JOURNAL TRANSCRIPT

---

0O22-3565/91/2591-0279$03.OO/O THE JOURNAL OF PHARMACOLOGY

Copyright

Vol. 259, No. 1

EXPERIMENTAL THERAPEUTICS Society for Pharmacology and Experimental

AND

C 1991 by The American

Printed

Therapeutics

Membrane-Mediated Effects of the Steroid 17-a-Estradiol Adrenal Catecholamine Release1 M. G. LOPEZ, Departamento Accepted

F. ABAD,

C. SANCHO,

de Farmacologla,

for publication

Facultad

R. DE PASCUAL,

do Medicina,

A. BORGES,2

Universidad

R. MAROTO,

Aut#{244}nomade Madrid,

Arzobispo

W.

DIXON3

Morciio,

on

and

4, 28029,

in U.S.A.

A. 6.

Madrid,

GARCIA

Spain

June 1 4, 1991

ABSTRACT

respectively.

However,

secretion

evoked

line (3 M for 30 sec) was not affected and was affected to a variable extent

The target

classical

receptors. nucleus crease

mechanism involves

tissues

of steroid

the

activation

The receptor-steroid to modulate gene in both

RNA

and

by pulses of methacho-

by 1 zM of 1 7-a-estradiol by 10 M 17-a-estradiol.

hormone

new

protein

synthesis.

mechanisms,

possibly

(see

review

by binding by

in neuronal

cortex steroids are known to influence of adrenal medulla catecholamine-synthetizing inoff and Axeirod, 1971). Also, corticoids suppress of axonal-like processes in cultured chromaffin et at., 1978). However, these effects are likely by a slow genomic mechanism. It was, therefore, whether

steroid

hormones

also

affect,

the catecholamine secretory process in adrenal This question is especially relevant because, situations, both cortical steroids and adrenal Received I

This

for publication work

was supported

FISS (Spain). 08504 and University and

2 Present Universidad 3 Present Pharmacy,

February

Also

of Kansas

mem-

the

enzymes (Molthe emission cells (Unsicker to be mediated interesting to in

short

from

term,

Fundaci#{243}n Areces,

CICYT

National

Institute

on Drug

Abuse

Grant

Biomedical

Research

Support

Grant

RR

DMPP, dimethylphenylpiperazinium;

massively. It is therefore the secretory activity

influence

In this study catecholamine steroid

was

sponsive

we found secretion

selected

DA

5806.

for

17-f-estradiol, has been

to

17-a-estradiol

et at. (1978)

LaBella

plausible of chromaffin

that evoked

17-a-estradiol by various

several

reasons.

a large

assay compounds showed

and

target

number

membrane.

Moreover,

large

opiates

adrenal

of steroids

in

the

adrenal

peptides in the

medulla

modulation (Lemaire

in an

in [3H]nalsuggests that in the cell

chromaffin

cells

et at.,

(Viveros

re-

and two and 17-a-

competition

medullary

of opioid

to be involved

seem

secretion ura

amounts

blocks This However, receptors.

only 17-a-estradiol (17-a-dihydroequilin

significant

steroids cells.

tissues

is inactive. with opioid

oxone radioceptor binding assay. This observation 17-a-estradiol may interact with opiate receptors crete

that

selectively secretagogues. In

17-a-estradiol shown to interact tested

opiate radioreceptor structurally related

1979)

se-

and

of catecholamine

et at., 1980;

Kumak-

et at., 1980).

chromaffin cells. during stressful catecholamines

Address: Departamento de Farmacologia, Facultad de Medicina, de la Laguna, Tenerife, Spain. Address: Department of Pharmacology and Toxicology, School of University of Kansas, Lawrence, Kansas 66045-2500.

ABBREVIATIONS

released

dihydroequilenin)

expression

19, 1991.

in part by grants

by The

the

1990).

Adrenal

explore

to the cell in an in-

However,

are might

steroids exert some of behavioral effects inthrough nongenomic

to receptors

Schumacher,

in its cytosolic

complex translocates expression which results

rapidity with which gonadal and adrenal their neuronal, electrophysiological and dicates that these hormones also act branes

action

of high-affinity

Catecholamine secretion evoked by higher concentrations of methacholine (1 00 M for 60 sec) was reduced by 50% by 10 M 1 7-a-estradiol. 17-a-Estradiol decreased secretion evoked by pulses of 1 20 mM K for 1 0 sec to a similar extent in the perfused bovine and cat adrenal gland. The 45Ca uptake into bovine chromaffin cells in culture stimulated by DMPP (1 00 M for 10 sec) or high K (59 mM for 10 sec) was almost inhibited completely by 100 zM 1 7-a-estradiol. The rapid action precludes a classical genomic mechanism and suggests effects at the cell membrane.

