We have studied the regulation of intracellular pH (pHi), and HCO3(-)-dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pHi was 6.96 in both nominally CO2/HCO3(-)-free, HEPES-buffered saline (6.96 +/- 0.14; n = 48) and in a saline containing 5% CO2/24 mM HCO3- (6.96 +/- 0.18; n = 48) (at pH 7.4). Inhibition of the Na+/H+ exchange by amiloride (2 mM) caused a significant decrease of pHi in nominally CO2/HCO3(-)-free saline. Addition of CO2/HCO3- in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na+ also caused a fall of pHi, and addition of CO2/HCO3- in Na(+)-free saline evoked a further fall of pHi, while the outward current was reduced or even reversed. The stilbene 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM) reduced the pHi recovery from the CO2/HCO3(-)-evoked acidification, and blocked the prominent intracellular acidification upon removal of CO2/HCO3-. Removal of external Cl- had little effect on these pHi changes. Lowering the external pH from 7.4 to 6.6 in CO2/HCO3(-)-containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO2/HCO3-. The results suggest that the CO2/HCO3(-)-dependent current is partly due to a reversible bidirectional, electrogenic Na(+)-HCO3- cotransporter, which helps to regulate pHi in these cells. In addition, a prominent Na+/H+ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steady-state pHi.

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We have investigated the pro- and anti-inflammatory effects of ricinoleic acid (RA), the main active principle of castor oil, in an experimental model of blepharitis induced by intradermal injection of carrageenan in the guinea-pig eyelid and its possible capsaicin-like mode of action on acutely dissociated rat dorsal root ganglia (DRG) neurons in vitro. Topical treatment with RA (10-100 mg/guinea-pig) or capsaicin (1-10 mg/guinea-pig) caused eyelid reddening and oedema. At lower doses (0.3-3 mg/guinea-pig and 0.009-0.09 mg/guinea-pig for RA and capsaicin, respectively) both drugs significantly potentiated the eyelid oedema induced by carrageenan. The tachykinin NK1 receptor antagonist FK 888 (0.59 mg/kg s.c.) abolished the potentiation of carrageenan-induced eyelid oedema induced by either RA or capsaicin. The neutral endopeptidase inhibitor, thiorphan (1.3 mg/kg i.v.) significantly enhanced the potentiation of carrageenan-induced eyelid oedema produced by RA. This potentiating effect was abolished by FK 888. Repeated (8 days) topical application of RA (0.9 mg/guinea-pig) or capsaicin (0.09 mg/guinea-pig) inhibited the carrageenan-induced eyelid oedema. This anti-inflammatory effect was accompanied by a reduction (75%-80% of SP and 46%-51% of NKA) in tachykinin content of the eyelids, as determined by radioimmunoassay. In dissociated rat DRG neurons, RA (0.1 mM for 5 min) significantly inhibited the inward currents induced by application of capsaicin (1 microM) and/or low pH (5.8), without inducing any currents by itself or changing voltage-dependent currents. Moreover, after 24-h incubation, RA (0.1 mM) significantly decreased the capsaicin (1 microM)-induced calcitonin gene-related peptide (CGRP) release from rat DRG neurons, whereas acute drug superfusion did not evoke CGRP release by itself. Summarizing, RA possesses capsaicin-like dual pro-inflammatory and anti-inflammatory properties which are observed upon acute and repeated application, respectively. However, unlike capsaicin, RA does not induce inward current in DRG neurons and it is devoid of algesic properties in vivo.