Stimulation of a sodium influx by cAMP in Helix neurons

Brain Research, 276 (1983) 289-296

289

Elsevier

Stimulation of a Sodium Influx by cAMP in Helix Neurons J. B. ALDENHOFF, G. HOFMEIER, H. D. LUX* and D. SWANDULLA

Department of Neurophysiology, Max-Planck-lnstitutefor Psychiatry, Kraepelinstr. 2, D-8000Munich 40 (F.R. G.) (Accepted February 15th, 1983)

Key words: cAMP - - intracellular injection - - intracellular sodium and chloride - - sodium current - - voltage clamp - - snail neuron

Brief pressure injections of aqueous solutions of cAMP in identified neurons of Helix pomatia caused depolarizations which lasted for tens of seconds. In voltage-clamped neurons an inward current of similar duration was induced which saturated at 10 juA/cm2 cell surface. In the range of negative membrane potentials with little voltage-dependent activation, this current was not accompanied by a change in membrane conductance. The inward current was not produced by injection of ATP, ADP, adenosine, inosine or cGMP. cAMP derivatives produced longer-lasting effects. Prolongation of the inward current was also observed after inhibition of the phosphodiesterase by IBMX. Drugs which block active transport had no effect on the response to cAMP injection. The inward current depended on extracellular sodium, and was maximal when all other mono- and divalent cations were replaced by Na ÷. The cAMP-induced current was accompanied by a transient increase in [Na+]i, but there was no change in [Cl-]i. Li ÷ could largely substitute for Na ÷; Ca 2+ was less effective. Addition of Mg2+ or Ca2+ to solutions containing a high Na+-concentration inhibited the response. Internal acidification with HC1 reversibly enhanced the inward current. These data indicate that the depolarizing effect of cAMP can be accounted for by an inward movement of Na-ions, and that the effect is augmented by H+-ions. INTRODUCTION It is known that adenosine 3',5'-cyclic m o n o p h o s phate ( c A M P ) exerts its actions at the intracellular side of the m e m b r a n e (for review see Siggins20). W e therefore used intracellular injection of aqueous solutions of c A M P and related c o m p o u n d s to directly study the action of the nucleotide on electrical properties of the m e m b r a n e s of snail neurons. Preliminary reports 2 described a transient m e m b r a n e depolarization after c A M P injection, which is due to the induction of an inward current, as seen in voltage clamp experiments. Surprisingly, this inward current was not associated with a change in m e m b r a n e conductance and did not r e s p o n d to changes in membrane potential. Thus it differs from the current described in Aplysia neurons by Pellmar 19 and in Helix neurons by D e t e r r e et al. 6. These observations have p r o m p t e d us to investigate the ionic mechanisms underlying the c A M P response. This study is confined to the short-term effects of c A M P application; we m a k e no a t t e m p t to follow effects which m a y develop over tens of minutes. ( F o r this see D r a k e and Treist* To whom correspondence should be addressed

0006-8993/83/$03.00© 1983 Elsevier Science Publishers B.~¢.

man 7 and Levitan and Norman14). W e present evidence that c A M P stimulates an influx of sodium ions. It is likely that m a n y of the changes in m e m b r a n e behavior that have been observed following external application of c A M P derivatives or of agents that increase the intracellular level of c A M P (for review see N a t h a n s o n Is) may, in fact, reflect nucleotide-induced changes in internal ionic composition. MATERIALS AND METHODS

Preparation and recording The e x p e r i m e n t s were done on the larger neuronal somata of the subesophageal ganglia of the snail, Helix pomatia. All connective sheaths were r e m o v e d after exposure to pronase (1 mg/ml saline for about 5 min). The recording set-up was as described previousThe m e m b r a n e potential was m e a s u r e d differentially b e t w e e n the intracellular microelectrode and a 3 M K C l - a g a r reference electrode s e p a r a t e d from the bath ground and located near the p r e p a r a t i o n .

lyl0,12.

