH influences calcium signaling, I have injected HCl, and in some experiments CaCl2, into snail neurons while recording intracellular pH (pHi) and calcium concentration ([Ca2+]i) with ion-sensitive microelectrodes. Unlike fluorescent indicators, these do not increase buffering. Slow injections of HCl (changing pHi by 0.1–0.2 pH units min−1) first decreased [Ca2+]i while pHi was still close to normal, but then increased [Ca2+]i when pHi fell below 6.8–7. As pHi recovered after such an injection, [Ca2+]i started to fall but then increased transiently before returning to its preinjection level. Both the acid-induced decrease and the recovery-induced increase in [Ca2+]i were abolished by cyclopiazonic acid, which empties calcium stores. Caffeine with or without ryanodine lowered [Ca2+]i and converted the acid-induced fall in [Ca2+]i to an increase. Injection of ortho-vanadate increased steady-state [Ca2+]i and its response to acidification, which was again blocked by CPA. The normal initial response to 10 mM caffeine, a transient increase in [Ca2+]i, did not occur with pHi below 7.1. When HCl was injected during a series of short CaCl2 injections, the [Ca2+]i transients (recorded as changes in the potential (VCa) of the Ca2+-sensitive microelectrode), were reduced by only 20% for a 1 pH unit acidification, as was the rate of recovery after each injection. Calcium transients induced by brief depolarizations, however, were reduced by 60% by a similar acidification. These results suggest that low pHi has little effect on the plasma membrane calcium pump (PMCA) but important effects on the calcium stores, including blocking their response to caffeine. Acidosis inhibits spontaneous calcium release via the RYR, and leads to increased store content which is unloaded when pHi returns to normal. Spontaneous release is enhanced by the rise in [Ca2+]i caused by inhibiting the PMCA.

Resistance of Retinal Extracellular Space to Ca2+Level Decrease: Implications for the Synaptic Effects of Divalent Cations

Ion-sensitive microelectrodes were used to measure the variations of [Ca2+]o induced by application of low Ca2+ media in the superfused eyecup preparation of the Pseudemys turtle. The aim of the experiments was to evaluate the possibility, suggested by previous studies, that in the deep, sclerad, layers of the retina [Ca2+]omay remain high enough to sustain chemical synaptic transmission even after prolonged application of low-Ca2+ saline. It was found that, at depths of 100–200 μm from the vitreal surface, [Ca2+ ]o did not fall below 1 mM even after application for periods of 30–60 min of nominally Ca2+-free media, and it was >0.3 mM after 30-min application of media containing EGTA and with a Ca2+concentration of 1 nM. Previous studies in isolated salamander photoreceptors have shown that a reduction of [Ca2+]o to 0.3–1.0 mM may result in a paradoxical increase of Ca2+ influx into synaptic terminals due to the reduced screening of negative charge on the external face of the plasma membrane. On the basis of these results, the persistence or enhancement of synaptic transmission from photoreceptors to horizontal cells observed in various retinas treated with low-Ca2+ media may be accounted for within the classical Ca2+-dependent theory of synaptic transmission without invoking a Ca2+-independent mechanism.

Methionine Sulfoximine, a Glutamine Synthetase Inhibitor, Attenuates Increased Extracellular Potassium Activity during Acute Hyperammonemia

Hyperammonemia causes glutamine accumulation and astrocyte swelling. Inhibition of glutamine synthesis reduces ammonia-induced edema formation and watery swelling in astrocyte processes. Ordinarily, astrocytes tightly control extracellular K+ activity [K+]e. We tested the hypothesis that acute hyperammonemia interferes with this tight regulation such that [K+]e increases and that inhibition of glutamine synthetase reduces this increase in [K+]e. Ion-sensitive microelectrodes were used to measure [K+]e in parietal cortex continuously over a 6-h period in anesthetized rats. After i.v. sodium acetate infusion in eight control rats, plasma ammonia concentration was 33 ± 26 μmol/L (± SD) and [K+]e remained stable at 4.3 ± 1.6 mmol/L. During ammonium acetate infusion in nine rats, plasma ammonia increased to 594 ± 124 μmol/L at 2 h and to 628 ± 135 μmol/L at 6 h. There was a gradual increase in [K+]e from 3.9 ± 0.7 to 6.8 ± 2.7 mmol/L at 2 h and 11.8 ± 6.7 mmol/L at 6 h. In eight rats, L-methionine-D,L-sulfoximine (150 mg/kg) was infused 3 h before ammonium acetate infusion to inhibit glutamine synthetase. At 2 and 6 h of ammonium acetate infusion, plasma ammonia concentration was 727 ± 228 and 845 ± 326 μmol/L, and [K+]e was 4.5 ± 1.9 and 6.1 ± 3.8 mmol/L, respectively. The [K+]e value at 6 h was significantly less than that obtained with ammonium acetate infusion alone but was not different from that obtained with sodium acetate infusion. We conclude that acute hyperammonemia impairs astrocytic control of [K+]e and that this impairment is linked to glutamine accumulation rather than ammonium ions per se.