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JOURNAL

Copyright

HI5TOcHE.MI5TRY

OF

© 1978 by The

CYCLIC

AND

Histochemical

Inc.

3’,5’-NUCLEOTIDE

LOCALIZATION

Vol. 26, No. 6, pp. 441-451, 1978 Printed in U.S.A.

CYTOCHEMISTRY

Society,

PHOSPHODIESTERASE;

IN

RAT

PHOSPHODIESTERASE

IN

N. T. FLORENDO, University

of Tennessee

Center

for publication

INTERSTITIAL RAT

RENAL

J. A. PITCOCK,

for the Health

September

CELLS1

MEDULLA

E. E. MUIRHEAD

AND

Sciences;

Tennessee Received

CYTOCHEMICAL

RENOMEDULLARY

and

Baptist

Memorial

Hospital,

Memphis,

27, 1978 (MS

77-203)

38146

29, 1977, and in revised

form

February

The localization of cyclic 3’, 5’-nucleotide phosphodiesterase activity in rat renal papillae was examined by utilizing cytochemical methods. Renal medullary interstitial cells had predictable phosphodiesterase activity predominantly on the cytoplasmic border of dilated cisternal membranes. Cells of the collecting tubule and loop of Henle contained diffuse reaction product. Capillaries had reaction product localized in pinocytic vesicles. Addition of theophylline resulted in no deposition of reaction product in interstitial cells and in cells of the collecting tubule and loop of Henle, suggesting an inhibition of phosphodiesterase activity. Since the membranes of dilated cisternae of renal medullary interstitial cells have been shown to be related to prostaglandin synthesis and probably to the anti-hypertensive function of these cells, the finding of phosphodiesterase activity on these membranes suggests a possible role of cyclic AMP in these two functions. Evidence terstitial

cell

function function lipids

suggests

that

(RIC)

exerts

the

renomedullary

an

in hypertensive

animals

may be mediated (17, 19, 20). The

in-

and

that

ing

of renal prostaglandins2 of enzymes involved

cyclic AMP prostaglandin

to cells implicated would suggest a role

AMP

utilized therefore zymatic methods, matic

in these

however, activity

permit in

intact,

only

allowing of enzymatic certain cells.

The degradation sine monophosphate

thus

far

localization

The

in for have are en-

undisrupted

AMP

‘This work was supported Health Service HL 19287-02. 2 Francoise-Marie, personal

by United

States

tissue

AND

of us

(10).

METHODS followed

with

1) utilizes

minor

5’ AMP the

is

as a

Exogenous

5’ nucleo-

and

captured

inorganic

by

lead

ions

in the incubation medium to form an electron product visible under the electron microscope the

site

methods

of phosphodiesterase

of fixation

employed

as

AMP

by endoge-

activity.

Cytochemistry: Adult male Wistar erage weight 350 g) were anesthetized neal injections of sodium pentobarbital Two

The

cyclic

to adenosine

latter

procedure

to 5’ AMP

phosphodiesterase. and

the

modifications.

exogenous

is hydrolyzed

converts

at or near

cells to specific

at, or adjacent localization.

which

included opaque

dissection

were of

the

tried. entire

albino rats (avby intraperito(30 The

first

papillae

mg/kg). method from

the

in cold (0-4#{176}C)2% glutaraldehyde in 0.05 M cacodylate-nitrate buffer, pH 7.4, containing 0.25 M dextrose. Entire papillae were subsequently washed at least 3 to 4 hr in kidneys

AMP

cytochemical localization of phosshould therefore indirectly sug-

a role for cyclic of phosphodiesterase

substrate

phosphate

tissue

is established action of cyclic

(Fig.

tidase

of

activity

renal papillae to RIC, utiliz-

by one

method

et al. (10)

technique nous

of enzy-

identification

originated

cytochemical

of Florendo,

of cyclic AMP to 5’ adeno(5’ AMP) by 3’, 5’ cyclic

phosphodiesterase terminating the

(12, 21). The phodiesterase gest site

Studies

technique

of phosphodi-

in rat directed

MATERIALS

in

slices from rat renal medulla (8), and not capable of correctly localizing activity to certain cells. Cytochemical

thereby not but assignment areas within

nucleotide the event

cells.

the

localization

investigated attention

been

implicated as a source (9, 18). The localization

cyclic

cytochemical

esterase was with particular

this

by anti-hypertensive RIC have also

metabolism synthesis

reason

anti-hypertensive

the

to, the For this

same

and

fixation

buffer

by

and

immersion

cut

into

1-mm

strips. The second procedure of the kidney by perfusion with mm

aldehyde

Public

plished dominal

communication.

in

the

same

buffer.

for

60 mm

cubes

or

1- to

2-

employed fixation 200 nil of 2% glutarPerfusion

was

accom-

through the left ventricle or through the abaorta in retrograde flow. After perfusion, the

441

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FLORENDO,

442 Cyclic 5’

AMP

AMP

phosphate FIG.

