Cyclic GMP depresses hippocampal Ca2+ current through a mechanism independent of cGMP-dependent protein kinase

Neuron. Vol. 1, 693-699,

October,

1988, Copyright 0 1988 by Cell Press

Cyclic CMP Depresses Hippocampal Ca*+ Current through a Mechanism Independent of cGMP-Dependent Protein Kinase Diane Doerner and Bradley E. Alger

ond messenger in these muscarinic

Department

begun to investigate the cGMP system

of Physiology

University

of Maryland

Baltimore,

Maryland

School

using the whole-cell

of Medicine

pathways. We have in more detail,

voltage-clamp technique in acutely

isolated hippocampal neurons. Specifically, we attempted

21201

to determine

if the cGMP system

affects Cal+ currents

in hippocampal pyramidal cells and, if so, how its effects

Summary

are mediated.

Cyclic CMP depresses Baz+ current through highvoltage-activated Ca*+ channels (Iti.) in acutely isolated hippocampal neurons. The effect is produced by intra-, but not extracellular, cGMP or by 5’ CMP The membrane-permeant derivative, 8-Br-cCMP, produces a reversible suppression. The effect of 8-Br-cGMP is similar to phorbol ester-induced IO* depression, except that Ica. depression due to 8-Br-cCMP is not blocked by protein kinase inhibitors H-8 or H-7, whereas phorbol ester effects are. The data suggest that cGMP depresses Ica. by a cGMP-kinase- and protein kinase C (PKC)independent mechanism. Cyclic AMP, which enhances Ica*, and the cyclic nucleotide phosphodiesterase inhibitor, IBMX, both antagonize IQ* depression induced by 8-Br-cCMP, but not that due to phorbol esters. Cyclic IMP, a more potent activator of phosphodiesterase than of cGMP-dependent protein kinase, is also a powerful depressant of Ia*. We conclude that cGMPinduced depression of Ica. is mediated by activation of cyclic nucleotide phosphodiesterase with consequent reduction of intracellular CAMP

like the CAMP system in which the effects of CAMP ap-

An interesting

aspect of the cGMP system is that, un-

pear to be exclusively dependent

kinase,

mechanisms

(Nestler

a cGMP-dependent

Neurotransmitters phatidylinositol tracellular Muscarinic

and hormones (PI) hydrolysis

cGMP

commonly

production

agonists

that enhance

as well

in hippocampus

late both the PI and cGMP systems, cently,

the physiological

stood.

We and others

effects of stimulating with

either

roles

enhance in-

(Berridge,

1981).

vigorously

stimu-

and yet, until

of neither

have studied of PKC

were underPI-PKC

(Baraban

system

et al.,

retina,

1985;

involves

activation

role of the cGMP sys-

vestigate which biochemical

depression

kinase,

hibitors,

such as H-7 and H-8,

of Ica* induced by phorbol

dependent

slice,

excites cGMP

neuronal

causes

K+ conductance

firing.

but the mechanism of this effect is entirely

19771,

phosphodiesterase

or some

other

site. Similarly,

regulates its own release via an autoreceptor, mimicked (Nordstrom

by cGMP

also via an unknown

and Bartfai,

19841, In-

carinic agonists

and cGMP

however, supporting

ACh

an effect

mechanism

1981). Itis significant

that mus-

produce the same effects,

the possibility

with IBMX.

Cyclic depres-

sion of Ica*. Cyclic IMP mimics the suppressant

effect of

cGMP These data imply that activation of CAMP phosphodiesterase mediates cGMP-induced depression of Ica*.

Results Cyclic GMP Deoresses ICa. Under

whole-cell

recorded with

voltage-clamp,

CsCHsSO,-filled

that cGMP is a sec-

Ba2+ current

was

patch electrodes

from

acutely isolated adult hippocampal 1986) bathed in control The

neurons

inward

elicited by steps to various depolarizing potential

of -70

has re-

et al., 1988) and found to consist exclu-

is seen in tissue-cultured acutely isolated neurons. activated Ca2+ current

current. current

but ordinarily

The

not in adult

The peak of the high-voltageconsists

of one component

though inactivation characteristics logical properties

smaller,

(T-type current)

defined by activation kinetics (Kay and Wong,

nents (Doerner

was

potentials from

mV. Caz+ current

low-voltage-activated

versus multiple

5 pM

current

in these cells (Kay and Wong,

of high-voltage-activated

transient,

(Kay and

saline containing

high-voltage-activated

1987; Doerner

fluence could be exerted at the level of the K+ channel, the Ca2+ channel,

can

AMP does not block the phorbol ester-induced

sively

unknown.

