A. den Hertog

The action of isoprenaline on the smooth muscle of the guinea‐pig taenia coli.

1. The beta‐action of catecholamines on the smooth muscle of guinea‐pig taenia coli was investigated by observing the effects of isoprenaline (2 X 10(‐8)‐7.2 X 10(‐6) M) in the presence of an alpha‐blocker, phentolamine (6.3 X 10(‐6) M). Electrical and mechanical activity were recorded with the double sucrose gap method. Calcium and potassium fluxes were determined using the 45Ca and 42K isotopes. 2. Isoprenaline suppressed spontaneous spike generation, reduced the size of evoked phasic contractions, and caused a small hyperpolarization of the membrane without change in membrane resistance. These effects were abolished by a beta‐blocker, propranolol (6.8 X 10(‐6) M). 3. The hyperpolarization induced by isoprenaline was smaller in quiescent, nonstimulated muscle than in active, frequently stimulated preparations. It occurred with the same time course as the reduction in the size of evoked phasic contractions. Both effects were dose dependent and reached a maximum at 7.2 X 10(‐7) M‐isoprenaline. 4. Hyperpolarization by direct current application did not reduce the size of evoked phasic contractions until excitation threshold was reached. In the presence of isoprenaline, repolarization of the membrane to its original level by depolarizing current application did not restore the reduced phasic contractions to their original size. 5. The slopes of the current‐voltage relation in the absence and presence of isoprenaline were parallel, confirming the absence of a change in membrane resistance. Isoprenaline also did not affect membrane resistance when applied in the modified ionic environments used. 6. In different external K concentrations (0.60‐29.5 mM) the relationship between the size of the electrotonic potential and the magnitude of the isoprenaline‐induced hyperpolarization was linear. A similar, direct relation was seen between isoprenaline hyperpolarization and membrane resistance when the latter was increased by lowering external chloride to 13.3 mM. 7. Excess Cao (7.5 mM) hyperpolarized the membrane and reduced membrane resistance, but the hyperpolarization by isoprenaline was larger than in normal solution, being inversely related to the membrane resistance. The hyperpolarization was directly related to the external Ca concentration, suggesting that the magnitude of the response to the beta‐action might depend on the cytoplasmic Ca concentration. 8. In low external Na (18 mM‐Nao) the beta‐action was scarcely affected. Complete replacement of Na with choline increased membrane resistance, muscle tone and phasic contractions; in this condition the effects of isoprenaline were abolished. 9. When the Na pump was blocked by exposure to zero K, to ouabain, or to both simultaneously, isoprenaline remained highly effective. However, prolonged exposure to ouabain abolished the beta‐action. 10. Isoprenaline (1.4 X 10(‐6) M) increased 45Ca efflux by about 20%, while 45Ca influx was not changed, and 42K efflux remained constant. 11...

A comparison of the effect of temperature, metabolic inhibitors and of ouabain on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres

1. A comparison has been made of the effect of cooling, metabolic inhibitors and of ouabain on the electrogenic component of the sodium pump in mammalian non‐myelinated nerve fibres.

Some further observations on the electrogenic sodium pump in non-myelinated nerve fibres

1. A study has been made of the hyperpolarization that follows a period of electrical activity (post‐tetanic hyperpolarization) and of the hyperpolarization which develops when potassium is readmitted after bathing the desheathed vagus nerve of the rabbit in potassium‐free Locke solution during 15 min (potassium‐activated response).

Ion fluxes during the inhibitory junction potential in the guinea‐pig taenia coli.

1. Contribution of different ions to the inhibitory junction potential (i.j.p.) in the guinea‐pig taenia coli was studied by measuring the 42K, 24Na and 36Cl fluxes, the membrane resistance and the influence of various external ion concentrations. 2. The membrane resistance, as measured by the electrotonic potential, decreased transiently during the i.j.p. A maximal reduction of the electrotonic potential of about 50% was found at the top of the i.j.p. 3. The i.j.p. amplitude could be reduced by raising the external potassium concentration. Extrapolation of the relationship observed shows that the inhibitory response would be abolished at 115 mM potassium. Similar experiments were made in chloride‐free medium, chloride being replaced by isethionate. Amplitude and time course of the response were not different in chloride containing Locke solution and chloride‐free medium. 4. The half‐times of 42K, 24Na and 36Cl effluxes during rest were 29, 10 and 9 min respectively. The 42K‐efflux from the preparation was markedly increased to about three times the resting efflux during field stimulation. In low‐chloride solution a similar effect on 42K‐efflux was observed during field stimulation. Only a slight increase in the chloride efflux was observed but the sodium efflux was not affected during field stimulation. 5. From the results presented it is concluded that the inhibitory junction potential is caused by a selective increase in potassium permeability of the smooth‐muscle cell membrane.

