low Volume Perfusion-Superfusion System for Measurement of Transmitter Release From Blood Vessels In Vitro

DYNES BUDAI,*

N. BUCHHOLZ,

JOHN

has been developed

An in vitro technique constriction

and transmitter

excessive arteries

dilution

roller

pump

vial filled

signals were of

solenoid

was dampened

partly

and detected lease

with

amplified,

[3H]norepinephrine.

and

tritiated

mance novel

Key

Drug

Oscillation

by delivering sensitive was

norepinephrine

was elicited

liquid

setup

pharmacological

Words:

us to measure

formance

liquid

media

by4-8

blood ear

Rewith

vasoconstric-

norepinephrine

pulses.

responses released

by

by high-perfor-

range (80-100

data acquisition

Rabbit

Endogenous

preloaded

the contractile

in the low picogram on isolated

electrical

electrical

was determined

computerized

Superfusion;

overflow;

was

stimulation system.

minimum

efflux evoked

Endogenous

experiments

Perfusion;

[3HlNorepinephrine

tissue

(r = 0.98) between

(HPLC)

permits

by electrical

by a computerized the

by the

a 4-mL buffer

and recorded

release.

chromatography

experimental

generated

through

Transducer-generated

into the perfusion

ear

and perfused-

and removal were performed in the baseline

after

of vasoTo avoid

of rabbit

chambers

transducers.

allowed

linear correlation

segments

tissue

Krebs’ solution

measured

This system

recording

blood vessels.

6612-mg

in 0.5-mL

administration

valves.

digitalized,

stimulation

in vitro

substances,

mm Hg and [3H]norepinephrine

was a good

electrical

small perfused

air. Vasoconstriction

by pressure

norepinephrine

tions of l-2 There

released

were mounted

in a closed system.

by timer-controlled

for the simultaneous

release from

of the

or rat tail arteries

superfused

AND SUE PIPER DUCKLES

pg). Our

and automated

vessels. artery;

norepinephrine

Rat release;

tail

artery;

High-per-

chromatography

INTRODUCTION

In many conventional cardiovascular pharmacology techniques, isolated blood vessels are incubated in tissue baths of 5-50 mL. While this arrangement is adequate to detect mechanical responses, substances released from relatively small tissues are diluted by the large volume of the tissue bath, precluding quantitation of transmitter release. In the early L3Hlnorepinephrine release studies of Su and Bevan (1967,1970), Krebs’ solution was allowed to flow over strips of rabbit main pulmonary From the Department of Pharmacology, College of Medicine, University of California, Irvine, California. Address reprint requests to: Sue Piper Duckles, Ph.D., College of Medicine, Department of Pharmacology, University of California, Irvine, CA 92717. Received May 8, 1989; revised and accepted July 21, 1989. * Permanent address: Central Research Laboratory, Szent-Cyorgyi Albert University School of Medicine, Szeged 6720, Somogyi B. u. 4. Hungary. 41 Journal

of Pharmacological

0 1990 Elsewer

Science

Methods Publishing

23, 41-49 Co.,

Inc.,

(1990) 655 Avenue

of the Americas,

New

York,

NY 10010

42

0. Budai et al.

artery that were placed vertically between a pair of platinum-wire electrodes. The superfusate dripped from the strip directly into a collecting tube, achieving minimum delay and dilution of the released transmitter. It has long been known that the central artery in the rabbit ear is particularly sensitive to vasoconstrictor drugs. Various methods have been introduced to perfuse isolated rabbit ears and assay vasoconstrictor substances (Gaddum, 1959; Page and Green, 1948; Schlossmann, 1927). In the pioneering experiments of de la Lande and Harvey (19651, central arteries were isolated from the rabbit ears, cannulated at their proximal ends, and suspended freely in organ baths. By combining the superfusion technique of Su and Bevan and the approach of de la Lande and Harvey, Allen et al. (1973) developed a technique to estimate release of 13Hlnorepinephrine from the adventitial and luminal surfaces of the isoiated ear artery mounted vertically in an empty tissue bath. The medium entered the arteries through a cannula at the lower end, left at the upper end, and trickled down over the external surface. The perfusion-superfusion solution dripped directly into collecting tubes. Another significant modification of the isolated, perfused rabbit ear artery preparation was introduced by Steinsland et al. (1973). Their low-volume apparatus allowed simultaneous, but separate, intraluminal perfusion and extraluminal superfusion of the artery. In the present study, our goal was to design a method for the simultaneous measurement of vasoconstriction and norepinephrine release in small (6-12 mg) isolated blood vessels perfused and superfused in a closed system, incorporating computerized data acquisition and automation. A study of sympathetic neurotransmission in the isolated rabbit ear artery using in part the described procedure has been previously published (Budai and Duckles, 1988). METHODS Tissue Preparation