Ica. in hippocampal

be prevented by addition of excess CAMP or blocking of

of a Ca2+-

(Cole and Nicoll,

which block depression

esters. We also demonstrate

pyramidal cells. The depressant effect of 8-Br-cGMP

In the hip-

depression

as the effects of 8-Br-

that CAMP enhances or stabilizes

cently been characterized

powerfully

is responsible

cGMP cannot be inhibited via potent protein kinase in-

a holding

pocampal

mechanism

does not appear to be mediated

via a cGMP-dependent

and the hippocampus cGMP

conductance

We now report that cGMP has a depressant effect on Ica*. This

tem. In the cerebral cortex in vivo (Stone et al., 1975) et al.,

of an ionic

for the effects seen.

TTX.

there are virtu-

(Hoffer

then

our second goal in studying the cGMP system was to in-

agonists (Worley

in oculo

which

(Fesenko et al., 1985; Haynes and Yau, 1985). Therefore,

Wong,

ally no data on the physiological

phosphodiesterase,

leads to a decrease in CAMP levels; and the third, in the

Malenka et al., 1986; Doerner et al., 1988) or muscarinic et al., 1987). However,

1984). The first is

kinase; the second is by activation

re-

the physiological

the hippocampal

activators

phos-

may act via at least three

and Greengard,

of a cyclic nucleotide

cyclic nucleotide

Introduction

mediated by a specific CAMP-

cGMP

as

19871, al-

and some pharmaco-

suggest the presence of two compoet al., 1988).

components

Since the issue of single has not been resolved,

we

Neuron 694

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ON awm .

Y y.,“.”

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.

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Y

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20

.

100 -

a

;Ir

.

l

4 tp

--I

c

50 -

0 cQYP (In)

l

-

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16

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24

12

Mlnut.0

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20

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Mlnut~a

r 100 6-Br-ct3YP 0 Control 6-Br-cQYP

l

Figure 1. Suppression of Whole-Cell 8-Br-cCMP

Ba’+ Current through Ca )+ Channels (denoted Ic,.) by cCMP and the Membrane-Permeant

Analog,

(A) Inclusion of cGMP (1 mM) in the patch pipette caused a rapid decline in whole-cell Icd.amplitude following an initial 5-6 min exchange period during which peak Ica. increased due to equilibration of the electrode contents with the cytoplasm (open circles). Sample traces, taken at 6 and 22 min from the cCMP (in) records, are shown in the inset. Vertical calibration, 200 PA; horizontal, 100 ms. Bath application of cCMP to another neuron (filled circles, arrow) had no effect on Ica. amplitude. _ (8) A 52% decrease in peak Icdl was observed in this neuron 6 min after bath application of 1 mM 8-Br-cGMP The effect was entirely reversed by lo-15 min of washing with control saline. Sample traces are shown in the inset. Vertical calibration, 80 PA; horizontal, 100 ms. The cell was recorded for 15 min prior to the illustrated record. (C, D) 8-Br-cGMP suppressed Icd. over the entire voltage range tested without an apparent shift in the whole-cell l/V relationship or reversal potential. Peak ICa., evoked by various depolarizing clamp steps from a holding potential of -60 mV, is plotted vs. test potential before (open circles) and 8 min after (filled circles) bath application of 1 mM 8-Br-cGMF! Sample traces, taken at the numbered positions, are shown in D before (top row) and after (middle row) addition of 8-Br-cGMP Vertical calibration, 85 PA; horizontal, 100 ms. In all experiments, unless otherwise specified, Ica. was evoked by depolarizing voltage-clamp steps to 0 mV from pre-pulse to -70 mV in TTXITEA-containing saline, sampled at 20 set intervals, and plotted as absolute peak amplitude vs. time. With the exception of (A) and those experiments in which a cyclic nucleotide was included in the patch pipette, all cells were recorded for 12-15 min prior to the illustrated record to allow adequate time for complete equilibration of the patch electrode contents with the cell. Drugs were present in the recording chamber for the period indicated by the heavy horizontal bars above the data points, Control rate of ICd. run-down was estimated by a computer-assisted linear regression fit of peak ICn. amplitudes recorded during the 3-5 min immediately preceding drug application and is indicated in some figures by a thin, unlabeled solid line.

focused

on the kinetically

TTX-insensitive low

carried

[Na+],,

(Doerner that

inward

carrier

certain

Ba2+, to

present

K+ currents, periods.

Ca2+ channels emphasize

current

necessary

peak.

blocked

by

Cd2+

Ba2+ was used as

experiments

prevents

since

it

Ca2+-dependent

and allows

longer,

our

results

pertain

more sta-

to

Ica* develops

membrane

patch,

period,

Ica* becomes

Ca2+

constant

per minute included

per

solution,

electrode

of lea* increase

min,

minutes

ran down n =

presumably

reflecting

rupture

of

the time

10 min

stable,

the current the current

when

filling

much

(mean

the

lA,

rapidly

(n = 12). This depressant

at

initial time

(8.4 + 2.8%

open

circles).