CALCIUM RELEASE FROM SEPARATE RECEPTOR-SPECIFIC INTRACELLULAR STORES INDUCED BY HISTAMINE AND ATP IN A HAMSTER-CELL LINE

1. The specificity of intracellular Ca2+ stores to Ca(2+)‐mobilizing agonists was studied in DDT1 MF‐2 vas deferens cells of the Syrian hamster. 2. Application of histamine (100 microM) or ATP (100 microM) to the DDT1 MF‐2 cells caused an initial increase of intracellular Ca2+ followed by a lower phase as measured by using Indo‐1 as fluorescent probe at 22 degrees C. The basal Ca2+ level (146 nM) was enhanced to 309 nM by histamine and to 379 nM by ATP. 3. A transient rise in intracellular Ca2+ lasting for about 2 min was measured in the presence of histamine or ATP in the absence of extracellular Ca2+. The basal Ca2+ level (78 nM) was increased to 128 nM by histamine and to 145 nM by ATP. 4. A transient hyperpolarization was elicited in single cells as measured with microelectrodes by both agonists under Ca(2+)‐free conditions with a similar time course as the change in internal Ca2+. The hyperpolarization observed in the presence of histamine amounted to 23 mV and 31 mV with ATP. The histamine‐induced responses were abolished by the H1 histaminoceptor antagonist mepyramine (10 microM) and the responses evoked by ATP were blocked by the P2 purinoceptor antagonist suramin (300 microM). 5. A second internal Ca2+ response could only be evoked under Ca(2+)‐free conditions by applying a higher agonist concentration or after replenishing the intracellular stores with Ca2+ from the extracellular space. 6. A second addition of an optimal concentration (100 microM) of the agonist to the cells under Ca(2+)‐free conditions did not evoke mobilization of internal Ca2+ or hyperpolarization, but resulted in a rise of the cellular inositol (1,4,5)‐trisphosphate content (Ins(1,4,5)P3) as determined by a radioligand binding assay. 7. The cells responded to both agonists (100 microM) with a transient Ca2+ response if successively applied at a maximal effective concentration (100 microM) under Ca(2+)‐free conditions. 8. Simultaneous stimulation of H1 histaminoceptors and P2 purinoceptors resulted in the absence of external Ca2+ in an additional increase in internal Ca2+ represented by the amplitude and area of the response and in an increased response area of the hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

On the metabolic basis of nervous activity.

1. A study has been made of the metabolic substrates that can support the active extrusion of sodium ions from mammalian non‐myelinated nerve fibres. The post‐tetanic hyperpolarization obtained in chloride‐free Locke solution, which reflects the electrogenic component of the sodium pump, was used as the index of metabolic activity.

Different mechanisms of Ca2(+)-handling following nicotinic acetylcholine receptor stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization in C2C12 myotubes.

1 The increase in intracellular Ca2+ on nicotinic acetylcholine receptor (nAChR) stimulation, P2U‐purinoceptor stimulation and K+‐induced depolarization was investigated in mouse C2C12 myotubes by use of fura‐2 fluorescence to characterize the intracellular organisation of Ca2+ releasing stores and Ca2+‐entry process. 2 Stimulation of nAChRs with carbachol induced a rapid rise in internal Ca2+ (EC50 = 0.85±0.09 μm), followed by a sustained phase. The Ca2+ response evoked by carbachol (10 μm) was completely blocked by the nAChR antagonist, pancuronium (3 μm), but was not affected by the muscarinic antagonist, atropine (3 μm), or under conditions when Ca2+ entry was blocked by La3+ (50 μm) or diltiazem (10 μm). Addition of pancuronium (3 μm) during the sustained phase of the carbachol‐evoked response did not affect this phase. 3 Stimulation of P2U purinoceptors with ATP (1 mM) induced a somewhat higher biphasic Ca2+ response (EC50 of the rapid phase: 8.72±0.08 μm) than with carbachol. Pretreatment with La3+ abolished the sustained phase of the ATP‐induced Ca2+ response, while the response was unaffected by diltiazem or pancuronium. 4 Stimulation of the cells with high K+ (60 mM), producing the same depolarization as with carbachol (10 μm), induced a rapid monophasic Ca2+ response, insensitive to diltiazem, pancuronium or La3+. 5 Under Ca2+‐free conditions, the sustained phase of the carbachol‐ and ATP‐evoked responses were abolished. Pre‐emptying of depolarization‐sensitive stores by high K+ under Ca2+‐free conditions did not affect the carbachol‐ or ATP‐evoked Ca2+ mobilization and vice versa. Preincubation of the cells with ATP in the absence of extracellular Ca2+ decreased the amplitude of the subsequent carbachol‐induced Ca2+ response to 11%, while in the reverse procedure the ATP‐induced response was decreased to 65%. Ca2+ mobilization evoked by simultaneous addition of optimal concentrations of carbachol and ATP was increased compared to levels obtained with either agonist. 6 Preincubation with high K+ under normal conditions abolished the sustained phase of the ATP‐evoked Ca2+ response. The carbachol response consisted only of the sustained phase in the presence of high K+. 7 The carbachol‐induced Ca2+ response was completely abolished under low Na+/Ca2+‐free conditions, while under low Na+ conditions only a sustained Ca2+ response was observed. The ATP‐ and K+‐induced responses were changed compared to Ca2+‐free conditions. 8 ATP (300 μm) induced the formation of Ins(1,4,5)P3 under Ca2+‐free conditions with a comparable time course to that found for the rise in internal Ca2+. In contrast to ATP, carbachol (10 μm) did not affect Ins(1,4,5)P3 levels under Ca2+‐free conditions. 9 It is concluded that the Ca2+ release from discrete stores of C2C12 myotubes is induced by stimulation of nAChRs, P2U‐purinoceptors and by high K+. Only the P2U‐purinoceptor and nAChR activated stores show considerable overlap in releasable Ca2+. Sustained Ca2+‐entry is activated by stimulation of nAChRs and P2U‐purinoceptors via separate ion‐channels, which are different from the skeletal muscle nAChR‐coupled cation‐channel.

