2--7--bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl-ester and propionic-acid

A growing body of evidence hints at intracellular free protons to be involved in the modulation of electric activity of cortical neurones. In this study we demonstrate that application of the weak acid propionate (2.5-20 mM) transiently lowers intracellular pH (pH(i)) of BCECF-AM loaded CA3-neurones in hippocampal slices. The predictability of this acidification prompted us to use propionate as a tool to investigate effects of pH(i) on spontaneous bioelectric activity (SBA) and epileptiform activity (EA, induced by bicuculline, caffeine or low magnesium) of CA3 neurones: SBA and EA were transiently suppressed by 2-20 mM propionate - coinciding with the transient neuronal acidification. As activation of Na(+)/H(+)-exchangers (NHE) is involved in the recovery from neuronal acidosis and NHE-inhibition alone is known to increase the activity of intracellular free protons of hippocampal neurones, we tested the effect of the NHE-blockers amiloride (0.5-1 mM) or HOE642 (200 microM) on SBA and EA of CA3-neurones. Long-term application of NHE-inhibitors alone continuously suppressed SBA and EA, which recovered during additional exposure to the weak base trimethylamine (5-10 mM). Simultaneous administration of propionate and NHE-blockers intensified the inhibition of neuronal activity. Together, these results indicate that intracellular acidification inhibits bioelectric activity of hippocampal CA3-neurones. This supports the hypothesis that pH(i) contributes to the control of cortical excitability.

Topics: Animals; Epilepsy; Fluoresceins; Guanidines; Guinea Pigs; Hippocampus; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Methylamines; Neurons; Propionates; Sulfones

Intracellular pH (pHi) regulation was studied in neurons from two chemosensitive [nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM)] and two nonchemosensitive [hypoglossal (Hyp) and inferior olive (IO)] areas of the medulla oblongata. Intrinsic buffering power (betaint) was the same in neurons from all regions (46 mM/pH U). Na+/H+ exchange mediated recovery from acidification in all neurons [Ritucci, N. A., J. B. Dean, and R. W. Putnam. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R433-R441, 1997]. Cl-/HCO-3 exchange mediated recovery from alkalinization in VLM, Hyp, and IO neurons but was absent from most NTS neurons. The Na+/H+ exchanger from NTS and VLM neurons was fully inhibited when extracellular pH (pHo) <7.0, whereas the exchanger from Hyp and IO neurons was fully inhibited only when pHo <6.7. The Cl-/HCO-3 exchanger from VLM, but not Hyp and IO neurons, was inhibited by pHo of 7.9. These pH regulatory properties resulted in steeper pHi-pHo relationships in neurons from chemosensitive regions compared with those from nonchemosensitive regions. These differences are consistent with a role for changes of pHi as the proximate signal in central chemoreception and changes of pHo in modulating pHi changes.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Ammonium Chloride; Animals; Animals, Newborn; Chemoreceptor Cells; Fluoresceins; Homeostasis; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Medulla Oblongata; Neurons; Propionates; Rats; Solitary Nucleus

The mean extracellular pH (pHe) within solid tumours has been found to be lower than in normal tissues. Agents which cause intracellular acidification at low pHe might have selective toxicity towards cells in tumours. Weak acids (or their anions) with pKa values in the range of 4-6 have a higher proportion of molecules in the uncharged form at low pHe and can diffuse more rapidly into cells. The effects of organic acids including succinate, monomethyl succinate and malonate to acidify cells have been evaluated under conditions of different pHe in the acidic range. These weak acids caused intracellular acidification of murine EMT-6 and human MGH-U1 cells in a concentration and pHe dependent fashion. At concentrations of 10 mM and above, these acids also caused in vitro cytotoxicity to these cells at low pHe (< 6.5). The rate and extent of cellular acidification caused by these weak acids, and their cytotoxicity at low pHe, were enhanced by exposure to amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA), agents which inhibit Na+/H+ exchange, and hence the regulation of intracellular pH. Acid dependent cytotoxicity was also investigated in a murine solid tumour using the endpoints of growth delay and colony formation in vitro following treatment in vivo. Agents were tested alone or with 15 Gy X-rays to select a population of hypoxic (and presumably acidic) cells. Achievable serum concentrations of succinate were about 1 mM and no antitumour activity of succinate was detected when used in this way. It is concluded that weak acids are selectively taken up into cells, and can cause selective cellular acidification and toxicity, at low pHe in culture. Weak acids that are normal cellular metabolites are not toxic in vivo, but weak acids carrying cytotoxic groups offer the potential for selective uptake and toxicity under the conditions of low pHe that exist in many solid tumours.

Topics: Amiloride; Animals; Butyrates; Butyric Acid; Carboxylic Acids; Cell Hypoxia; Cell Survival; Female; Fluoresceins; Humans; Hydrogen-Ion Concentration; Malonates; Mammary Neoplasms, Experimental; Mice; Propionates; Sarcoma, Experimental; Sodium-Hydrogen Exchangers; Succinates; Succinic Acid; Tumor Cells, Cultured; Urinary Bladder Neoplasms