Exogenous phosphate

+

PITCOCK

5

5’

nucleotidase

adenosine

ions

electron

lead

perfusion

procedure.

For

both

techniques,

the

buffer

containing

3 mM

cyclic

AMP,

3 mg/mi

C. atrox venom, 50 mM nitrate and incubated for metabolic shaking water preincubate the solution ylline to dissolve before

theophylline and 2 mM lead 30 mm at 37#{176}C in a Dubnoff bath. It was necessary to at 37#{176}C to allow the theophthe addition of the tissue. 2)

Cyclic

control.

AMP

substrate

Tissue

slices

were

C. at room temperature. The slices were transferred to TMS buffer containing 3 mg/mi C. atrox venom and 2 mM lead nitrate and incubated for 30 mm at 37#{176}C in a shaking water bath. preincubated

in

atrox

for

venom

3) Endogenous slices

were

room and

TMS

buffer

containing

30 mm

5’-nucleotidase preincubated

activity in TMS

temperature, incubated

5 mg/mi

divided as follows;

control.

buffer

into

two

a) incubation

for

0.05

osmium ethanol

phosphate

+

opaque

precipitate

equal

at

portions

of tissue

M cacodylate-nitrate tetroxide and

sections were and examined

in

embedded

buffer, the

same

pH

buffer,

in Araldite

7.4, fixed

slices

in 2%

dehydrated

or Epon

812.

taming the presence of reaction product, the thin sections were lightly stained with lead nitrate for photography. Unstained sections were also photographed. Biochemistry: Adult male Wistar albino rats (average weight 350 g) were anesthetized by intra-peritoneal injections of sodium pentobarbital (30 mg/kg). The kidneys were excised, the entire papilla was removed, placed in cold (0-4#{176}C)TMS buffer and homogenized in a Potter-Elvejhem homogenizer (5 v buffer: 1 v papilla). Studies were performed under conditions similar to those used for cytochemical localization of enzymatic activity. Phosphodiesterase activity was determined by the procedure of Butcher and

Sutherland

in Thin

cut, stained lightly with uranyl acetate in the electron microscope. After ascer-

(5)

of the method measuring

except

by Lowry

inorganic

that

and

a minor

Lopez

modification

(16) was used

for

phosphate. RESULTS

Biochemistry:

The

results

depicted

in Fig-

production depicting

of dephospho-

ure 2 demonstrate tectable inorganic

the linear phosphate

diesterase activity line represents

in rat renal papillae. inorganic phosphate

without represents presence mately release

added theophylline. inorganic phosphate

The

The

solid release

broken line release in the

of theophylline. There is approxia 75% decrease in inorganic phosphate at 40 mm of incubation suggesting an

inhibition addition

of phosphodiesterase of theophylline.

Cytochemistry: buffered glutaraldehyde orous

Tissue 30 mm

in TMS buffer containing 3 mM cyclic AMP and 2 mM lead nitrate for 30 min at 37#{176}C in a shaking water bath, or b) incubation of tissue slices in TMS buffer containing 3 mM 5’ AMP and 2 mM lead nitrate for 30 mm at 37#{176}C in a shaking water bath. After incubation, the tissue was rinsed three times

in

AMP

1. Endogenous phosphodiesterase hydrolyzes the cyclic phosphate bond of cyclic AMP to form 5’ AMP. 5’-nucleotidase added in excess in the form of Crotalus atrox snake venom, liberates inorganic which reacts with lead ions to form an electron opaque precipitate.

buffer wash procedure averaged 3.5-hr. The tissue slices were first incubated for 30 mm at room temperature in a solution containing 60 mM Ti-is maleate buffer, pH 7.4, 2 mM MgC12 and 0.25 M sucrose (TMS buffer) containing 5 mg/mi of lyophilized Crotalus atrox venom (Sigma Chemical Co., St. Louis, Mo.) as a source of exogenous 5’ nucleotidase. The slices were then transferred to TMS buffer containing either 3 mM, 1.5 mM, or 0.75 mM cyclic AMP, 3.0 mg/mi C. atrox venom and 2 mM lead nitrate and incubated at 37#{176}C in a Dubnoff metabolic shaking water bath for 30 mm. Control reactions run simultaneously with the above reactions were as follows. 1) Theophylline control. Tissue slices were preincubated in TMS buffer containing 5 mg/mi C. atrox venom for 30 mm at room temperature. The slices were subsequently transferred to TMS

