CO2 reduces the cGMP concentration of the vertebrate photoreceptor to the levels normally observed on illumination

Vol.

96, No.

September

BIOCHEMICAL

2, 1980 30,

AND

BIOPHYSICAL

RESEARCH

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Pages

1980

REMOVING BICARBONATE/CO2

785-792

REDUCES THE cGMP CONCENTRATION OF THE

VERTEBRATE PHOTORECEPTOR TO THE LEVELS NORMALLY OBSERVED ON ILLUMINATION Edward

P. Meyertholen,

Department

of Biological

Received

August

Meegan

Sciences,

J. Wilson

Purdue

and Sanford

University,

E. Ostroy

West Lafayette,

Indiana

11,198O

SUMMARY: Illuminating the toad retina or removing bicarbonate/C02 from the incubation solution reduces the cGMP concentration of the rod outer segment by approximately 65%. In the absence of bicarbonate/C02 the maximum light-induced changes in cGMP concentration were reduced by 75%. Electrophysiological experiments done under these conditions showed approximately a 45% linear reduction in receptor potential amplitude at all measured light intensities with no major changes of cell sensitivity or certain other properties. In many respects decreasing the cellular cGMP concentration by eliminating bicarbonate/ CO2 did not mimic the effect of light on the phototransduction or light adaptation properties of the vertebrate photoreceptor.

INTRODUCTION: intensely ual

The biochemistry

studied

transduction

decreases

models

duction

3).

phototransduction

(4,

5),

dibutyryl

measuring observing

centration

processes role

linking

testing

processes

of the

by illumination. of cGMP within

from vertebrate Thus its

role

change

effects

dark,

physiological

a major

concern

with

phototrans-

of cGMP in the have

in cGMP concentration

we are levels

8).

with

cGME', inhibitor)

We report

can be used

photoreceptor

here

to reduce

to the

level

normally

able

to alter

the

without

vis-

light-induced

of perfusion

(7,

the perfusate

in the

with

a phosphodiesterase

the photoreceptor

of bicarbonate/CO2

decrease

has been

of the photoreceptor

light-induced

electrophysiological

of cGMP into

has been

this

cell

associated

of the

the possible

adaptation of the

(1)

cGMP, or IMX (isobutylmethylxanthine,

cGMP concentration attained

of

the rate the

the

GMP) concentration

The methods

photoreceptor

The possible

have been proposed

or light

(6)) or injection elimination

to elucidate

and adaptation.

and several

involved

in attempts

in cGMP (cyclic

(2,

of the vertebrate

requiring

that the

coninjection

0006-291X/80/180785-08$01.00/0 785

Copyright 0 1980 by Academic Press, Inc. $1 rights of reproduction in any form reserved.

Vol.

96, No.

BIOCHEMICAL

2, 1980

or the addition

of extrinsic

effect

changes

of these

AND

artificial

BIOPHYSICAL

RESEARCH

inhibitors.

COMMUNICATIONS

We have also

on the electrophysiological

properties

observed

the

of the photo-

receptor. METHODS AND MATERIALS: Marine toads (Bufo marinus) were dark adapted for 24 -hours before all experiments. The composition of the bicarbonate solution was: Na+ 108 mM, Cl- 90.5 mM, HC03- 24 mM, K+ 2.5 mM, S04- 0.6 mM, Caft 1.0 mu, Mg* 1.6 mM, glucose 5.6 mM, and N-2-hydroxyethylpiperaxine-N'-Z-ethanewas equilibrated with a gas mixture of 95% sulfonic acid 3 mM. The solution 0215% CO2 and the pH was adjusted to 7.8. The solution without bicarbonate/ CO2 was identical to the control solution except that 24 mM NaCl was substiwas equilibrated with 100% Oz. The tuted for 24 mM NaHC03, and the solution small difference in free Ca* concentration between the two solutions due to bicarbonate binding (1.0 mM vs 0.9 mM) was found to have minimal effects on the measurements. For extracellular recording, 10 mM Na aspartate was added to both solutions. The methods for extracellular and intracellular recording have been described previously (9, 10). For the cGMP measurements the retina was excised free of pigment epithelium under infrared illumination. Incubations were carried out for 2 to 30 min in approximately 20 ml of solution. Full bleaching was attained by exposing the retina to room illumination for 5 minutes. To break off the rod outer segments the retina was transferred to a beaker containing 0.5 ml of the appropriate solution, shaken gently for 10 set and the retina was then removed. To stop all reactions, 0.5 ml of 30% TCA (trichloroacetic acid) was then added. To insure disruption of the rod outer segments, the mixture was frozen and thawed twice. The mixture was then centrifuged at 27,000x g for 15 min. The supernatant was washed four times with 5 ml of water-saturated ether to remove the TCA, and the acid free solution was air dried. The residue was reconstituted with 0.3 ml of 50 mM acetate buffer (pH 6.2) and the cGMR content was measured by the radioimmunoassay technique (Collaborative Research, Waltham, MA (11)). The TCA precipitates were dissolved in 0.3 ml of 1.0 N NaOH and protein amounts were determined using the method of Lowry --et al. (12). RESULTS:

As presented

outer

segments

bating

solution

ate/CO2 ing

there

is

reduced

were

of light There

or eliminating

protein). not

respectively

incubated

completely

elimination In the

from

decreases in

eliminated

that by the

removal

786

incu-

of bicarbon-

pmoles

cGME/mg protein.

Illuminat-

2 caused

and 28 (+2) -

toad

similar pmoles

solution photoreceptor

of bicarbonate/CO the effect

from the

in the presence

incubating

of the

photoreceptor

dark,

in cGMP concentration

the absence

indicates

error)

the

of toad

of bicarbonate/CO2

to 26 (+3) -

bicarbonate/CO2

further

This

cGMP concentration

the bicarbonate/CO

on the cGMP concentration

were

retinas

by the

77 (2 5, standard

cGMP concentration, removing

1 the

and by illumination.

the retinas

Thus

in Fig.

of light

on the

of bicarbonate/CG2.

in

cGMP/mg protein.

mimicked outer

on illumination 2, (to

decreases

the

effect

segment. of the

15 (51)

pmoles

cGMP concentration Approximately

cGMP/mg is 25%

Vol.

96, No.

2, 1980

Figure

(E) not

1.

BIOCHEMICAL

permanently

still

injuring

of the photoreceptor

cubating tional

in

8lOPHYSlCAL

to be present.

the This

cGMF', we performed

retinas is

also

seemed to slightly

were

illustrated reduce

the

determined

by the

final

ments

indicate

that

the effect

of the

incubating

These

experiments

show that

of transfer

between

cGMP concentration

were

observed

was of some interest

electrophysiological

responses

the

changes

of the

solution

the

photoreceptor.

787

the

addi-

the

final

on cGME' is

in cGIQ concentration

effect

in-

These

solutions.

2 to 30 minutes

to observe

Although

No time-dependent

solutions. from

incubating

the ability

the different

cGMP concentrations,

were

also

1.

we were

of experiments

between

in Fig.

that

altering

a series

transferred

concentrations

It

COMMUNICATIONS

To be certain

or permanently

the photoreceptor,

the dark,

transfer

appears

to regulate

solutions.

in 5 minutes

RESEARCH

Cyclic GMP Concentrations of Rod Photoreceptor Outer Segments. Error bars indicate standard error of the mean. First four bar graphs present effects of various solutions with differing lightdark conditions. Second four bar graphs present effects of incubating only dark-adapted retinas in one solution for five minutes and transferring the retinas to a second solution for five minutes.

of the effect

in which,

AND

reversible.

occur effects

experi-

withon the

of incubation.

of bicarbonate/CO2 Representative

on the experi-

Vol.

96, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

11CI 1OC I

-

SO ,

-

80

Bkarbamte

/CO2 -/

, -

70 3 =a iy) 5i 40 M

30

20

10

0

60

5.5

50

4.5

Intensity

Figure

ments

Representative Extracellular Experiment Comparing the Effect of Bicarbonate/CO2 and Light on the Distal PI11 Responses of the Toad Retina (Bufo marinus). Light stimuli were 300 msec in duration with 3 minetweentimuli. The unattenuated stimulus light intensity (500 nm) was 1.4 x 10S5 W/cm2. Background light intensity (500 nm) was 1.4 x lo-11 W/cm2. Intensities were selected so that no stimuli were presented above cone threshold. The amplitudelight intensity relationship (V-log I) was determined after 84 min in the initial perfusion medium without bicarbonate/CO2 (perfusion rate 8 ml/min). The perfusate was then changed to include bicarbonatelC02. The bicarbonate/CO2 V-log I curve was determined after 84 minutes in that perfusate and the curve + background light after 107 min. The linear compression curve illustrates a 57% decrease of the bicarbonate/CO2 curve. This experiment is representative of 10 such solution changes with 8 separate retinas. (Observed decreases without bicarbonate/Cog, range 40-65X, average 53% -+ 6 standard error.)