290 Clamp current was delivered to the cell through a separate microelectrode, by a clamp amplifier with

without electric artifacts as concluded from the ab-

up to + 140 V output (Datel 300 A). Clamp amplifi-

sence of a change in m e m b r a n e current if solutions such as 110 m M KCI or even distilled water and oil

cation was about 5 × 103, increasing to 105 below fre-

were injected in the studied range of injection vol-

quencies corresponding to the m e m b r a n e time con-

umes. Electrophoresis of c A M P gave principally similar results, but the resolution of the onset of the

stant, thus more critically controlling the m e m b r a n e potential. Clamp currents were measured in the bath

c A M P - i n d u c e d effects was rather poor due to the relatively longer duration of the electrophoretic appli-

by a virtual ground IV-converter. The temperature of the bath was held constant at 20 __ 0.1 °C with a

cation. Since there is also the problem of the effective

thermoelectric device and a thermistor in a feedback configuration. In experiments with zero [CI-]o and

transport number in electrophoretic application, pressure injection of the drugs was generally preferred.

with an intracellular Cl--selective electrode, the po-

H ÷ or CI- were, however, injected electrophoretically from separate electrodes, with current flow against

tential electrode contained a mixture of 0.6 M K2SO 4 (85%) and 1.5 M KC1 (15%) 5, and the current carry-

the voltage clamp to avoid current across the membrane.

ing electrode was filled with K-aspartate. Ion-selective microelectrodes, filled with either Na+-ion exchanger21 or CI--ion exchanger (Corning

Injected substances

477315) were fabricated by standard procedures 16. The electrode readings were calibratedS,21 in 80 m M

10 m M aqueous solutions, partly with the addition of

K+-isothionate solutions containing various concentrations of NaC! in the range of 0.5-50 mM. Elec-

66 m M KC111: K+-salt of adenosine 3',5'-cyclic monophosphate (cAMP), (Boehringer M a n n h e i m ) ,

trodes for acid injection contained 0.1 N HC1, those

Na+-salt of N-6'-monobutyryl adenosine 3',5'-cyclic monophosphate ( m o n o b u t y r y l - c A M P ) , N6,O2'-dibutyryl adenosine 3',5'-cyclic m o n o p h o s p h a t e (dibuty-

The following substances were pressure injected as

for C1--injection 3 M KCI. Injection procedure

ryl-cAMP), 8-bromo-adenosine 3',5'-cyclic mono-

Drugs were injected by a fast and quantitative pressure injection method12 with a resolution of 10-11 1 that compared well with an average cell volume of 5

phosphate (8-bromo-cAMP), adenosine 5'-diphosphate ( A D P ) , guanosine 3',5'-cyclic m o n o p h o s p h a t e (cGMP), adenosine 5'-triphosphate (ATP), further-

× 10-9 1. With single injections, quantities on the or-

more adenosine and inosine, all obtained from Sigma. The injected solutions had a pH of 7.4 adjusted by the addition of either K O H or HCI.

der of 1% of the cell volume were injected by pressure pulses of 0.1-1 s duration, which were thus instantaneous on the time scale of the induced effect. Tests of the pressure injection method for side effects were extensively done before 12. The injection was

Solutions

Composition of the bathing media are given in Ta-

TABLE I Bathing solutions

All solutions contained in addition 10 mM of glucose, pH was 7.8 throughout. HEPES, N-2-Hydroxyethylpiperazine-N'-ethanesulfonic acid (Serva); Tris, Trihydroxyaminomethane; TMA, Tetramethylammonium. mM

(.1) Normal Ringer (2) Saccharose sol. (3) TMA sol. (4) Na sol. (5) Ca sol. (6) Mg sol. (7) Li Ringer (8) CI-free Ringer

Na +

K+

80 --110 ---80

4 . -. --4 4

Ca 2+

M g 2+ T M A +

Tris +

Li +

CI-

HEPES

o-Gluconate- Saccharose

10

5 . -. -70 5 5

-20 5 5 5 5 -.

------80

114 20 115 115 145 145 114

5 ---

----

-200 --

-

-

-

.

. --

.

. 70 -10 10

-110 ---.

.

.