Ten

a cGMP-containing

was only 51% of maximal amplitude

down

+ s.e.m.)

min, after which

the cell with

in control

with

1 mM, was

cGMP,

solution,

more

Figure

running

0.8%

lasted 4-7

5; e.g.,

after impaling

rent (n = 4), while for S-10 min after

relatively

to equilibrate

1983). After this initial

rate of 1.8 f

(n = 15). However, in the

the current

contents

(Marty and Neher,

interior

period

We refer to inward current it is carried by as “lea*,” although

that

for the electrode

thecell

a gradual,

1987), indicating

regulation.

Typically, the

in the

inactivation,

recording

through channel

Ba2+ and

Ca 2+ channels.

it passes through

Ca2+ channel ble

by

inward

in our cells is resistant to

et al., 1988; Kay and Wong,

the charge blocks

simple

current

cells with was 86%

peak cur-

no cGMP

after

of maximum

effect was due to intracellular

Cyclic CMP Depresses Hippocampal Ca*+ Current 695

cGMP When

A

600l* ‘.%* 400

applied extracellularly,

. -

l .**

-

intracellular 5’ GMP had no effect (n = 3; data not

PDBU

l.**

.

6-l)r-6OYP

” . -,

l

~“**&:.“*~.

shown). The 8-bromo

l***

2

derivative

more membrane (Figure

2ooF 3

2 1

application

was 38 + 3%. The cGA4P analog was ef-

(mean percent reduction

[n = 41). When

8-Br-cGMP

was washed from the bath,

Figure IC illustrates

a typical

I/V relationship

before

(open circles) and after (filled circles) adding 8-Br-cGMF! It

6-Br-CQMP

l

5-8 min of

in Ica* at 10 PM,

its effects were readily reversible.

B . 1

of Ica*

due to I

8% [n = 41; at 20 PM, 24% [n = 41; and at 100 PM, 25%

20

16

12

6

to be

We ob-

caused a rapid depression

1A). In 21 cells, the mean depression

mM 8-Br-cGMP

MlllUtOS

400

is thought

fective in the range of 10 to 1000 uM within

-I

4

of cGMP

permeant than cGMIP itself.

served that 8-Br-cGMP

I

I mM cGMP had

no effect (n = 3; e.g., Figure lA, filled circles). Similarly,

H-6

is apparent that &Br-cGMP

reduces IcaB throughout

a

wide voltage range with no effect on ICa* activation or ap-

.

parent reversal potential and no change in the leakage conductance. The traces in Figure ID were taken from the same cell as in Figure IC at the numbered points on the l/V curve before (top) and after (middle) adding 8-BrcGMPto

the bath. The same effects have been recorded

from 20 other cells. 3

6

9

12

15

H-8 and H-7 Do Not Affect cGMP-Depressed Ica* The above observations

MhJlOS e

the cGMP-induced

b

100

dependent

raised the issue as to whether

depression

is mediated via cGMP-

kinase. As a test of this hypothesis,

plied H-7 and H-8, isoquinolinesulfonamide

* = 60 -0 ‘a g 60 s

which

I

cl 02 .-

2

4o 20

1ti

-..

H-8

..m.

-I

6

II

-.-

.. . .