Materials Perfusion Monitoring Both

and Methods

of Cat Adrenal Glands For Continuous of Catecholamine Release

adrenal

glands

of

the

cat

were

isolated

on-Line and

prepared

for

retrograde perfusion as shown earlier (Garcia et a!. 1980). The glands were perfused at room temperature (22 ± 2#{176}C) with Krebs-bicarbonate solution of the following composition (in millimolar): NaCl, 1 19; KC1, 4.7; CaC12, 2.5; MgSO4 - 7H20, L2; KH2PO4, 1.2; NaHCO3, 25; and

HEPES, 4-(2-hydroxyethyl)-1

-piperazineethanesulfonic

acid. 279

Downloaded from jpet.aspetjournals.org at ASPET Journals on May 9, 2016

The effects of 17-a-estradiol on the secretion of catecholamines from the perfused bovine and cat adrenal gland and bovine chromaffin cells in culture elicited by dimethylphenylpiperazinium (DMPP), methacholine and high potassium were studied. In perfused cat adrenal glands, secretion of catecholamines evoked by pulses of DMPP (1 M for 30 sec) was decreased by 1 7-aestradiol at concentrations of 1 and 1 0 M by 50 and 80%,

280

Lopez etal.

Vol. 259

Bovine adrenal .

(DMPP)

Bovine

adrenal

(DMPP)

A.

100

40

S C 0 U

a. 80

0

30 S

(a a) (I) (0

S S S

60

20

a) a)

S

C a)

40

E

C

S 0

E

(0

10

.C

0

(5

20

.c C.)

S (0

(5

a)

0

(0

C)

P1

0 Cirol.

-6 Log

.5,5

.5

P2

P3

P4

P5

P6

.4,5

(17-alpha-estradiol)

B. S U)

40

a. as

30 S U) S S S

20

S

glucose,

11. The

pH being

fmal

After late

a 90-mm

hermetic

initial

was

was perfused through at a rate of 1 ml/min;

fluid

with

each

chamber

the chambers were mitigate the pulses

and

bubbled

perfusion,

perfusing

electrodes;

space

solution

02

95%

gland

was

placed

alytical

Systems

carbon

working

less-steel

tial

LC-4B

between two helical silver of air to reduce the dead by the peristaltic pump. The full

generated

the gland

Omniscribe

recorder.

Equilibration

an Ag/AgCl

electrode.

V and

the

by means

of a LKB

Once

solutions

detector

equipped

with

electrode

and a stain-

were

current

adjusted

the

signal

base

line

of norepinephrine

oxidized was was

glassy

at a poten-

recorded

in an

stable.

(10’#{176}to

10

g/ml)

were

administered

through the gland (internal standard) or directly to the detector (external standard). In order to analyze the released catecholamines separately, aliquots were recovered after passing through the detector. Dopamine, norepinephrine and epinephrine were separated using a Series 10 Perkin Elmer high-performance liquid chromatography pump, provided with a 100-id Reodyne (71125S) loop injector. A

C18 (3-il particle

bondapak

umn,

size) reverse

phase

column,

without

precol-

used. The mobile phase consisted of (per liter): NaH2PO4 H2O, 9.66 g; EDTA (Tritriplex), 3.72 g; 1-heptanesulfonic acid sodium salt, 1 g; and methanol, 20 ml. The pH was adjusted to 3.1 with ortophosphoric acid. The solution was filtered (0.22 m Millipore filters) and degassed by vacuum during 15 mm. Standard solutions of dopamine, epinephrine, norepinephrine and dihydroxibencilamine were used to calibrate the system; 10 to 100 ng/ml of dihydroxibendilamine were always added to the samples to have an internal reference standard. system

was

The conditions were

as

described

of the electrochemical above.

Catecholamine

detector release

and the recording was

10

(5

P1

peristaltic

reference

Catecholamines

oxidation

S 0 C (5 S S

held

and maintained

amperometric

electrode,

auxiliary

of + 0.65

E

in a metacri-

the system allowed the study of secretion from both glands, but only one was stimulated at a time. The liquid flowing from the perfusion chamber rapidly reached an amperometric detector through the thin polyethylene tubing; the dead space between the gland and the detector was less than 100 l and the fluid took approximately 5 sec to reach the detector. Electrochemical detection of standard and released catecholamine was performed using a Bioanpump

C

5% CO2, the

and

7.4.

quantitated

P2

17-n.E

P4

P5

Cd 1mM

10tM

Fig. 2. A, catecholamine

release in response to DMPP, 100 M, in a bolus injection of 2 ml in perfused bovine adrenal glands is shown. Six consecutive pulses are given to one same gland at 1 5-mm intervals. The data are expressed as total catecholamines released per pulse, in micrograms measured fluorometrically, and correspond to the mean ± S.E.M. of four experiments. B, effects of 10 M 17-a-estradiol (1 7-a-E) and 1 mM Cd to DMPP secretory responses are shown. P1 and P2 correspond to control pulses; during the third pulse (P3) and 1 5 mm before it, 1 0 M 1 7-a-E is perfused. After this, the gland is washed and two more control pulses are given at 15-mm intervals to study recovery. During the last pulse (P6) the effects of the inorganic calcium channel blocker, is tested. The data are means ± S.E.M. of four glands. *P < .01 with respect to P2. by measuring

or curve areas, and expressed as nanoamor nanograms of total catecholamine release per minute ofcollection period (1 ml). In these conditions the sensitivity of the method was around 1 ng/ml (6 pmol/ml) for epinephrine and norepinephrine. peres

(oxidation

peak

heights

current)

Perfusion of Adrenal Glands For Discontinuous of Catecholamine Release Perfusion

of cat

adrenal

glands.