MUIRHEAD

phosphodiesterase

papillae were dissected and immersed in the same buffer. The perfusion techniques yielded variable resuits ranging from good to poorly fixed papillae. Most of the experiments were performed on tissue fixed by the immersion technique but these were confirmed by the

AND

incubation

activity

Despite and

by

brief fixation subjection

procedures,

the

the

in 2% to rig-

distinctive

morphological features of RIC remain identifiable (Fig. 3). Large lipid droplets and dilated cisternae, features characteristic of the cells, are quite evident. Plasma as well preserved but of the

collecting

as endothelial Complete strate.

RIC:

cated most membranes The some

membranes are not are stifi discernible.

tubule,

the

cells are mixture, Electron predictably of dilated

opacities are unidentifiable

ioop

Downloaded from jhc.sagepub.com at UNIV OF MASSACHUSETTS on April 5, 2015

as well

also

opaque

deposits

on or cisternae

adjacent (Figs.

localized cytoplasmic

also

of Henle

identifiable. 3 mM cyclic

quite Cells

AMP

subare

lo-

to the 4 and 5).

randomly structures

in not

PHOSPHODIESTERASE

IN

RAT

RENAL

qualities quality

7

443

but these are of true reaction

membranes tic vesicles

w

MEDULLA

of vesicles were also

not of product.

compatible more electron

the plasma membrane but granular in quality as those

= 0.

the “metallic” In capillaries, with pinocydense than

these were not as of definite reaction

(1,

0 I

a.

product. product

In unstained sections is absent throughout

inherent

density

of the

is qualitatively tallic” appearance ...0

without

.Q

20

I0

30

MINUTES FIG. 2. Phosphodiesterase activity in renal medulla determined under identical conditions utilized to depict cytochemical localization of enzymatic activity. The addition of theophylline decreases inorganic phosphate release by seventy-five per cent.

necessarily The

adjacent

opacities

to the

appear

cisternal

evident along membranes with less opacities along velope (Fig. 4). The uous with the dilated able

sections.

Membranes

of electron opacities tions, reaction product

Collecting

Electron

randomly collecting

over ducts

of the

did

(Fig. 4). clearly

more

loop

of

Henle

to be localized membranes appeared

secalong

and

vas-

occurred

area of Henle

of the cells.

pattern of reaction

at both

apical

of collecting duct to have a predictable

opacities vesicles

opacities

within

and

of Henle

cells

localization suggestive

of electron of pinocytic

Complete AMP: At

mixture, 1.5mM and 0.75mM these substrate concentrations

Control, morphologic bation was when snake vesicles This tase

to vesicles (Fig. 6A).

most cyclic there

rat

were

venom: tubule

predictably

absent.

however, did This may or non-spe-

incubation with 3 mM 5’ AMP: The picture obtained under this incuindistinguishable from that obtained venom was omitted. Only pinocytic

contained

occasional

electron

too may represent non-specific or endogenous 5’-nucleotidase

phodiesterase

opacities. phosphaactivity.

granule

complex

appears

of the phos-

to be localized

characteristic

Other intracellular membrane are product.

of RIC

organelles consistently

Theophylline,

diesterase,

at

and devoid

an inhibitor

inhibits

the

formation

(4, 22).

the plasma of reaction of phosphoof lead

phos-

phate precipitates at this site. Although isobutylmethyixanthine has been utilized as another inhibitor of phosphodiesterase activity (3), the results previous

with theophylline studies (10, 23)

quate in demonstrable

in this study and in were more than ade-

completely abolishing phosphodiesterase

absence

of endogenous

cisternal electron

membranes opqaque

tion, ically

observed. The some granular

activity

that in RIC demonstrable

or adjacent to the membranes of dilated cisternae. The dilated cisternae and the juxtaposed lipid granules form the dilated cisternae-lipid

discrete dilated

were have

collecting

This study demonstrates kidney, cytochemically

omitted 5’AMP

opacities membranes

snake

RIC,

DISCUSSION

appeared to be no electron opacities observed. Contro4 theophylline inhibition: When 50 mM theophylline were included in the incubation mixture (Fig. GC and Fig. 7) no discernible electron cisternal