6

9

12

in

blocking

cGMP-kinase-

in biochemical

tests (Hidaka inhibi-

Control 15

had no effect on 8-Br-cGMP-induced

1~~~depres-

sion (n = 9; e.g., Figure 2A). In six cells treated with 100

0 H-7 3

potent

tor (K, = 0.48 PM); however, at doses from 2 to 100 PM,

. . H._... l -•*

quite

et al., 1984). H-8 is the most potent cGMP-kinase

t

f

are

induced phosphorylation

we ap-

derivatives,

16

Mlnutsr Figure 2. Protein Kinase Inhibitors, H-7 and H-8, Prevent Phorbol Ester-Induced Suppression of Ica. but Do Not Block cCMPMediated Responses (A) H-8 (100 pM) had no apparent effect on Ic,. over the course of an 8-9 min control period, but completely prevented the suppressant effects of PDBu (1 pM). In the absence of H-8, PDBu typically reduced Ica. amplitude 30%-60% within 2-5 min of bath application (see C). Ica. was substantially reduced following application of 1 mM 8-Br-cCMP reaching a maximal 67% suppression after 5-6 min of exposure to the drug. Sample traces are shown in the inset. Vertical calibration, 160 PA; horizontal, 150 ms. (8) Intracellular H-7 does not block cGMP-mediated responses. Whole-cell ICa- was recorded with a patch pipette containing 50 uM H-7. Ica. run-down in the presence of H-7 was estimated at approximately 0.75%/min. Within 8 min of bath application of 1 mM 8-Br-cGMP peak Ica. was reduced by 41% from the control value predicted by the estimated run-down. inset vertical calibration, 130 PA; horizontal, 100 ms. (C) In the absence of H-7 in the electrode solution, peak Ic.,. in a typical cell, normalized to the initial control value, was decreased 57% within 4 min of bath application of 500 nM PDBu (filled circles). Sample traces are shown in the inset. Vertical calibration, 100 PA. horizontal, 120 ms. Addition of 50 pM H-7 to the electrode ef-

uM H-8, the mean depression caused by 8-Br-cGMP

was

33 + 9%.

less

H-7,

a related derivative with somewhat

potency in blocking cGMP-dependent uM), was similarly

kinase (K, = 5.0

ineffective (n = 6; e.g., Figure 2B).

Since H-7 and H-8 also block CAMP-dependent and PKC at similar

concentrations

[H-7] and 15 uM [H-8];

kinase

(K, CPKC] = 6.0 uM

K, [CAMP-dependent

nase] = 3.0 uM [H-7] and I.2 uM [H-8]),

protein ki-

these results

suggest that none of these kinases mediate the effects of &Ml?

H-7 and H-8 Block the Depressant Effects of PKC . Actwators on I,-=. In contrast

to their

Ica* depression,

H-7

lack of effect on cGMP-mediated and

H-8

entirely

prevented

or

reversed the effects of PKC activators on ICa* (n = 27; see also Doerner

et al., 1988). HA1004,

a relatively inac-

fectively blocked the suppressant effects of PDBu in another neuron (open circles). Icd- rundown in the presence of H-7 did not deviate from the control rate following bath application of 500 nM PDBu, although the drug remained in the recording chamber 15-20 min.

Neuron

696

A

~-W-CAMP

15’r

CAMP

I

i

12

9

6

3

I

Mlnuter Figure 3. Cyclic AMP pocampal Neurons

Enhances

3

Icd. in Acutely

Isolated

tive derivative (K, [PKC] = 40 PM) with similar these (PDBu)

we used

at 100 nM-1

12

9

12

B 150

IBMX 6-Br-cGMP

In

phorbol-12,13-dibutyrate

3

t.rM to activate PKC (Nishizuka,

6 Minutes

1984). PKC is an intracellular hibitor should

enzyme,

and a putative PKC in-

act on the inside of a cell. We recorded

four cells with 50 pM H-7 in the patch pipette and observed that the effects of phorbol

esters were entirely

prevented

although

(e.g.,

Figure

2C).

H-8,

about 30

times more potent in blocking cGMP kinase than PKC, nevertheless

does block PKC with a K, of about 15 PM

(Hidaka et al., 1984). As shown that at high concentrations

in Figure 2A, we found

(60 PM) H-8 did block the

effects of PKC activators on Icae(n = 5). The mean ICae suppression 5.4%

9

tullnutes

chemical

was ineffective (n = 4, data not shown).

experiments,

6

Hip-

Peak Icd., evoked by depolarizing clamp steps to 0 mV from a prestep potential of -70 mV in each of five neurons was normalized, pooled, and plotted vs. time, before and after bath application of 1 mM &Br-cCMP Each point represents the mean f s.e.m. Control run-down was estimated by a linear regression fit of the pooled values immediately prior to CAMPapplication. A slight (
properties,

6-Br-cGMP

caused by 200 nM PDBu

in cells filled

nearly 40% These

with

in cells without

experiments

was only

60 uM H-8,

H-8 (see Figure 5).

are important

that H-7 and H-8 are efficacious, affect cGMP is significant,

11.7 k

compared with in demonstrating

hence their failure to

and that despite similarities,

Figure 4. cCMP-Mediated Suppression of I‘.,. Is Blocked Elevated Intracellular CAMP or Pre-Exposure to IBMX

by

(A) Inclusion of CAMP in the patch pipette depresses 8-Br-cGMP responses. Peak Ic.,. was measured at 30 set intervals in each of six 1 mM CAMP Data neurons using patch electrodes containing points represent normalized and pooled ICa. amplitudes, plotted here as mean f s.e.m. vs. time. A 9% decrease in peak I(‘,. resulted, on average, from bath application of 1 mM &Br-cGMl? In theabsence of CAMP, cCMP typically caused a 30%50% reduction in Ica.. (B) Pretreatment of neurons with IBMX also prevents cCMPmediated suppression of Icdl. Peak Ica. amplitudes following a S-10 min pretreatment with 1 NM IBMX and after subsequent addition of 1 mM 8-Br-cGMPwere normalized, pooled, and plotted as mean * s.e.m. vs. time for each of five neurons. In the majority of cells, IBMX entirely blocked the suppressant effect of 8-Br-cGMP on IL.,.. In a few cells, enhancement of lc,1. was observed following prolonged exposure to IBMX, as reflected by the overall increase in the variability of the values over the course of the experiment.

there are also differences between the mechanism of action of cGMP and PKC on Ic.,*.