Cats

of both

Monitoring sexes

weighing

1.5 and 3 kg were anesthetized with sodium pentobarbitone (40 mg/kg). Both adrenal glands were isolated and prepared for retrograde perfusion as described previously (Garcia et at., 1980). The glands were perfused at a rate of 6 ml/min with Krebs-Tris solution (composition in millimolar: NaC1, 144; KC1, 5.9; MgCI2, 1.2; CaC12 2.5; Tris, 10; and glucose, 11) pH 7.4, bubbled continuously with pure oxygen. Glands were perfused initially with this solution for 60 mm for between

Downloaded from jpet.aspetjournals.org at ASPET Journals on May 9, 2016

Fig. 1. Inhibition by 17-a-estradiol of catecholamine release from perfused bovine adrenal glands. After equilibration, glands were challenged 7 times at 20-mm intervals with DMPP (1 00 MM for 10 sec). The release of total catecholamines obtained during the third stimulation was taken as 100% in each individual experiment (control; Ctrol). Then, secretion obtained in subsequent challenges, in the presence of increasing concentrations of 17-a-estradiol, was expressed as percent of control in each individual gland. At each concentration, 1 7-a-estradiol was present 20 mm before and during stimulation with DMPP. Data are means ± S.E.M. of six glands.

Cat adrenal

(DMPP)

281

and Secretion

17-a-Estradlol

1991

Later, glands were stimulated by switching from normal solution to another containing a secretagogue (high K, DMPP or methacholine). Perfusion of 17-a-estradiol in Krebs’ solution was started 30 mm before each stimulation. Perfusion of bovine adrenal glands. Bovine adrenal glands were brought each morning from the slaughterhouse in cold Krebs-Tris solution. The fatty tissue surrounding the gland was dissected carefully and the gland was washed 2 or 3 times inserting a syringe in the adrenal vein with freshly oxygenated Krebs-Tris at 37#{176}C, in order to remove blood cells and to make sure that there were no leakages. After this a cannula was inserted in the adrenal vein and was tied up tightly with a string. Deep incisions, down to the medulla, were performed with a blade around the gland to allow the outflow of the perfusing liquid. equilibration.

Krebs-Tris

The gland was then connected to a peristaltic WI) and perfused at a rate of 10 ml/min.

pump

(Gilson,

Middleton,

Before starting the experiment the glands were perfused to 90 mm with Krebs-Tris bubbled continuously with pure

37’C in order

to reach

by bolus

injections

duced solution

Quantitation bovine tion,

the

perfusate

1 -I

--

Us-ESTM.

(-log

$0 Ma.

(MD

before

detection of catecholamine release from perfused cat adrenal glands. The records represent the electrochemical signal obtamed from perfused adrenal glands in which the perfusate from the gland flows dlrecfly on the amperomethc detector. Secretion was evoked by pulses of DMPP (1 iM for 30 sec, represented by #{149}). 17-a-Estradiol was added to the perfusion fluid at times represented by the horizontal bar.

fluorometric

Calcium

uptake

into

from

bovine

were isolated

N. A sample

of the perfusate

background

bovine adrenal

chromaffin

assayed

was and

Gordon

in bottles Madrid,

catechol-

amines. Chromaffm

cells

by Moro et aL

as described

The

at

a concentration

of

Eagles Medium with penicillin (50 I.U./ml)

containing cells

cell fraction from Percoll gradients in suspension under smooth continuous an MCS-140 microcarrier stirrer (from Techne

with

Spain)

Modified

calf serum ml).