illustrating

recording similar

2.

the

techniques results.

of the

data

are

obtained

presented

On removal

tude was observed effect

40

(-Lag)

both

in Figures

extracellular 2 and 3.

of bicarbonate/C02,

(57% of maximum,

removal

with

Fig.

of bicarbonate/CO2

Both

a decrease

2; 55% of maximum, was then

788

and intracellular

compared

techniques

gave

in response

ampli-

Fig.

3).

with

the effect

This of

Vol.

96, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

18

16

6

4.5

Figure

a steady

3.

linear

35 CLog)

3.0

25

Representative Intracellular Experiment Comparing the Effect of Bicarbonate/CO2 and Light on the Photoreceptor Responses of the Light stimuli were 200 msec in duraToad Retina (Bufo marinus). tion with 15 Kbzstimuli. The unattenuated stimulus light intensity (500 nm) was 2.3 x 10S6 W/cm2. The background light intensity (500 nm) was 1.5 x lo-lo W/cm2. Perfusion rate was approxiAfter a minimum of 5 minutes in the initial permately 4 mlimin. fusion solution, bicarbonate/C02, a V-log I curve was determined. The V-log I curve without bicarbonate/CO2 was started 9.5 min after changing solution. The linear compression curve illustrates a 55% decrease of the bicarbonate/C02 curve. This experiment is representative of 3 individual cells which were studied completely and 7 cells which were studied under various conditions and whose data were averaged. The only observed difference between the averaged cells and the data presented here was that some of the cells in solution without bicarbonate/CO2 showed a somewhat greater sensitivity.

background

maximum signal tion

40 Intensity

light

amplitude.

of bicarbonate/CO2 compression

selected

so that

The effects produced

of the original

are

decreases Signal

789

it

induced

clearly

the

same reduction

different.

in signal Amplitude-Light

amplitude

in

While

elimina-

which

were

Intensity

relation-

a

Vol.

96,

No.

ship

(see

light

Linear

caused

ties

Compression

significant

consistent

tebrate for

with

receptor

previous

Using

changing

to perfusate

cell

8 of 11 changes,

ate/CO2

caused

cells).

and Ostroy

effect

of light

responses.

in membrane

potential

Whereas

steady

background

(2 of 3 comparisons

of receptor

in the

time

(3 of 3 comparisons),

course

of the photoresponses

to peak

elimination sons;

maximum latency caused

total

DISCUSSION:

no clear

under

conditions

receptor,

are

comparable (6)

cGM-P, or dibutyryl concentration changes

in

presumably

that

without

reduce

started

at our

that We have

time-

total

effect

of the

(3 of 3 compariElimination

of bicar-

(2 of 3 comparisons)

of sensitivity

by Lipton

solution their

or

levels

--et al.

Since IMX,

altered

the

cGMP.

of cGMp and went

(6).

added

However,

injected

we

of the photo-

experiments

(7).

and then

that

and then

not observed

and Miller

media higher

latency,

in the

latency

to peak

obtained

the same range.

perfusion

the

decreases

the cGMP concentration

is possible

in their

compared

amplitudes),

of 1.8).

changes

mM bicarbonate

by Nicol

of

(2 of 3 comparisons).

to the results

reported

15

report

in the

the only

in the

time

with

the

also

decreases

similar

a factor

on the

effects

cGI@, it

latency

set,

bicarbon-

of the photoreceptor

and decreases

decreases

responses

used a 0.13

within

mM bicarbonate they

effect

The amplitude

--et al.