Possible Role of CAMP in the cGMP Effect

increase in Ic,* (Figure 3). Additionally,

In cardiac cells, cGMP depresses

ments,

activation thus

of a CAMP-dependent

decreasing cellular

Fischmeister,

1986).

stores

ICa+by increasing the

diffusion

phosphodiesterase,

of CAMP (Hartzell

For this hypothesis

and

to be viable in

hippocampal pyramidal cells, it would be necessary for CAMP to have an enhancing

in which

via

from the recording electrode, we observed a

prolonged period of Ica. increase (“run-up”) at the start of the recording session most experiments

(data not shown).

ICa*typically

5-10 min (presumably

effect on ICa*. Although

in other experi-

we applied CAMP intracellularly

Whereas

due in part to the time taken for

this seemed quite likely, since CAMP enhances Ica* in a

the cell to fill with Cs+ and blocking of K+ currents

number of cell types (Tsien,

occur)

1983), including

hippocam-

pal dentate granule cells (Gray and Johnston, had not yet been demonstrated application of 8-Br-CAMP

1987),

it

in pyramidal cells. Bath

ordinarily

produced a modest

before

in

grows larger for the first

beginning

CAMP in the electrode

its gradual

run-down,

it was not unusual

for Icd* to

continue to increase for 20 min or more. Moreover, min after reaching its maximum,

to

with 10

ICa*, in cells infused

Cyclic GMP Depresses Hippocampal CaL+ Current 697

100

*

PDBu

60

3 2

r- L

60

I ;

40

20

-

-

1+

I +

CAMP

A

6-Br-cGMP

100 -

_‘-. :

*

SO-

3 f

1

%A m. m.

.-

SO-

0

.

a. .-

:

-

l

t l

5 l l ..

f

._w

i-

l *

h-f l

-

-

.y%% 2

Figure 5. Comparison of the Inhibitory Effect of Elevated Intracellular CAMP on Phorbol Ester- and cCMP-induced Suppression of tea*

Under control conditions, with no CAMP in the patch electrode, bath application of 200 nM PDBu suppressed Ica. by 37.3 * 7.2% (n = 10) (let? panel, open bar). Inclusion of 1 mM CAMP in the recording electrode did not alter the magnitude of this effect (average suppression due to 200 nM PDBu; 34.5 f 5.1%, n = 10; left panel, filled bar). In 14 control ceils, with no CAMP in the recording electrode, bath application of 1 mM 8-Br-cCMP reduced Ica. by 36.2 f 5.0% on average (right panel, open bar). The effect of 8-B,cGMP was significantly reduced in those cells recorded with 1 mM CAMP in the patch electrode (average reduction; 16.0 f 3.3%, n = 6; p < 0.05; right panel, filled bar).

with

1 mM CAMP was significantly

cells without

exogenous CAMP (1077 f

292 PA, n = 6,

75 pA, n = 19; p < 0.02).

These data fur-

that CAMP increases Ica* in

pyramidal cells. If cGMP were to act by reducing CAMP stores, then buffering should

intracellular

CAMP to a high concentration

prevent the effect of cGMP

To test this predic-

tion, we recorded from cells with electrodes containing 1 mM CAMP We found that in these cases the depressive effect of S-Br-cGMP

on Ica* was greatly reduced (Fig-

ures 4A and 5). The hypothesis

that cCMP acts via increased activa-

tion of CAMP-dependent phosphodiesterase that inhibition

of this phosphodiesterase

also implies

would also in-

hibit cGMP effects. To test this prediction, we treated five cells with the phosphodiesterase prior to applying 8Br-CAMP,

inhibitor

cGMP. By itself,

crease of about 20%

u P

1600

4

greater than that in

versus 509 f

ther support the hypothesis

B

IBMX

in ICa*although,

IBMX (1 p,t%I)