were

kept

in

an

incubator

1 million

cells

per

ml

of

10% heat inactivated and streptomycin

fetal (50 tg/

(Heraeus,

Spain

Madrid,

under an atmosphere of 5% CO2 at 37#{176}C. Experiments were carried out with 48- to 72-hr cultured cells. The cells were centrifuged at 120 x g during 10 mm and the pellet was resuspended in Krebs-HEPES (concentration in millimolar: NaC1, 144; KC1, 5.9, MgCl2, 1.2; CaCl2, 0.5; HEPES, 10 and glucose, 11), pH 7.4, at 37#{176}C to a final concentration of 2 million cells per 0.5 nil/tube. The cells were kept in a bath at 37#{176}C with continuous agitation. 17-aEstradiol was incubated during 30 rain. After incubating the drug, Ca uptake was studied by adding 1 ml of CaCl2 (21.2 mCi/mg, Amersham, Des Planes, IL) at a concentration of 4 Ci/ml so that the final concentration in the tube with the cells was 2 jCi/ml, in the presence or absence of high K (59 mM) or DMPP (100 M) during 10 sec. The stimulation was stopped by adding 1 ml of Krebs-HEPES containing 10 mM LaC13 and no Ca and the cells were filtered through Whatman GF/C filters and washed 4 times with Krebs-HEPES containmg 10 mM LaCl3. 2 mM EGTA, but no Ca at 4#{176}C. The filters

a) U)

a.

as a)

Cl) (0 a)

a) a) C

E (0 0 C C.)

a)

(0 C.)

were put into vials, scintillation liquid (Ready-Micro, lerton, CA) was added and radioactivity was counted counter (Packard 1500 Tri-Carb, Downers Grove, IL). P1

17.cz-E.

P3

10 iM

Fig. 4. In one gland three consecutive pulses are given: P1 corresponds by DMPP (1 0 M for 30 sec), in the second pulse (P2) the blocking effects of 10 17-a-estradlol (1 7-a-E) are shown and P3 is a control pulse after a wash-out period of 30 mm to study reversibility of secretion after treatment of the gland with the steroid. Catecholamine secreon is expressed as micrograms per pulse. Each bar Is the mean ± S.E.M. of five glands. These experiments were performed In perfused cat adrenal glands and total secretion of catecholamines is measured fluorometrically (ordinate). *P < .01 compared to P1.

to control secretion evoked

(1971).

of total

cells.

medulla

just

catecholby the

(1990). The epinephrine-enriched was used. The cells were kept

Dulbecco’s

(DMPP)

of 0.05

pulse

Fluoresence unite were converted into micrograms amines by using appropriate standards of the pure

Ltd.,

adrenal

release from perfused cat and the glands were stimulated for secrein cold tubes acidified with perchloric

was taken to measure release. Total catecholamine release method described by Shellenberger

agitation

Cat

collected

concentration

the secretory

amine

Fig. 3. On-line

was

10 sec a Krebs’

during

K or DMPP.

of catecholamine glands. When

adrenal

acid to a final :i_-’

high

containing

Catecholamine

of 2 ml or perfusing

60

Drugs and reagents and 17-a-estradiolwere

used. purchased

MO). Dulbecco’s

Modified

Eagles

biotics

were

from

GIBCO

(Grand

macia

(Piscataway,

NJ).

DMPP from

Ful-

iodide, methacholine chloride Sigma Chemical Co. (St. Louis,

Medium,

Island,

Beckman, in a scintillation

calf serum

fetal

NY)

and

Percoll

and antifrom

Phar-

17-a-Estradiol was dissolved in absolute ethanol (Merck Sharp & Dohme, West Point, PA) to a concentration of 10_2 M; further dilutions were done in Krebs’ solution; 10_2 M concentrations of DMPP were prepared in water and ethanol, respectively; final concentrations used

were prepared Statistical

directly in the Krebs’ solution analysis. Statistical analysis

used. of means

were

performed

Downloaded from jpet.aspetjournals.org at ASPET Journals on May 9, 2016

a

equilibrium.

during

oxygen at secretion was in-

282

L#{244}pezetal.

VoL 259

Cat adrenal

(K’)

Fig. 5. On-line detecon of catechdamine release from perfused cat adrenal glands. The records represent the electrochemical signal obtained from perfused adrenal glands in which the perfusate from the gland flows directly on the amperometric detector. Secretion was evoked by pulses of methacholine (3 for 30 sec, represented by

likkL

K .

S

C

C

#{149}C

‘C

C

C

#{149}

-%

-J---

I

17s-ESTRADIOL

Hog

(MD

with the computer program MICROSTAT (copyright 1985, Madrid, Spain), using the Student’s t test. The cance between means was taken at P < .01.

Ecosoft, Inc. level of signifi-

Results of 17-a-Estradiol on the Nicotinic-Mediated Response in Bovine and Cat Adrenals

EffeCts

Secretory

Bovine adrenal. (initial equilibration

After perfusing the gland for 60 to 90 mm period), the basal catecholamine output in bovine adrenal glands amounted to 5.6 ± 1.5 ig/2 mm (n = 6). Stimulation with DMPP (100 M for 10 sec) caused a release of 131.6 ± 21.1 ag/pulse. These stimulations were applied to each gland several times, at 20-mm intervals. In these conditions, the secretory response to DMPP was quite constant. Figure 1 shows that 17-a-estradiol decreases catecholamine release in a concentration-dependent manner. At the maximum concentration used (30 tM), 60% blockade of secretion was achieved. The calculated IC,o for 17-a-estradiol was around 10 tiM. In

a different

blocking

effects

set of the

of experiments, hormone

was

the

reversibility

tested.