8A).

without

Pinocytic vesicles of capillaries, show occasional electron opacities. represent endogenous 5’nucleotidase cific phosphatase activity.

and lipid devoid

deposits

be no selective in some instances

Electron

membrane

that of the “meproduct as seen

apparatus

In unstained stands out

the entire cytoplasmic (Fig. GB) and loop

appear

and basal plasma cells. Capillaries

Golgi

membranes, and predictably

opaque

There appeared to deposition although product

be

border of the cisternal memRibosomes are not evident in

ducts,

culature:

to

of dilated cisternae the outer nuclear en-

latter appears to be contincisternae as seen in favor-

and mitochondria, plasma granules are consistently

the cytoplasmic branes (Fig. 8A). this preparation.

membranes.

qualitatively

(Fig.

incubation

loop

8B) reaction section. The

osmium-fixed

different from of reaction

theophyffine

Control,

40

(Fig. the

with instead endogenous located

cytochemically activity.

5’nucleotidase is suggested deposits when

activity by the lack snake venom

cyclic AMP as substrate of cyclic AMP is used. at

5’-nucleotidase the plasma

Downloaded from jhc.sagepub.com at UNIV OF MASSACHUSETTS on April 5, 2015

activity membrane

The in of is

or when In addiis typwhen

I

-

V.

.a.T.1-.

.,.-4J

C.

-.

0’

a..

.

C

Ltion of KIC showing granule-cisternal membrane complex. (A) shows a process of an interstitial cell. These are frequently opposed to tubular cells or capillaries. (B) shows portions of the outer nuclear envelope dilated to form a cisterna similar to the more peripheral dilated cisternae. The reaction product is barely discernible at this magnification. No reaction product is seen in the extracellular matrix. 3 mM cyclic AMP substrate; (A) xlO,000; (B) x12,250. 444

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PHOSPHODIESTERASE

IN

RAT

RENAL

MEDULLA

445

t.

FIG.

4.

of dilated Occasional

demonstrable technique action ity at inorganic

Portion of RIC showing electron opaque deposits most consistently at, or adjacent to, the membranes cisternae. The Golgi complex, mitochondria and lipid droplets are devoid of reaction product. opacities are found randomly distributed over the cytoplasm. 3 mM cyclic AMP substrate; X17,500.

cytochemically suggests that the

product cisternal

(6, 7). Thus electron opaque

reflects phosphodiesterase membranes Determination

phosphate

release

under

similar

this reactivof

The

significance randomly

of RIC, collecting not known. Since

of

the

distributed duct this

controls, it is possible sent phosphodiesterase

electron over

opaque the

of Henle cells in appropriate

that this activity.

may The

de-

also repredeposition

role

for

cyclic

hormone

AMP activity

as on

and basal duct may of the sug-

a mediator the

of

collecting

tubules (11). In a majority of cases, however, collecting duct and ioop of Henle cells had electron opaque deposits randomly distributed over

cytoplasm

and ioop is absent

gested antidiuretic

cyto-

chemical conditions with or without the addition of theophylline supports the presence of phosphodiesterase adtivity in rat renal papillae. posits

of reaction product in the apical plasma membranes of the collecting have functional significance in view

is

the cells. The significance bution of reaction product the cytoplasmic reaction known. Since these are controls, it is possible uted reaction product

Downloaded from jhc.sagepub.com at UNIV OF MASSACHUSETTS on April 5, 2015

of the random distriin tubular cells and product in RIC is not absent in appropriate

that the randomly may also represent

distribphos-

446

FLORENDO,

PITCOCK

AND

MUIRHEAD

.,.-.

.

1 .#{149}..

: Fi;. product membrane

5. High magnification most convincingly does not contain

phodiesterase ity

in

rat

of a lipid granule-cisternal at the cytoplasmic border electron opacities. 3 mM

activity. kidney

Phosphodiesterase

may

exist

in both

activparticulate

and soluble forms as demonstrated in homogenates of entire kidney (13). It is possible that the

FIG. with

6. pinocytic

(A)

Portion vesicles.

of a blood 3

mM

membrane complex of the membrane cyclic AMP substrate.

cisternal occasionally ties of fraction

illustrating of the dilated x31,500.

membrane

opacities

observed

plasma

tubular cells represent of phosphodiesterase

vessel

presence cisterna.

