Intracellular pH in rat isolated superior cervical ganglia in relation to nicotine‐depolarization and nicotine‐uptake

Abstract

Summary1. The intracellular pH (pHi) of rat isolated superior cervical ganglia incubated in normal Krebs solution (pH,=7-37) was estimated to be 7-33 from the uptake of a weak acid, 14C-5,5-dimethyloxazolidine-2,4-dione (DM0). Addition of 30 ,uM nicotine for 30 min reduced the DMO-estimated pHi by 015 units to 7'18. This effect was prevented by hexamethonium (25 mM) or by depolarizing the ganglion with K+ (124 mM).2. 3H-Nicotine (30 pAM) was concentrated within the ganglia to an intracellular/extracellular concentration ratio (CI/C0) of 5'54 in normal Krebs solution and 4'61 in 2 5 mm hexamethonium. This would suggest an intracellular pH of 6&54 and 6'63 respectively. In ganglia previously depolarized by K+ the corresponding values for Ci/C0 were 4'02 (minus hexamethonium, estimated pHi 6'95) and 4417 (plus hexamethonium, estimated pHK 6'94).3. A multicompartment cell interior comprising an acid cytoplasm (pH-6'6) and more alkaline nucleus and mitochondria is proposed to explain the difference between the values of pHi estimated from the uptake of DMO and nicotine. It is suggested that the fall in pH, during nicotine-depolarization results from metabolic stimulation following Na+ entry.

Intracellular pH and the distribution of weak acids and bases in isolated rat superior cervical ganglia.

“…For example, one might subdivide the tissue into its various gross compartments: neurones, fibres, glial cells, etc. However, since the aim of pH1 measurements is usually to estimate the cytoplasmic pH, it seems more helpful to treat the cell population as relatively uniform (in the absence of contrary evidence), and then simply divide the total intracellular space into two pH compartments: the cytoplasm, which in the tissue as a whole comprises the bulk (88 %) of the intracellular volume (Brown & Halliwell, 1972), and the residual space occupied by membrane-bound intracellular organelles. Taking the relative volumes of these two compartments (Vcyt = 88%, Vres = 12%) and setting PHacid = PHDMO = 7-3 and pHbase = 6-55, eqns.…”

“…In previous experiments on isolated sympathetic ganglia (Brown & Halliwell, 1972), the DMO method indicated an intracellular pH (pHi) of 7*3 at an extracellular pH (pHo) of 7.4. The weak base nicotine, however, distributed as though the effective pH, was 6*6.…”

“…It has previously been established that neither nicotine nor DMO are significantly metabolized 599 600 D. A. BROWN AND J. GARTHWAITE in isolated ganglia (Brown et al 1971;Brown & Halliwell, 1972). To check for metabolism of the other compounds, ganglia (usually four) were incubated for 1 or 2 hr at 25 0C in the presence of the test compound.…”

“…Although many of the compounds have well known pharmacological actions, these were controlled either by addition of antagonists (e.g. hexamethonium with nicotine; see Brown & Halliwell, 1972), by using concentrations below those with pharmacological activity (tested by electrophysiological recordings: cf. Brown et al 1971), or when an effect on pH1 was suggested, by testing their effects on the distribution of nicotine or DMO.…”

“…The experimental routine for measuring uptake of labelled markers was essentially as previously described (Brown, Halliwell & Scholfield, 1971;Brown & Halliwell, 1972) except that in preparation for scintillation counting, the tissue was dissolved in 'Soluene' (Packard) and 'Instagel' (Packard) was used as scintillant. When the uptake of [L4C]trimethylamine was measured, however, some modification was necessary as the free base form of trimethylamine is volatile (b.p.…”