Figure

of the 2A

shows

that quick injections of DMPP (2 ml of a 100 M solution into the adrenal vein) caused a net catecholamine secretory response of 35 ± 4.1 ag/pulse from control bovine adrenal glands. The response decayed by 20% at the sixth pulse (P6). In experimen-

tel

glands,

17-a-estradiol

(10

tM,

present

15 mm

before

and

during the secretory pulse) caused a 61.6% blockade of secretion. Upon washing out the compound, secretion recovered to the initial values (fig. 2B). Cd ions (1 mM), present before P6, reduced the DMPP secretory response to about 20% of the initial response. Cat adrenal. On-line electrochemical detection of catecholamine release from perfused cat adrenal glands, evoked by pulses

shown

with

in effects of eter were response;

low

concentrations

of DMPP

(1 iM

for

30 sec),

is

figure 3. Once the secretory response stabilized, the two concentrations of 17-a-estradiol on this paramtested. At 1 M, the hormone halved the secretory 10 iM decreased secretion by over 80%. After perfus-

ing

the

gland

response

for 50 mm

to DMPP

with

recovered

Krebs’

solution,

the

secretory

fully.

In a second group of experiments, secretion was triggered by higher concentrations of DMPP (10 tM for 30 sac). Because the rates of catecholamine release were higher, they could be measured fluorometrically. Three pulses of DMPP were applied to paired glands from the same animal. During P1, secretion amounted to 809 ± 164 ng of catecholamines (fig. 4). In the presence of 17-a-estradiol (10 SM), secretion was reduced to 41% of that obtained in P1. On washing out the hormone, the secretory rate (P3) recovered to figures even higher than those obtained in P1. Thus, as in the bovine adrenal, 17-a-estradiol blocks nicotinic secretory responses in a reversible manner.

of 17#{149}a#{149}Estradiol on Muscannic-Mediated

Effects

in Cat Adrenals

Responses

(3 zM for 30 see) given to cat adrenal connected on-line to an electrochemical detector, triggered quick secretory responses (fig. 5). The responses are blocked by atropine (not shown), suggesting that they are due to muscarmnic receptor stimulation (Ballesta et at., 1989). At 1 ,.M, 17-a-estradiol affected these secretory responses very little; Pulses

of methacholine

glands,

10 M

partially

decreased

catecholamine

release.

Because

the

methacholine secretory responses underwent a gradual desensitization (four first records in fig. 5), it is difficult to conclude that 17-a-estradiol was affecting the muscarmnic-mediated response. Therefore, experiments using higher concentrations of methacholine,

with

fluorometric

estimations

of catecholamine

release, were performed. Figure 6 shows the secretory responses evoked by three pulses (P1, P2 and P3) of methacholine (10 tM for 60 sec) applied at 30-mm intervals. This concentration evoked secretion of 173 ± 8 ng in the three pulses; 17-a-estradiol (present during P2) did not modify this secretory response. A higher concentration of methacholine (100 iM for 60 see) evoked a much more marked secretory response (1512 ± 248 ng, n = 7) of catecholamines. This response was reduced by 50% in the presence of 10 M

17-a-estradiol.

After

terminating

the

perfusion

with

17-

Downloaded from jpet.aspetjournals.org at ASPET Journals on May 9, 2016

1__-.

.).

17-a-Estradiol

1991

Cat adrenal A. Methacholine

.

Bovine

283

and Secretion

adrenal

(K’)

10tU 100

(a 3

a. 0 I-

S UI S

80

C 0 U

S S

0

S

C

E S

60 S

0 C U S

U)

(0 S

a)

S U

40

I-

P1

17#{149}cz-Estradlol

as

P3

C

E Cs

0

20

C U

S S

100 tM

C-)

(a 3

V

a.

‘

‘

Ctrol.

as

.

.

-6

-5,5

.5

-4,5

S

(a

Log

S S

(17-alpha-estradlol)

S

Fig. 7. Effects of 17-a-estradiol on potassium-evoked secretion of catecholamines from perfused bovine adrenal glands. Glands were stimulated with 118 mM K for 10 sec. 17-a-Estradiol was added to the perfusion fluid 20 mm before the secretion test pulse. The data correspond to the mean ± S.E.M. of six experiments. Ctrol, control.

S C

E S 0

C

U S

P1

i7.a-EstradoI

P3

Fig. 6. In perfused cat adrenal glands, the effect of 10 M of 17-aestradiol on secretion evoked by the muscailnic agonist methacholine at two concentrations is shown. In the experiments carried out in A, secretion is triggered by 10 M of methacholine, whereas in B a 10-fold higher concentration of agonist is used (n = 7). P1 and P3 correspond to control pulses (in the absence of the steroid) whereas P2 represents the effeCtS of 10 M 17-a-estradiol on secretion evoked by methacholine. The pulses were appiled at 30-mm intervals. The hormone was added to the perfusion fluid 20 mm before the secretion test pulse. Data correspond to the means ± S.E.M offour glands. * < compared to P1.

pletely. release Cat see) (5.2

Cd ions (1 mM) blocked K-evoked by over 80% (P6 in fig. 8B) adrenals. The application of K pulses

to perfused

± 0.9

estradiol out the

cat

adrenals

a large

ig of catecholamines).