6

Figure 6. clMP Mimics the Suppressant Effects of cGMP on ICa+ (A) Inclusion of clMP (1 mM) in the recording pipette caused a rapid decline in ICa. amplitude following an initial 4 min equilibration period in a typical neuron, closely resembling the effect of intracellular cGMP (see Figure 1A. open circles). Sample traces, taken at the numbered positions, are shown in the inset. Vertical calibration, 200 PA; horizontal, 100 ms. (B) Brief bath application of the membrane-permeant analog, 8-BrclMP (1 mM; indicated by the left horizontal bar), caused a rapid reduction in peak IQ-, which was rapidly reversed when the drug was washed from the perfusion chamber with control saline. Sample traces, taken during this initial application, are shown in the inset. Vertical calibration, 400 pA; horizontal 100~ms. A secondaryapplication of 8-Br-clMP (right horizontal bar) airer a recovery period produced a smaller response in this and three other cells. A slight relaxation of the 8-Br-clMP response with prolonged exposure to the drug was also typical.

caused an in-

this effect was variable. IBMX also inhibited

Cyclic IMP Depresses Ica. Cyclic IMP is a potent activator of cychc-nucleotide-dependent

phosphodiesterase,

stimulating

but is very

cGMP-dependent

al., 1973). When

protein

tion,

In contrast to its marked effects on cGMP CAMP in the

ICa. as intracellular

recording electrode had no effect on phorbol

and Figure 6A). After an initial

cGMP on I,-,.. This

experiment

that CAMP simply

occludes

stabilizing

ICa., and further

PKC systems

ester-in-

(Figure 5). Figure 5 also illustrates effect of &Br-

rules out the possibility the effect of cGMP

by

indicates that the cGMP and

are independent.

clMP

during

produced virtually

et

the identical

the

solu-

effects on

period of Icd* increase

current

amplitude

declined until 10 min after impalement

rapidly

it was only 32 +_

(n = 4). Bath application of the mem-

brane-permeant derivative, 8-Br-clMP reversible

in

cGMP (see Figure lA, open circles,

equilibration,

13% of maximum

inefficient

kinase (Miller

included in the electrode filling

lack of Effect of CAMP on Phorbol Ester-Induced Ica. Depression

that CAMP did reduce the depressive

16

as in the case of

the effects of cGMP on Ica* (Figure 48).

duced ICa-depression

12

Minutes

depression

produced a rapid,

of ICaB(n = 7; Figure 68). The ef-

fects of clMP were less pronounced with repeated appli-

Neuron 698

cations and tended to “fade” with maintained

applica-

tion that, in heart, cGMP only affected ICa that had first been enhanced

tion (Figure 66).

not depressed Discussion Although well

increase

in intracellular

established

biochemical

levels of cGMP

consequence

carinic receptor activation (El-Fakahany physiological the

significance

mammalian

brain,

cGMP depresses tion

is a

of

mus-

et al., 1988), its

has not been determined We

have demonstrated

that

ICa*in hippocampal cells. A similar

has been demonstrated

and Fischmeister,

in cardiac cells

1988).

neuron

In both hippocampal

pyramidal cells and heart cells, the conclusion drawn that a cGMP-dependent

ac-

(Hartzell

1986) and recently in Aplysia

R15 (Levitan and Levitan,

in

has been

kinase is not involved in

by

kinase,

in activating cGMP-

is much less effective than

cGMP in reducing ICa.We were not able to use the intracellular

perfusion

system

used in cardiac cells to in-

fuse cGMP, which

is not readily membrane

and so we could

not make this

pocampus.

permeant,

comparison

Instead, we used well established

in hipinhibitors

CAMP)

was

very

effectively

The similar

effects of PKC activators and cGMP sug-

gested the possibility

that these two biochemical systems

might be linked in a single pathway in their regulation of Ca*+ channels. activity, (Zwiller

PKC does enhance guanylate cyclase

and increase

cGMP

production,

mediated the ultimate effects of PKC activation on hippocampal Ic,*. Alternatively,

cGMP

Ica*. However,

our experiments

demonstrating

Icd* depression

phorbol

due to cGMP, but not depression

esters,

provides

The common

conclude that the two systems

on Ca*+ channels,

tests of this hypothesis

ICa* depressant

is in opposition

cGMP-dependent

kinase in brain apart from thecerebel-

CAMP systems)

action of the PKC and

discussed

by Nishizuka

its

suggested

that

effects

hip-

depressive

an

of PKC and

(1984).

tems can be appreciated in the case of the Ca*+-dependent

produce

or en-

represents

CAMP at high

regulation in hippocampus.

might

This

The interplay among the three second messenger sys-

1984).

cGMP-induced cGMP

to the stabilizing

control (opposition

levels via the recording electrode largely prevented the This

other

We

and Greengard,

ICa* depression.

although

are necessary.

example of bidirectional

intracellular

due to

evidence against the second

pathway. We tentatively act independently

data indicating that there is very little

also showed that buffering

ICa.