-

--5 5

-

---114

-

----

291 ble I. In Ringer free of alkali metal and alkaline earth metal salts TMA-CI (tetramethylammonium-CI) was the substituent. In one experiment saccharose replaced these salts. Solutions containing varying amounts of Na +, Ca 2+ or Mg 2+ were obtained by appropriate mixtures of solutions 3-6 of Table I. Li +Ringer had all Na + exchanged for Li + and Cl--free solution was prepared from gluconate-salts (Sigma). Osmolarity measured with a freeze point osmometer was 220 mOsmol in all solutions. Na +- or Li+-con taining solutions were pH-buffered by HEPES, adjusted by addition of NaOH. Otherwise, Tris was used and was adjusted by HC1. 10-5 M ouabain (Sigma), 1 mM furosemide (Hoechst), 1 mM SITS (4-acetamide-4'-iso-thiocyanostilbene-2,2'-disulfonic acid, BDH), 1 mM amiloride (gift from Sharp & Dohme), 1 mM DNP (dinitrophenol, Sigma), 50/zM IBMX (isobutylmethylxanthine, Sigma) were dissolved in normal Ringer. RESULTS

Characteristics of the cAMP response cAMP injections elicited a transient membrane depolarization in current-clamped cells and an inward current under voltage clamp (Fig. 1). This effect was present in all larger neurons of the ganglia, including cells F1, 2, 34, 76 and 77 of Kerkut et alA3. These responsive neurons also had sodium-dependent action potentials of relatively short duration (5-10 ms). Very little response to cAMP injection was observed in U-cells 15, in which electrogenesis is predominantly calcium-dependent, as manifested by long spike durations (20--40 ms) and large overshoots (+60 mV). Injection of cAMP at the usual holding potential of --50 mV induced an inward current (IcAMP) which

10s Fig. 1. Effect of pressure injection (arrow, duration about 0.1 s) of cAMP in snail neurons. A: neuron in current clamp with membrane potential (VM) of--50 mV. B: the neuron in voltage clamp at holding potential (HP) o f - - 5 0 mV. Recording bandwidth DC to 0.1 kHz.

2nA

Fig. 2. Effect of increasing injection volumes of cAMP: IcAMP saturates with injection volumes between 3 and 10% of the cell volume. The saturation is indicated by the fiat peak current slope. This steady peak current is prolonged with larger injections. HP is --50 mV, the response in this and the subsequent figures is low pass filtered at about 10 Hz.

developed gradually, peaking at 11.2 + 9.6 s (n = 200) after the injection (range: 4--25 s). The current decayed from its peak with a somewhat slower time course (half-time: 16.8 + 12.0 s). For lower injection volumes, the magnitude of the peak current grew as the injection volume was increased. However, as shown in Fig. 2, the peak current soon reached a plateau level, and larger injections only prolonged the response. This indicated saturation of the response, which occurred at a current density of 10/~A/cm a cell surface. Usually, injected volumes were 1% of the cell volume, which induced a current of 3.7 + 2 nA (n = 200). The membrane conductance was determined by measuring the current response to hyperpolarizing or depolarizing pulses of small (10-20 mV) amplitude. Little, if any, change in membrane conductance could be detected throughout the duration of the injection-induced current flow (Fig. 3). The possible voltage dependence of the cAMP response was investigated by measuring the peak response to carefully adjusted injections (variation in volumes < 5%) at various negative membrane potentials between--120 and --20 mV. The cAMP-induced effect showed no significant change with voltage within this range of membrane potentials with little voltageactivated membrane conductances. It was thus not possible to measure a membrane conductance or to deduce a reversal potential for IcAMP (Fig. 4). If a

292

A

10s

B

~

t8M 6 4 2

Fig. 3. Celt membrane resistance (RM) before and during I~,~aP. cAMP injection is indicated by arrow. I~AMPis 11.5 nA at peak. A: shows current responses (Is) to hyperpolarizing voltage steps (Vs) from --50 to --70 mV. B: trace of computed R M = Vs/Is before and during IcaMP. Rra during I~Arapis similar to that before IcAMP.With a Na+-equilibrium potential (ENa) of +80 mV (see Discussion) a Na+-specific conductance calculates to g~ra= IcAM~(ENn--HP)= 0.09/~S. Correspondingly, the RMof 7.0 + 0.2 M~ at rest is expected to decrease to 4.3 Mff]. The observed value of R Mduring peak I~AMPis 7.1 + 0.2 M~2. conductance change occurred, it was invaribly associated with a'n abrupt rather than gradual increase in the inward current, as is typical for an injection artifact.