(10

jIM),

secretion

the hormone for secretory response

Effects Cells

caused

In

the

decreased

by

30 mm with normal recovered partially

of 17-a-Estradiol

on Calcium

catecholamine (120 mM

for 10

secretory

response

presence

of 17-a-

40%.

After

washing

Krebs-Tris (78 ± 4%)

Uptake

solution, (fig. 9).

into Chromaffin

Ca

uptake into resting or stimulated cells is shown in 10. DMPP (100 iM for 10 see) increased Ca uptake from 0.09 ± 0.01 to 0.21 ± 0.03 fmol/cell (n = 6); at 10 tiM, 17a-estradiol blocked DMPP-evoked Ca uptake by 86%; at 100 M, blockade of Ca uptake was almost complete (90%). K stimulation (59 mM for 10 sec) also enhanced Ca uptake to 0.22 ± 0.03 fmol/cell (n = 6). Again, 10 M 17-afigure

a-estradiol,

the

fusing

Krebs’

with

secretory solution

response for

recovered

partially

on per-

30 mm.

Effects of 17-a-Estradiol on Potassium-Evoked Catecholamine Release Bovine adrenals. Pulses of high K solutions (118 mM K for 10 sec in the presence of 0.5 mM Ca) increased catecholamine release from 5.5 ± 09 ig (basal) to 110.1 ± 29.6 big/pulse (n = 7). 17-a-Estradiol decreased this response in a concentration-dependent manner (fig. 7). In another group of experiments, the reversibility of the blocking effects of 17-a-estradiol on K-evoked secretion was studied. Secretion was triggered by injections of a high K solution (2 ml of 118 mM K and 2.5 mM Cai. The secretion in P1 (54 ± 2.5 g of catecholamines) decayed slowly in subsequent pulses given at 20-mm intervals; however, secretion in P6 was still about 70% of that seen in P1 (fig. 8A). In the presence of 17-a-estradiol (10 tiM), catecholamine release decreased by 60% (P3 in fig. 8B). On washing out the hormone, the secretory response to K injections recovered almost com-

estradiol

M

inhibited

a further

the

net

reduction

Ca

in Ca

entry

uptake

by 83%.

However,

was not

at

100

seen.

Discussion We blocks

in

have

found

response

especially

to clear

extent with high muscarinic-mediated

ing

that

catecholamine

from

estradiol

these is located

the

steroid

secretion

from

some

secretagogues.

upon

nicotinic

K

stimulation,

hormone adrenal

17-a-estradiol chromaffin

Although stimulation,

this and

cells

effect

to

is

a lesser

it is dubious in the case of The obvious hypothesis emergeffects is that the target for 17-a-

secretion.

selective at the

plasma

membrane

of the

chromaffm

cell. If the drug were acting at some intracellular site secretory machinery, the responses to all secretagogues be blocked to the same extent.

on the would

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Vol. 259

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Fig. 9. Effects of 17-a-estradiol (1 7-a-E) on potassium-evoked secretion of catecholamines from perfused cat adrenal glands. Secretion was evoked by depolarization with a solution containing 120 mM KF for 10 sec. P1 isthesecretionevoked by 120 mM Kfor 10 sec. P2 is secretion evoked by K4 in the presence of 17-a-E. P3 is secretion evoked by K4 after removal of 17-a-E from the perfusion fluid and perfUSion with Krebs’ solution for 30 mm. 17-a-E was added to the perfusion fluid 20 mm before and during the secretory response. The data are the means ± S.E.M. of seven experiments. *P < .01 compared to P1.

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Fig. 8. Effects of 17-a-estradiol on potassium-evoked secretion of catecholamines from perfused bovine adrenal glands. A, secretion evoked by six consecutive pulses of I 1 8 mM K in a bolus injection of 2 ml every 20 mm. B, after 2 control pulses (PI and P2) the effects of 1 0 MM 17-aestradlol are shown. P3 and P4 correspond to another two pulses with K in P5, potassium-evoked secretion is abolished by I mM of the inorganic calcium antagonist, Cd. The data are the means ± S.E.M. of four experiments. < .01 compared to P2. DMPP and high K are known to increase Ca uptake into chromaffin cells through the recruitment of voltage-sensitive Ca channels (Artalejo et at., 1987; Gandia et at., 1991). Because 17-a-estradiol blocked Ca uptake evoked by these two secretagogues, it seems clear that the steroid might be acting directly

on

those

channels.

ated with the nicotinic for the hormone. This blocking

DMPP-evoked

In

addition,

receptor might

could explain

the

ionophore

associ-

be also a second target its higher potency in

secretion.