Induction of Na+/K(+)-ATPase activity by long-term stimulation of nicotinic acetylcholine receptors in C2C12 myotubes.

1 To investigate the role of long‐term stimulation of nicotinic acetylcholine receptors (AChRs) on the regulation of membrane potential, non‐contracting C2C12 myotubes were stimulated for 1–4 days with carbachol (10 μm) and membrane potentials were measured by the intracellular microelectrode technique after washing out of the drug. 2 The membrane potential (– 45.7 mV) gradually increased by 10.1 mV to − 55.8 mV during 4 days treatment, which was caused by enhanced electrogenic Na+/K+‐pumping. 3 The concentration‐dependent enhancement of Na+/K+‐ATPase activity in long‐term carbachol‐treated myotubes (4 days, EC50 = 5.3 μm) was prevented by co‐treatment with the competitive nicotinic AChR antagonist, pancuronium but not by the muscarinic antagonist, atropine. 4 Enhanced Na+/K+‐ATPase activity still developed in carbachol‐stimulated myotubes during cotreatment (4 days) with the nicotinic AChR‐channel blocker, chlorpromazine (1 μm). Membrane depolarization as such, obtained by incubation in high K+ medium (40 mm, 4 days) did not enhance Na+/K+‐ATPase activity. 5 Non‐treated myotubes possessed a high‐affinity ouabain binding site (Kd = 119 nm) in association with the low Na+/K+‐pumping activity. Long‐term stimulation of myotubes (4 days) with carbachol or with a combination of carbachol and chlorpromazine was accompanied by the development of an additional low‐affinity ouabain binding site (Kd = 13 μm). 6 Binding of monoclonal antibodies directed against either α1‐ or α2‐subunit of Na+/K+‐ATPase were both increased in myotubes treated with carbachol (4 days). 7 These results support the concept that nicotinic AChRs regulate Na+/K+‐ATPase activity, independent of the functionality of the receptor‐operated ion‐channel.

The effects of lithium on excitable cell membranes II. The effect on the electrogenic sodium pump of non-myelinated nerve fibres of the rat

Abstract The effect of low doses of lithium (1–5 mM) on the electrogenic component of the sodium-potassium pump was measured. The preparation was the desheathed vagus nerve of the rat mounted in a single or double sucrose-gap apparatus. Lithium reduced the maximum amplitude of the potassium-activated response (PAR). This effect was caused by competitive antagonism between lithium and potassium and not by increased membrane conductivity. It is concluded that the effect of lithium on the electrogenic sodium pump possibly illuminates its mode of action in manic depressive illness.

The effect of diphenylhydantoin on the electrogenic component of the sodium pump in mammalian non-myelinated nerve fibres

Abstract A study has been made of the effect of diphenylhydantoin on the electrogenic component of the sodium pump in non-myelinated fibres of the desheathed rabbit vagus nerve. Acute or chronic administration of this drug did not affect the excitability, nor the membrane permeability, nor the activity of the sodium pump of the nerve fibres.