the rate at which IcAMP decreases is determined by the enzymatic breakdown of cAMP. Within a period of some minutes after adding IBMX at a concentration of 5 0 # M to the bathing medium 14, the mean duration of the response was prolonged by about 20% (Fig. 5); no further effects were seen at Later times. The maximum prolongation seen with 50/~M IBMX was by a factor of two. With IBMX the response increased in half of the cases by 20% or less. More drastically prolonged inward currents could be observed with the injection of some cAMP-derivatives which are able to cross the membrane and are therefore commonly applied to study c A M P effects (for review see Siggins20). These agents are known to be resistant to breakdown by phosphodiesterase 17 Injection of monobutyryl-cAMP produced an inward current that rose very slowly, reaching its peak after 2 min. The peak currents were always considerably smaller (about 50%) than those seen with the unsubstituted compound. The half-time of recovery was about 5 rain; considerably longer than that of IcAMP-

Effect of cAMP-derivatives and IBMX

Similar results were obtained with the injection of 8-bromo-cAMP, which also produced a very slow rise to peak and half-time of decay of 20 min. Slowing was even more pronounced with dibutyryl-cAMP, which induced responses with times to peak of 10 min, and durations of one hour or more after a single injection. Injections of this compound from one batch of two resulted in a slow recovery that followed a quickly attained peak. We attributed the early peak to impurities of c A M P in the sample.

Since I B M X inhibits phosphodiesterase, it would be expected to cause a prolongation of the response if

Effect of ion transport blockers

Specificity of the cAMP response The specificity of the response was tested by the injection of substances involved in the metabolism of c A M P TM. The substances used were adenosine, inosine, A D P and ATP. They all failed to produce responses in cells which did respond to cAMP. Injection of c G M P 9 was also ineffective.

A

The substances tested were oubain, furosemide, SITS and amiioride. None of these had a discernible blocking action on IcAMp. Abolishing oxidative phosphorylation by D N P for up to 30 min was also not ef-

B

mV -15 -20 -25

-60

Membrane potential (mV) -40 -20 0

2~ EtD

4

-50

6~

5nA[

i

20s

-----O

O"-O'-O--

D O t~ t-

Fig. 4. IcAMPat different membrane potentials. A: Icar¢a, is elicited with constant injection volumes at the indicated preset clamp potentials (Cl--free Ringer). B: plot of peak inward current vs potential.

t

"

, 10.

oA

10S Fig. 5. Effect of the phosphodiesterase inhibitor IBMX at 50 ~M in normal Ringer.

293

A

A ~ / / ~ NR

lOs

2n

110 mM TMA

NR

110mM Na +

A

"I-MA * 1 0 r a M

C a 2+

A

B

lOs

10s Fig. 6. Effect of replacing external cations by TMA. Left side:

controls in normal Ringer (NR). A: Ic~d~P disappears when all cations are replaced by TMA except those introduced as impurities. B: 10 mM Ca2÷ in TMA solutions (0 Na +, 0 K+). The steady state current at HP of--50 mV shifted reversibly by +0.5 nA (A) and +1 nA (B) when test solutions were introduced. A change of holding potential to produce a current offset of + 1 nA did not affect IcAMPin normal Ringer. fective, with the exception of a slightly increased response in 2 out of 6 experiments.

Changes in extracellular and intracellular ionic composition Comparisons of the responses of single cells to cAMP injections in various Ringer solutions were made with carefully controlled injection volumes (variations < 5%), and only responses that were totally reversible were chosen for study. The dependence of IcAMP o n extracellular cations was determined by varying the concentrations of NaCI, CaCl2 or MgCl 2 employing T M A to preserve osmolarity. The inward response was absent in pure TMA-solutions (110 mM/l) (Fig. 6A), and also with MgCI2 substituting for T M A at any concentration. In solutions that contained CaCl 2 but no NaCl, there was a relatively small IcAMP that grew in proportion to [Ca2+]o,

A

5 mM -----10 . . . . 20 - - ~ _ _ ~ ~ 4o 8o

Na*÷TMA

B

NR ~ - - ~

Li*R __.]2nA lOs

Fig. 7. Dependence of IcAMpon [Na+]o . A: IcAMPat increasing [Na+]o with TMA substitution for other cations. B: Na + is replaced by Li + (Li+R) in normal Ringer (NR). HP was --50 mV.