These effects are not explained in the frame of the classical genomic mechanism of steroid hormone actions through activation of high-affinity cytosolic receptors and their further translocation to the cell nucleus to induce new protein synthesis. These effects are known to take hours or even days to appear (MacEwen, 1991). Rather, the catecholamine blocking effects

are

better

cell membrane recent examples

explained

in the

newly

effects of steroids in neurons. of these nongenomic, rapid

emerging

There effects

P3

17-a-E. 10

concept

are several of steroids.

of

For instance, Majewska et at. (1986) found that two metabolites of the steroid hormones, progesterone and deoxicorticosterone, are potent barbiturate-like ligands of the ‘y-aminobutyric acid receptor chloride ion channel complex, inhibiting the binding of the convulsant t-butylbicyclosphosphorothionate to the -y-aminobutyric acid receptor complex. By applying steroids directly to specific neurons, immediate changes in their firing frequency are observed (Pfaff and Pfaffmann, 1969; Kelly et at., 1977; Nabekura et at., 1986; Hua and Chen, 1989). The activity of hormone-sensitive striated muscles can be enhanced within minutes of steroid treatment (Sachs and Leipheimer, 1988). As these rapid steroid effects are not prevented by protein synthesis inhibitors (Nabekura et at., 1986; Sachs and Leipheimer,

1988),

it is likely

that

they

result

from

a direct

action of steroids on the plasma membrane. Progestagen bovine serum albumin conjugates, which do not diffuse freely the plasma membrane into the cytosol, activate the release of dopamine from striatal neurons (Dluzem and Ramirez, 1989). Also, progesterone stimulates the release of luteinizing hormone from hypothalamic tissue (Ke and Ramirez, 1987). And, recently, progesterone has been shown to cause, within 30 mm of its application, an increase in oxytocin receptor binding in the ventromedial nuclei of the female rat hypothalamus. This rapid progesterone effect appears to be exerted on a membrane receptor, because it was produced in vitro as well as in vivo and was not mimicked by a variety of other steroids (Schumacher et at., 1990). It is unlikely that the inhibition by 17-a-estradiol of catecholamine

secretion

is associated

to

chronic

steroid

genomic

Downloaded from jpet.aspetjournals.org at ASPET Journals on May 9, 2016

I

S C

0 C

-

(K4)

S

S S

!

adrenal

cultured (A)

DMPP

bovine

chromaftin

100iM

S

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1I

0,2

S

to

a. 3 (5

0,1

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BASAL

17-a-E.

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iM

100

MM

mM

-I-

0

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S

a. 3

0,1

to

C.) It)

0,0

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. BASAL

.rmL 17.a.E. 10 MM

K

17.a.E. 100

MM

Fig. 10. The blockade of Ca uptake into cultured bovine chromaffin cells by 17-a-e$tradiol (1 7-a-E) at two different concentrations (1 0 and 100 tM) when these cells were stimulated by DMPP, 100 M (A), or K4, 1 18 mM (B), for 10 sec. The data express total uptake of Ca in femtomoles per cell and are the means ± S.E.M. of six expenments. *) < .01 when compared to the values of Ca uptake evoked by DMPP (A)

or K (B).

effects because, first, its effects are established and, second, they are readily reversible upon hormone.

Rather,

its

effects

might

be

in 5 to 20 mm

washing

ascribed

to

out the

the

novel

membrane rapid effects observed in different neuronal systems. The question still remains as to whether the effects of 17-aestradiol on chromaffin cell secretion is shared by other estrogenie and nonestrogenic hormones, and whether an opiate-like activity as that seen in the cardiovascular system (Dixon and Chandra, 1988) might be involved in its action. References ARTALEJO, C. R., G4uci A. G. AND AuNIs, D.: Chromaffin kinetics measured isotopically through fast calcium, fluxes. J. Biol. Chem. 262: 915-926. 1987.

cell

strontium

calcium

and

channel

barium

SCHUMACHER, concept in

M.: Rapid neuroendocrinology.

membrane

effects Trends

of steroid Neuroendocrinol.

hormones: Sci.

An emerging 13:

359-362,

1990. M., C0RINI, N., PFAFF, D. W. AND MACEWEN, B. S.: Behavioral effects of progesterone-associated with rapid modulation of oxytocin receptors. Science (Wash. DC) 250: 691-694, 1990. UNSICKER, K., KRI5cH, B., OTTEN, V. AND THOENEN, H.: Nerve growth factorinduced fiber outgrowth from isolated rat adrenal chromaffin cells: Impairment by glucocorticoids. Proc. Natl. Acad. Sci. U.S.A. 75: 3498-3502. 1978. VivzRos, 0. H., DILIBERTO, E. J., JR., HAZUM, E. AND CHANG, K. J.: Opiatelike materials in the adrenal medulla: Evidence for storage and secretion with catecholamines. Mol. Pharmacol. 16: 1101-1108, 1979. SCHUMACHER,

Send reprint requests to: Dr. Antonio logia, Facultad de Medicina, Universidad Morcillo s/n, 28029 Madrid, Spain.