in

of reaction The plasma

RIC

membrane the activity,

and

the

opaciparticulate while the

showing the presence of reaction product in vesicles most compatible substrate. x30,000. (B) Portion of collecting duct cell. Electron fashion without affinity for cellular organelles. 3 mM cyclic AMP substrate. on cytochemically demonstrable phosphodiesterase activity. No electron in the RIC. Pinocytic-type vesicles (arrows) still have darker membranes. depicted in Figure 6A. 50 mM theophylline plus 3 mM cyclic AMP substrate.

cyclic

opacities are distributed in random x 17,500. (C) Effect of theophylline opaque reaction product is identified These are not as granular as those x17,500.

the

AMP

Downloaded from jhc.sagepub.com at UNIV OF MASSACHUSETTS on April 5, 2015

r

II -

4

C

a

PC

I

...

.

.(

,:‘

:

-

..,

.b

.,,.

1

C

‘IP

*

1

$4

.:.

t.

..

C-”

Gc

4

.

I

‘Ih

447

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1a

448

1’

.-‘.

-

.‘

1 7. Higher

FIG. nuclear density

magnification and the

membrane

is unlike

cytoplasmic represent

that

of the reaction

opacity the

of a portion of Figure 6C demonstrating the presence of ribosomes formation of a dilated cisterna with a lipid droplet close by. The

of

soluble

RIC

product. and

tubular

phosphodiesterase

x60,000. cells frac-

tion. Another deposition

possible explanation for is the diffusion of reaction

these sites or the diffusion phate before reaction with electron opaque precipitates plasm. However, the absence ties

over

mediately

lipid

droplets

adjacent

product

is minimal.

this random product to

of inorganic phoslead ions forming within the cytoof electron opaci-

and

mitochondria

im-

to cisternal

membranes

con-

taining reaction product of diffusion or non-specific

on the outer quality of the

suggests that the effect absorption of reaction

Evidence of entire

also rat

is divisible

into

tively

Km

high

indicates

kidneys

Cytochemical ase activity

that

in homogenates

phosphodiesterase

two and

groups, one

one

having

demonstration in the caudate

activity having

AMP concentrations product is located

at

membrane

the

However,

post-synaptic with

the

post-synaptic

use

of lower

Km

(13).

of phosphodiesternucleus of rat brain

utilizing lower cyclic gests that the reaction associated

a rela-

a lower

and

sugmainly

minimally

organelles

cyclic

AMP

(3). concen-

trations in the present study causes no production of electron opaque reaction products. Previous studies utilizing this same technique

Downloaded from jhc.sagepub.com at UNIV OF MASSACHUSETTS on April 5, 2015

31

.t

.

.t

p

.‘

V.

..

C

..2”-. )...

-L_

..

.

.

FIG. 8. (A) High magnification of an unstained section demonstrating the presence of reaction product on the membrane of a dilated cistema. The plasma membrane (P) is devoid of reaction product. 3 mM cyclic AMP substrate. x72,000. (B) High magnification of an unstained section demonstrating the membrane of a dilated cisterna and the plasma membrane (P). No reaction product is visible. 50 mM theophylline plus 3 mM cyclic AMP substrate. x77,000. 449

Downloaded from jhc.sagepub.com at UNIV OF MASSACHUSETTS on April 5, 2015

FLORENDO,

450 (10)

have

tivity

phosphodiesterase

predominantly

example,

at plasma

electron

activity tic

demonstrated

have

plasma plasma

opaque

been

membranes

of rat

this

deposits brain

technique

(10),

has

21),

in the

shown

in synaptic neuromuscular of

RIC with

membranes membranes

latter studies The dilated

in-

lamellae

of the

cytochemically activity

outer in the mus-

demonstrable predominantly and not has support

(23, 25). cisternae-lipid

synthesize prostaglandins (18). of the dilated cisternae-lipid to prostaglandin synthesis is

lipid

granules

become

changes in the comwith indomethacin,

larger

and,

In indo(24), the

in

some

in-

stances, appear to coalesce. inhibition of prostaglandin volve the dilated cisternae-lipid

This suggests that synthesis may ingranule com-

plex,

a suggestion

by the

tion somal

of prostaglandin fraction of rabbit

evidence activity

supported

demonstrating in microsomal

the micro(2). Direct

prostaglandin synthetic fractions of RIC in cul-

ture, however, has presence of a well

not been developed

lipid granule complex in the antihypertensive

also

(24). oped

demonstra-

synthesis in renal papillae

documented. The dilated cisternae-

appears function

rats, and well develan anti-hypertensive animals (24). Since one

mediator of this antihypertensive function is a lipid (19, 20), it is possible that the lipid granules of the complex play a role in the anti-hypertensive

function of RIC. In view of evidence

between

cyclic

AMP

The

and

prostaglandin

a connection synthe-

the

function

of the

complex. The is still obscure.

slices of medulla (8) suggests synthesis or exogenous pros-

supports

is largely

conjectural.

authors

the

wish

excellent during

to

thank

technical

this

this

Ms.

relation-

Linda

assistance

Reed she

ren-

study.