Fig. 8. Effect of replacing external cations by Na ÷. A. Left: control injection in normal Ringer (NR). Right: all cations are replaced by Na ÷. B. Left: the bathing solution contains 100 mM NaCl and 10 mM CaC12. Right: the bathing solution contains 100 mM NaCI and 10 mM MgCI2. HP was --50 inV. but saturated near 20 mM [Ca2+]o with a size of about 1/3 of that seen in normal Ringer (Fig. 6B). An increase in IcAMpwas always observed when [Na+]o was increased (Fig. 7A), and the largest responses were recorded in pure (110 mM) NaCl-solution. In this situation, the peak IcAMPwas up to 10 times larger than that in normal Ringer containing 80 mM NaCI and 15 mM divalent ions (Fig. 8A). The above observations suggested a depressing effect of divalent cations. This was verified by the finding that at a constant level of [Na+]o, IcAMPdecreased when either [Ca2+]o or [Mg2+]o was raised (Fig. 8B). Substitution of Li ÷ for Na + had only marginal effects on IcAMP;in the experiment of Fig. 7B, it decreased IcAMpby about 10% and prolonged the response which was not seen in other experiments. IcAMP did not depend on [Cl-]i, nor did it involve the movement of Cl-ions across the membrane. When the bath was perfused with Cl--free Ringer that had a normal cationic composition, the neurons initially depolarized to up to 0 mV, and then repolarized within the next 30 min to 1 h. Injections of cAMP into these cells, which were thus depleted of internal CI-, either in the depolarized state or after repolarization, produced effects that were not significantly different from those in normal Ringer. To further verify that IcAMP is not dependent on C1--ions, [Cl-]i was directly monitored with intracellular C1--selective electrodes. These failed to show any significant potential change during the cAMP responses (Fig. 9A). In these experiments, the sensitivity of the intracellular Ci--selective electrode was

294 A

A mM 17

116

HC

]2nA 30s

B

Ilia

IM ~ ~ [Na'],~ ~ - ' 4 " ~ ' ~ ' ~ .

-

C

mM ~ ]34 -32

Fig. 9. Changes in [Cl-]i and [Na+]i monitored with ion-selective microelectrodes (lower traces in A and B) during IcAMp. The CI- and Na+-potentials were measured as the voltage difference between the ion-selective electrode and the voltage-recording electrode. The half-time of recovery from the peak of the response (see B) was about 2 min. Dashed lines denote the average pre-existent CI-- and Na+-potentials. HP was --50

......

, ] 2nA 10s

Fig. 10. Effect of H+-injection on IcAMr,in normal Ringer. A: after a control injection, H-ions are electrophoretically applied with a current strength of 50 nA for 4 min by an intracellular HCl-electrode. B: cAMP injected immediately after the H ÷electrophoresis. C: control injection about 16 rain after H+-ap plication. Steady state current offset during H+-injection was about --1 nA returning to zero at times of the subsequent control. HP was --50 mV. Thus, following H+-injection, the size of the response to a given volume of c A M P was enhanced by up to one order of magnitude (Fig. 10).

mV. DISCUSSION

confirmed 'in situ' by electrophoresis of CI- from a (fifth) KCl-filled electrode. The above experiments strongly suggest that the net transient inward current induced by c A M P injection is at least partially due to the inward movement of Na+-ions. As shown in Fig. 9B, [Na÷]i, as monitored with a Na+-selective microelectrode, was indeed significantly increased during IcAMP. The Na+-specific signal rose slowly after c A M P injection as expected from a gradual increase in [Na+]i during the course of the inward current. In the case of Fig. 9B the injected c A M P solution was Na+-free which may have been responsible for the initial small fall of the Na+-signal. To diminish dilution effects, 10 small c A M P injections with an overall volume of 1% of the cell volume were applied in succession within a period of 12 s. A fall in intracellular pH drastically enhanced the response of the cell to cAMP. This was demonstrated in a series of experiments, in which cells were acidified by electrophoretic injection of H +. Although acidification itself induced inward currents these were comparatively small, even after sustained injection of H ÷ (see also24). The c A M P response, however, was clearly greatly augmented by such treatment.