G. Garcia,

Departamento

Aut#{243}noma de Madrid,

de FarmacoC/Arzobispo

Downloaded from jpet.aspetjournals.org at ASPET Journals on May 9, 2016

0,3 S U

J. J., BORGE5, R., GARCIA, A. G. AND HIDALGO, M. J.: Secretory and radioligand binding studies on muscarinic receptors in bovine and feline chromaffin cells. J. Physiol. (Lond) 418: 441-426. 1989. DixoN, W. AND CHANDRA, A.: Effect of 17 alpha-estradiol, a possible endogenous opiate antagonist, on cardiovascular responses in conscious, unrestrained rats. In Opioid Peptides and Blood Pressure Control, ed. by K. 0. Stumpe, K. Kraft and A. I. Faden, pp. 183-189, Springer-Verlag, 1988. DLUZEN, D. E. AND RAMIREZ, V. D.: Progesterone effects upon dopamine release from corpus striatum on female rats. II. Evidence for membrane site of action and the role of albumin. Brain Res. 476: 338-344, 1989. GANDIA, L., CASADO, L. F., LOPEz, M. G. AND GARCIA, A. G.: Separation of two pathways for calcium entry into chromaffm cells. Br. J. Pharmacol., 103: 1073-1078, 1991. GutcIA, A. G., HERNANDEZ, M., HORGA, J. F. AND SANCHEZ-GARCIA, P.: On the release of catecholamine and dopainine 3-hydroxylase evoked by ouabain in the perfused cat adrenal gland. Br. J. Pharmacol. 68: 571-583, 1980. HUA, S. Y. AND CHEN, Y. Z.: Membrane receptor-mediated electrophysiological effects of glucocorticoid on mammalian neurons. Endocrinology 124: 687-891, 1989. LA BELLA, F. S., KIM, R. S. S. AND TEMPLETON, J.: Opiate receptor binding activity of 17-a-estrogenic steroids. Life Sd. 23: 1779-1804. 1978. LEMAIRE, S., LEMAIRE, I., DEAN, D. M. AND LIvE’rr, B. G.: Opiate receptors and adrenal medullary function. Nature (Londj 288: 303-305, 1980. KE, F. C. AND RAMIREZ, V. D.: Membrane mechanism mediates progesterone stimulatory effect on LHRH release from superfused rat hypothalami in vitro. Neuroendocrinology 45: 514-517, 1987. KELLY, M. J., Moss, R. L. AND DUDLEY, C. A.: The effects of microelectrophoretically applied estrogen, cortisol and acetylcholine on medial preoptic-septal unit activity throughout the estrous cycle of the female rat. Exp. Brain Ru. 30: 53-64, 1977. KUMAKURA, K., KAROUM, F., GUIDO’FH, A. AND COSTA, E.: Modulation of nicotinic receptors by opiate receptor agonists in cultured adrenal chromaffin cells. Nature (Lond.) 283: 489-492, 1980 MACEWEN, B. S.: Non-genomic and genomic effects ofsteroids on neural activity. Trends Pharmacol. Sci. 12: 141-147, 1991. MAJEWSKA, M. D., HARRISON, N. L., SCHWARTZ, R. D., BARKER, J. L. AND PAUL, S. M.: Steroid hormone metabolites are barbiturate-like modulators of the Gaba receptor. Science (Wash. DC) 232: 1004-1007, 1986. M0LIN0FF, P. B. AND AXELROD, J.: Biochemistry of catecholamines. Annu. Rev. Biochem. 40: 465-500, 1971. MoRo, M. A, LOPEZ, M. G., GANDIA, L., MIcHELENA, P. AND GARcIA, A. G.: Separation and culture of living adrenalineand noradrenaline-containing cells from bovine adrenal medullae. Anal. Biochem. 185: 243-248, 1990. NABEKURA, J., O0MURE, Y., MINAMI, T., MIZUN0, Y. AND FUKUDA, A.: Mechanism of the rapid effect of 17$-estradiol on medial amygdala neurons. Science (Wash. DC) 233: 226-228, 1986. PFAFF, D. W. AND PFAFFMANN, C.: Olfactory and hormonal influences on the basal forebrain of the male rat. Brain Ru. 15: 137-156, 1969. SAcHS, B. D. AND LEIPHEIMER, R. E.: Rapid effect of testosterone on striated muscle activity in rats. Neuroendocrinology 48: 453-458, 1988. BALLESTA,

cells

0,3

0

285

and Secretion

17-a-Estradiol

1991