0.11

of the

observed

Lipton

were

change

time-course

with

that of the

with

with

we have

(3 of 3 comparisons);

of bicarbonate/CO2

bonate/C02 the

potentials

was observed

consistent

caused

potential

a depolarization

time-course

light

reported

the membrane

14 of 18 changes,

are

on the

on the ver-

results

to perfusate

experiments

and bicarbonate/CO2

the

it

caused

(in

In preliminary

(14).

with

recording,

hyperpolarization

intensi-

in maximum amplitude

affects

changing

light

of bicarbonate

and contrast

8 cells);

background

lower

changes

also

COMMUNICATIONS

the steady

at the

effects

bicarbonate/CO2

with

a cellular

Pinto

14, 15)

intracellular

without

The changes

of the

Bicarbonate/CO2

(16).

of the photoreceptor.

changes

The observed

(13,

RESEARCH

2 and 3),

amplitude

reports

potential

BIOPHYSICAL

in Figs.

changes).

an invertebrate

(in

AND

curve

signal

sensitivity

(i.e.,

are

8lOCHEMlCAL

2, 1980

cGMP

S-50

fold

they

used 24

Therefore

to even higher

Vol.

96, No.

2, 1980

levels.

We have not

definitive (14)]

compared

the cell

of cGMP. levels

induced

normally

decreasing

the

the

of light

the vertebrate

attained changes

the

remaining

However, cellular small

the

bicarbonate/CO2

from

the perfusate

as

on altering we do not yet adaptation

to reduce

the

by complete

concentrations light-induced

cellular

could

changes

it

is not

regards

However,

that

be interpreted

of cGMP some light-

did

not mimic

properties

concentration

of changes

electrophysiological as indicating

a critical

of cGMP do reflect

to the

In many respects

adaptation

to these

not

the

concentration

or light

of cGMP are

mech-

bicarbonate/C02

may indicate

also

understand

be produced.

by eliminating

critical

--et al.

way with

bleaching.

of cGMP can still

cGMP by Lipton

processes,

in a singular

The data

data

any and

data

are not

have

we [and Pinto

on the phototransduction

photoreceptor.

We do not that

Because

cGMP concentration

COMMUNICATIONS

depolarizations

or light

been able

cGMP in the photoreceptor

processes.

8).

the present

We have

concentration

effect

(7,

RESEARCH

experiments.

observed

phototransduction

to interpret

BIOPHYSICAL

comparable

on eliminating

and Miller

of the

to the

while

AND

to the hyperpolarizations

possible

of

for

observed

and Nicol

anisms

role

performed

explanations

Ostroy

(6)

BIOCHEMICAL

factor, some critical

that the step.

ACKNOWLEDGEMENTS: Supported in part by grants from the United States Public Health Service EYO0413 and GM07211 (E.M.). We thank Peter J. Stein for some preliminary experiments and Roberta A. Svoboda for her comments.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Miki,

N., Keirns, J. J., Marcus, F. R., Freeman, J., and Bitensky, M.W. 76:3820-3824. (1973). Proc. Nat. Acad. Sci. U.S.A. Farber, D. A., Brown, B. M., and Lolley, R. K (1978). Vision Res. -18: 497-499. Liebman, P. A. and Pugh, E. N. (1979). Vision Res. 19:375-380. J. Gen. zysiol. 73:629-653. Woodruff, M. L., and Bownds, M. D. (1979). Kilbride, P. and Ebrey, T. G. (1979). J. Gen. Physiol. 74:41%426. Lipton, S. A., Rasmussen, H., and Dowling J. E. (1977). 7. Gen. Physiol. 70:771-791. NicolFG. D. and Miller, W. H. (1978). Proc. Nat. Acad. Sci. U.S.A -75: 5217-5220. Nicol, G. D. and Miller, W. H. (1979). Nature 280:64-66. Gedney, C. and Ostroy, S. E. (1978). Arch. Biochem. Biophys. 188:105-113. J. Gen, Physiol. Lipton, S. A., Ostroy, S. E., and Dowling, J. E. (1977). 70:747-770 Stein=, A. L. (1973). Pbarm. Rev. 5:309-313.

791

Vol.

96, No.

12. 13. 14. 15. 16.

2, 1980

8lOCHEMlCAL

AND

Lowry,

BIOPHYSICAL

0. Ii., Rosebrough, N. J., Farr, J. Biol. Chem. 193:265-275. Winkler, B. S., Sims= V., and Benner, Visual Sci. 16:766-768. Pinto, L. H. and ztroy, S. E. (1978). Flaming, D. G. and Brown, K. T. (1979). Brown, H. M., and Meech, R. W. (1979).

792

A. L., J.

RESEARCH

and Randall,

(1977).

Invest.

COMMUNICATIONS

R. J.

(1951).

Ophthalmol.

J. Gen. Physiol. 71:329-345. Nature 278:852-85. J. Physiz 297:73-93.