The most interesting features of the cAMP-induced inward current are the absence of significant changes in membrane conductance and its failure to respond to variation of voltage in a wide range of negative membrane potentials. O u r data point to a participation of Na+-ions in the generation of IcAMe. Taking [Na+]i to be between 3.2 mM (Swandulla and Lux, unpublished) and 3.6 m M 23, the Na+-equilibri um potential under resting conditions is calculated to be between +75 mV and +80 mV. For the IcAMp of 11.5 n A in Fig. 3 from a cell with 0.14 ~S input conductance, an exlusive opening of Na+-channels should produce a conductance increase by about 60% at a holding potential o f - - 5 0 mV. In contrast, the conductance change during IcAMP was within background conductance variation of about 5% of the mean. One might thus conclude that a cAMP-induced Na+-conductance develops at the entire expense of another, such as a cAMP-sensitive K+-conductance, with the result of an unchanged net membrane conductance. Such mechanism implies indeed a weak voltage dependence of IcA~p with current reversal at ENa + [ EK [ (at about + 150 mV). It is noteworthy that in order to compensate for a hypo-

295

thetical rise of a c A M P - i n d u c e d Na+-conductance, the fall of a cAMP-sensitive K+-conductance should nearly reach the available resting m e m b r a n e conductance in cases with large IcAMP. T h e conclusion towards a decrease of a K+-conductance after internally elevated c A M P indeed c o r r o b o r a t e s recent suggestions of D e t e r r e et al. 6 (see also ref. 8). O u r conclusion that c A M P stimulates an influx of sodium ions is based on the following observations: [Na+]i increases during IcgMp , IcgMp increases with increasing [Na+]o, with m a x i m u m currents being recorded in pure NaCl-solutions, while the current disappears reversibly in alkali metal and alkaline earth metal free T M A - s o l u t i o n s . External divalent cations, such as Ca 2÷ and Mg 2÷, exert an inhibitory influence when a d d e d to pure NaCl-solutions. Nevertheless, Ca 2+ but not Mg 2+ is capable of carrying a small inward current when N a ÷ is absent. Li ÷ can effectively replace Na +. C1- does not participate in the process. Reduction of [Ca2+]o and [Mg2+]o does not raise intracellular p H 1. Thus, the increase of IcAMP by removal of divalent cations could well be due to a mechanism different from that which enhances the response in acid condition. I n j e c t e d H-ions may catalyze the nucleotide's action at particular sites or in-

REFERENCES 1 Ahmed, Z. and Connor, J. A., Intracellular pH changes induced by calcium influx during electrical activity in molluscan neurons, J. gen. Physiol., 75 (4) (1980) 403-426. 2 Aldenhoff, J. B., Hofmeier, G. and Lux, H. D., Depolarizing action of injected cAMP in Helix neurones, Pflagers Arch. ges. Physiol., 382 (1979) Suppl. R23. 3 Beavo, J. A. and Mumby, M. C., Cyclic AMP-dependent protein phosphorylation. In J. A. Nathanson, and J. W. Kebabian (Eds.), Handbook of Experimental Pharmacology, Vol. 58/L Springer, Berlin, 1982, pp. 363-392. 4 Braughler, M. J. and Corder, C. N., Reversible inactivation of purified (Na ÷ + K+)-ATPase from human renal tissue by cyclic AMP-dependent protein kinase, Biochim. biophys. Acta, 524 (1978) 455-465. 5 Deisz, R. A. and Lux, H. D., The role of intracellular chloride in hyperpolarizing post-synaptic inhibition of crayfish stretch receptor neurones, J. Physiol. (Lond.), 326 (1982) 123--138. 6 Deterre, P., Paupardin-Tritsch, D., Bockaert, J. and Gerschenfeld, H. M., Role of cyclic AMP in a serotoninevoked slow inward current in snail neurones, Nature (Lond.), 290 (1981) 783-785. 7 Drake, P. F. and Treistman, S. N., Mechanisms of action of cyclic nucleotides on a bursting pacemaker and silent neuron in Aplysia, Brain Research, 218 (1981) 243-254.