LITERATURE

Cell

CITED

8.

9.

10.

11.

Biology,

WB

Saunders,

New

York,

1970,

p

150 DeRubertis FR, Zenser TV, Craven PA, Davis BB: Modulation of the cyclic AMP content of rat renal inner medulla by oxygen. Possible role of local prostaglandins. J Clin Invest 58:1370, 1976 Dunn MJ, Howe D: Prostaglandins lack a direct inhibitory action on electrolyte and water transport in the kidney and the erythrocyte. Prostaglandi.ns 13:417, 1977 Florendo NT, Barrnett RJ, Greengard P: Cyclic 3’,5’-nucleotide phosphodiesterase: cytochemical localization in cerebral cortex. Science 173:745, 1971 Grantham JJ, Burg MB: Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules.

implicating

in

granule relationship

and cyclic

1. Adinolfi AM, Schmidt SY: Cytochemical localization of cyclic nucleotide phosphodiesterase activity at developing synapses. Brain Res 76:21, 1974 2. Anggard E, Bohman SO, Griffin III JE, Larsson C, Maunsbach AB: Subcellular localization of the prostaglandin system in the rabbit renal papilla. Acta Physiol Scand 84:231, 1972 3. Ariano MA, Adinolfi AM: Subcellular localization of cyclic nucleotide phosphodiesterase in the caudate nucleus. Exp Neurol 55:84, 1977 4. Bulger RE, Trump BF: Fine structure of the rat renal papilla. Am J Anat 118:685, 1966 5. Butcher RW, Sutherland EW: Adenosine 3’, 5’phosphate in biological materials. 1. Purification and properties of cyclic 3’, 5’-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3’, 5’-phosphate in human urine. J Biol Chem 237:1244, 1962 6. Dalton AJ, Haguenau F, Eds: The Membranes. Acaaemic Press, New York, 1968, p 102 7. DeRobertis EDP, Nowinski WW, Saez FA, Eds:

to be involved of these cells

The complex appears to be poorly develin cells incapable of diminishing the blood

pressure of hypertensive oped in cells demonstrating function in hypertensive

thought

metabolism suggests that

of synthesis this

is

of RIC RIC in

synthesis. in culture

involved

demonin RIC

stimulate an increase in cyclic AMP to oxygen. The close proximity of

sites but

dered

culture actively The relationship granule complex

of prostaglandin RIC grown

be

cisternae-lipid of the

evidence from prostaglandin

these

of a complex

in prostaglandin function,

may

taglandins in response

for

morphological functional

an inhibitor methacin-treated

AMP

(8), the activity

ACKNOWLEDGMENT

a distinguishing that may have

suggested by morphological plex in RIC cultures treated

in membranes

dilated mechanism

on

complex

feature significance.

particularly

ship,

associated in these

granule

MUIRHEAD

sis in slices of rat renal medulla stration of phosphodiesterase

The that

vesicles of nerve endings junction (23). Thus

the finding phosphodiesterase cisternal plasma

and

however,

AND

to be active antihypertensive

of cytochemactivity

of rod cells in mouse retina (25), tubular system of newt skeletal

cle (23), and in the newt

of

of thyroid cells (14), and in of most skin cell types (15).

same

been

indicative

(1, 3, 10,

localization phosphodiesterase

also

For

in post-synap-

tracellular membrane ically demonstrable segment transverse

ac-

membranes.

demonstrated

membranes membranes

Utilizing

PITCOCK

12. Greengard mechanism

Am

J Physiol

211:255,

1966

P, McAffe DA, Kebabian of action of cyclic AMP

Downloaded from jhc.sagepub.com at UNIV OF MASSACHUSETTS on April 5, 2015

JW: On the and its role in

PHOSPHODIESTERASE synaptic

transmission

GA, Advances 1. Raven 13.

14.

15.

16.

17.

Press,

in

Greengard

in Cyclic Nucleotide New York, 1972,

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