larly, the finding that CAMP loading of cells could block

with biochemical

(Nestler

that in-

appeared to rule out the first pathway. Simi-

hancing effect of the CAMP system.

lum and striatum

have been

of PKC had no effect on the cGMP-induced

cGMP-dependent

is consistent

could

involved at a step prior to PKC activation in depressing

cGMP system

conclusion

in rat brain

et al., 1985), and it seemed possible that cGMP

(Hidaka et al., 1984), H-8 and H-7, to rule out a role for kinase. This

reduced

cGMl?

depression

protein

ICa was

by

argument is based on the fact that 8-Br-cGMP, much more effective than cGMP

Resting

in hippocampal

cells, we found that resting ICa. (i.e., not stimulated

hibitors

dependent

In contrast,

exogenous

the mediation of cGMP effects on ICa-. In the heart, this which is

by the CAMP system. by cGMP

in

K+ current,

(Cole and Nicoll,

lAHP is subject

I,++

to complex

Besides depression by cGMP

1984) and PKC (Baraban et al., 1985;

pocampus, as in the heart, via activation of CAMP phos-

Malenka et al., 1986), it is also reduced by increases in

phodiesterase.

CAMP (Madison and Nicoll,

This

hypothesis

was supported

by our

data indicating that the effects of cGMP were blocked

ply that the muscarinic

by CAMP and the CAMP phosphodiesterase

PKC synergistically.

IBMX

and were

phodiesterase

mimicked

await biochemical cGMP

by clMP,

activator. Further studies.

inhibitor

a potent

For example, although 8-Br-

is a weak activator of cGMP-stimulated

nucleotide

phosphodiesterase

in frog ventricular

(H. C. Hartzell,

personal communication),

be determined

to what extent

brain phosphodiesterase. stimulation

phos-

tests of this hypothesis

Nevertheless,

interpretation

One apparent difference

reduction

pressed (Doerner

cGMP-induced

resultant reduction of CAMP and depression

PKC-induced

tissue

ofcyclic nucleotide phosphodiesterase,

vides a parsimonious

would

with

of Ica*, pro-

decrease

between the cardiac cells

although

ICawill

Hence, concurrent

activation of the PKC and cGMP sys-

tems would ensure maximal depression It is now well established

trisphosphate

that neurotransmitters

messenger

and diacylglycerol.

and Fischmeister,

effects of dual sec-

Experimental

Pmcedures

1987) and also in the present paper in hip-

pocampal pyramidal cells. It may be that in hippocampal cells

can

of inositol

1986). A similar,

effect occurs in dentate granule cells (Gray

and Johnston,

system

Our data represent a

ond messenger activation.

1983; Hartzell

of lAHP by mus-

carinic receptor agonists.

of CAMP In the heart, CAMP dramatically (Tsien,

also be sup-

suggest PKC effects are independent of CAMP reduction.

second example of the physiological

but smaller,

the

et al., 1988). The present experiments

and hippocampal pyramidal cells was seen in the action enhances lea*

IAHP; however,

of lAHP may Involve direct ac-

act via the dual second

of our data.

both cGMP and

by cGMP, involving

reduction in CAMP per se would enhance residual IAHP. tion on the K+ channels,

stimulates

utilizes

ICa depression

cyclic

it remains to

8-Br-cGMP

CAMP reduction,

1986). Our present data im-

system

ica is more

nearly

maximal

state, so that there is little opportunity

in the “resting” for further

in-

crease. In agreement with this suggestion is the observa-

Acutely isolated pyramidal neurons were prepared from juvenile guinea pig hippocampus according to the methods of Kay and Wong (1986). Briefly, tissue chunks (1 mm3) were gently agitated in buffered saline containing 1.8 mg/ml DPCC-trypsin for 2-3 hr, triturated through fire polished pipettes, then allowed to settle in