crease the n u m b e r of available sites. D i r e c t participation of H-ions in IcAMp is unlikely since the current is inward. Since |cAMP causes an increase in [Na+]i, it will und o u b t e d l y lead to activation of the A T P - d e p e n d e n t N a ÷ - K + - p u m p . H o w e v e r , it is unlikely that this effect determines the time course of IcAMP. Thus, IcAMP develops and decays within tens of seconds, while the time course of the o u t w a r d current that follows N a ÷injection takes tens of minutes u n d e r c o m p a r a b l e conditions 22. Braughler and C o r d e ~ r e p o r t e d that the N a ÷, K +A T P a s e from renal tissue was reversibly inhibited by a c A M P - d e p e n d e n t protein kinase. H o w e v e r , the inward current observed by us is hardly due to a transient b l o c k a d e of the o u t w a r d current production by the N a + - K + - p u m p , since the response was insensitive to p u m p inhibitors such as oubain and D N P . It is known that c A M P activates a protein kinase by phosphorylation (for review see B e a v o and M u m b y 3) which, in turn, catalyzed reactions leading to a variety of cellular functions TM. Phosphorylation of m e m bra'ne-bound proteins m a y thus be responsible for the observed depolarizing action of intracellular elevated c A M P .

8 Gerschenfeld, H. M. and Paupardin-Tritsch, D., Ionic mechanisms and receptor properties underlying the responses of molluscan neurones to 5-hydroxytryptamine, J. Physiol. (Lond.), 243 (1974) 427-456. 9 Goldberg, N. D., O'Dea, R. F. and Haddox, M. K., Cyclic GMP, Adv. Cyclic Nucleotide Res., 3 (1973) 155-223. 10 Heyer, C. B. and Lux, H. D., Properties of a facilitating calcium current in pace-maker neurones of the snail, Helix pomatia, J. Physiol. (Lond.), 262 (1976) 319-348. 11 Heyer, C. B. and Lux, H. D., Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia, J. Physiol. (Lond.), 262 (1976) 349-382. 12 Hofmeier, G. and Lux, H. D., The time courses of intracellular free calcium and related electrical effects after injection of CaCI 2 into neurons of the snail, Helix pomatia, Pflagers Arch. ges. Physiol., 391 (1981)242-251. 13 Kerkut, G. A., Lambert, J. D., Gayton, R. J., Loker, J. E. and Walker, R. J., Mapping of nerve cells in the suboesophageal ganglia of Helix aspersa, Comp. Biochem. Physiol., 50A (1975) 1-25. 14 Levitan, I. B. and Norman, J., Different effects of cAMP and cGMP derivatives on the activity of an identified neuron: biochemical and electrophysiological analysis, Brain Research, 187 (1980) 415--429. 15 Lux, H. D. and Hofmeier, G., Properties of a unitary calcium- and voltage-activated potassium current in Helix po-

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18 19

20

matia neurons, PJTiigers Arch. ges. Physiol., 394 (19821 61-69. Lux, H. D. and Neher, E., The equilibration time course of [K*]o in cat cortex, Exp. Brain Res., 17 (19731 190-205. Meyer, R. B. and Miller, J. P., Analogs of cyclic AMP and cyclic GMP: general methods of synthesis and the relationship of structure to enzymic activity, Life Sci., 14 (t974) 1019-1040. Nathanson, J. A., Cyclic nucleotides and nervous system function, Physiol. Rev., 57 (1977) 157-256. Pellmar, T. C., Ionic mechanism of a voltage-dependent current elicited by cyclic AMP, Cellular and Molecular Neurobiology. Vol. 1/1 (1981) 87-97. Siggins, G. R., Regulation of cellular excitability by cyclic nucleotides. In 1. A. Nathanson, and J. W. Kebabian

(Eds.), Handbook of Experimental Pharmacology, Vol. 58/H, Springer, Berlin, 1982, pp. 3(/5-346. 21 Steiner, R. A., Oehme, M., Ammann, D. and Simon, W., Neutral carrier sodium ion-selective microelectrode for intracellular studies, Analyt. Chem., 51 (1979) 351-353. 22 Thomas, R. C., Membrane current and intracellular sodium changes in a snail neurone during extrusion of injected sodium, J. Physiol. (Lond.), 201 (1969) 495-514. 23 Thomas, R. C., Intracellular sodium activity and the sodium pump in snail neurones, J. Physiol. (Lond.), 220 (19721 55-71. 24 Thomas, R. C., The effect of carbon dioxide on the intracellular buffering power of snail neurones, J. Physiol. (Lond.), 255 (19761 715-735.