Cyclic CMP Depresses Hippocampai Ca2+Current 699

polylysine-coated culture dishes. Cells were studied from lo-90 min after dissociation. Whole-cell currents were recorded under tight-seal voltage clamp (Hamill et al., 1981). using either a Dagan 8900 (Dagan Corporation) or an Axopatch IC (Axon Instruments) patch clamp. Low resistance (2-5 MOhm) patch electrodes were filled with (all concentrations in mM) 145 CsCHaSO,, 10 HEPES, 5 M&II, 11 EGTA, or 11 Cs,BAPTA (Molecular Probes, Inc), 1 CaCl,, 5 ATP, 10 TEA, and 0.1 leupeptin (Boehringer Mannheim). In some experiments, electrodes also contained 50 pM l-(5-isoquinolinesulfonyl)-2_methylpiperazine (H-7), 1 mM CAMP 1 mM cCMP 1 mM 5’ GMP or 1 mM clMP Series resistance error and liquid junction potentials arising from different pipette solutions were measured and compensated using either the Dagan or Axopatch systems. The extracellular solution contained (in mM) 125 NaCI, 10 BaCI?, 10 TEA, 5 CsCI, 2 MgCIa, 10 HEPES, 10 glucose, and 0.005 TTX (pH 7.4). All experiments were performed at room temperature (22”C-24°C). Unless otherwise specified, all drugs were obtained from Sigma Chemical Co. (St. Louis, MO). Cyclic AMP 8-Br-cCMP (10 pM-1 mM), 8-Br-clMP, cGMP H-7 (20 PM-50 pM), N-(2-[methylamino]-5-isoquinolinesulfonamide dihydrochloride (H-8; 2 uM-100 uM. Seikagaku America, Inc.), and N-(2-guanidinoethyl)-S-isoquinolinesulfonamide hydrochloride (HA1004; 100 pM, Seikagaku America, Inc.) were dissolved in water and frozen in aliquots for later use. Nucleotides were applied at 1 mM concentration unless otherwise noted. Phorbol-12,13-dibutyrate (PDBu: 100 nM-1 t&l) and %rsobutyl-1-methylxanthine (IBMX; 1 ph&lOO pM) were dissolved in DMSO, divided into aliquots, and frozen until use. DMSO was present in the recording chamber at concentrations not exceeding 0.1%. and in control trials had negligible effects on ICa.. Drugs not included in the patch pipette solution were added directly to the static recording chamber and, in some experiments, were removed by perfusion of the chamber with control saline. Data were digitized at 40 KHz, sampled at 500 Hz, and stored on microcomputer disk using the pClamp system (Axon Instruments). Experimental values are given as mean f s.e.m. Statistical significance was tested using a two-tailed t-test. Acknowledgments

adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons. Nature 327, 620-622. Hamill, 0. t?, Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 397, 85-100. Hartzell, H. C., and Fischmeister, R. (1986). Opposite effects of cyclic GMP and cyclic AMP on Ca2+current in single heart cells. Nature 323, 273-275. Haynes, L., and Yau, K.-W. (1985). Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones. Nature 317. 61-64. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984). lsoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23, 5036-5041. Hoffer, B., Seiger, A., Freedman, R., Olson, L., and Taylor, D. (1977). Electrophysiology and cytology of hippocampal formation transplants in the anterior chamber of the eye. II. Cholinergic mechanisms. Brain Res. 119, 107-132. Kay, A. R., and Wong, R. K. S. (1986). Isolation of neurons suitable for patch-clamping from adult mammalian central nervous systems. I. Neurosci. Meth. 16, 227-238. Kay, A. R., and Wang, R. K. 5. (1987). Calcium current activation kinetics in isolated pyramidal neurones of the CA1 region of the mdture guinea-ptg hippocampus. J. Physiol. 392, 603-616. Levitan, E. S., and Levitan. I. B. (1988). A cyclic GMP analog decreases the currents underlying bursting activity in the Aplysia neuron R15. J. Neurosci. 8, 116221171 Madison, D. V., and Nicoll, R. A. (1986). Actrons of noradrenaline recorded intracellularly in rat hippocampal CA1 pyramidal neurones, in vitro. J. Physiol. 372, 221-244. Malenka, R. C., Madison, D. V., Andrade, R.. and Nicoll, R. A. (1986). Phorbol esters mimic some cholinergic actions in hippocampal pyramidal neurons. J. Neurosci. 6, 475-480. Marty, A., and Neher, E. (1983). Tight-seal whosle-cell recording. In Single-Channel Recording, B. Sakmann and E. Neher, eds. (New York: Plenum), pp. 107-122.

We thank Dr. Michael P Walsh for his comments on a draft of this manuscript and Dr. H. Criss Hartzell for suggesting the clMP expertments and for permission to cite unpublished data. This work was supported by National institutes of Health grant NS22010 and an award from the Maryland Biotechnology Center.

Miller, J. P., Boswell, K. H., Muneyama, K., Simon, L. N., Robins, R. K., and Shuman, D. A. (1973). Synthesis and biochemical studies of various 8-substituted derivatives of guanosine 3.5’~cyclic phosphate, inosine 3’,5’-cyclic phosphate and xanthosine 3’,S’-cyclic phosphate. Experientia 12. 5310-5319.

Received June 29, 1988; revised August 22, 1988.

Nestler, E. J., and Creengard, P (1984). Protein kinases. In Protein Phosphorylation in the Nervous System (New York: John Wiley & Sons), pp. 17-80.

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