and Perfusion

Proximal 3-5-cm segments of the rabbit ear artery or 6-8 cm segments of rat tail artery were dissected from male New Zealand White rabbits (2.5-3 kg) or male Fischer 344 rats (250-300 g), respectively. Tissues were cannulated at both ends by 12-cm pieces of PE-90 (ear arteries) or PE-20 (tail arteries) polyethylene tubing (Becton Dickinson and Co., Parsipanny, NJ, USA) and placed into the low-volume perfusion system (Figure I). Aerated Krebs’ solution was delivered by a Gilson Minipuls 2 roller pump (Gilson International, Middleton, WI, USA). Automated drug administration and removal can be achieved by a timer-controlled solenoid valve. Pulsation in the perfusion pressure generated by the roller pump was dampened by a 4-mL glass vial turned upside down and filled with 1 ml Krebs’ solution. Air bubbles were captured by the same vial. The perfusion chamber is made up of three detachable parts, shown in greater detail in Figure 2. The identical top and bottom housings, which contain platinum stimulating electrodes and T connectors as inlet/outlet ports, are connected by a transparent Teflon tubing (2.8 x 70 mm, internal dimensions). Vessels were placed in the perfusion system by means of the long cannulating tubings (Figure 2). As

Transmitter

Release from Small Blood Vessels

FIGURE 1. Experimental design for monitoring perfusion pressure by computerized data acquisition system and collection of substances released from the perfused-superfused blood vessel.

shown

in Figure 2A, artery

of the Teflon

tubing.

housing (Figure 26,C). ing of the T connector and sealed

with

2E,F). After

first carefully

inserted

tubing was tightly attached

into the middle to the chamber

Third, the cannulating tubing was passed through one openof the bottom housing, and the excessive length was cut off of polyethylene

tubing

(Figure 2D).

end of the sealing tubing was connected The upper chamber housing was fixed

the cannula

tubing

were

the Teflon

a 5-cm length

fusion, the other the T connector. ylene

segments

Second,

in the T connector

cut longitudinally,

the final

was sealed length

To allow

super-

to the other opening of in a similar way (Figure

by a small piece of polyeth-

of the artery

segment

was set by

pulling out the cannulating tubing. Great care is taken throughout the entire procedure to avoid overstretching the tissue, and the final length of the tissue was set to equal the in situ length. The other end of the cannula was connected to the heat exchanger

made

perfusate,

the outlet

of coils of PE-120 polyethylene opening

of the upper

tubing.

T connector

To collect

fractions

led to a fraction

of the

collector.

The entire assembly was kept at 37°C in a water bath. Tissues were equilibrated for 60 min before each experiment was begun. The perfusion pressure was monitored with a Statham P23 AC transducer. The resulting electrical

signals were

cision Instruments,

digitalized

Inc.,

New

by MacLab

Haven,

analog to digital converter

CT) and recorded

by Macintosh

(World

Pre-

SE computer

43

46

D. Budai et al.

-PE2200r90Tc~Sing -

Seamg

-PI

--

Eiacimde

wng

I -

Teflon tubing

-

Artery segment

FIGURE 2. Detailed drawing of perfusion chamber shown on the left side of Figure 1. The procedure for mounting blood vessel segments in the chamber is illustrated in sequence from A to F. See methods for detailed explanation.

{Figure 1). Increases in the perfusion pressure were taken as a measure of the vasoconstriction and expressed in mmHg. The composition of the Krebs’ solution was (in mM): NaCI, 118; KCI, 4.8; Cat&, 1.6; Kl-12P04, 1.2; Nat-lCO,, 25; MgS04, 1.2; ascorbic acid, 0.3; and glucose, 11.5. Before entering into the vesseis, the medium was saturated with 5% COz in O2 and warmed to 37°C. Drugs can be added to the perfusion medium by switching perfusate solutions with a solenoid valve and timer resulting in both intra- and extraiuminal application (Figure If. Electrical

Stipulation

Platinum electrodes mounted in both perfusion chamber housings (Figures 1 and 2) were placed 5 cm apart at both ends of the tissue for administration of field

Transmitter Release from Small Blood Vessels stimulation

using a Grass S48 stimulator.

Parameters

used for excitation

cular nerves in the rabbit ear artery were frequency of 8 Hz, amplitude pulse duration of 1 ms. Rat tail artery segments were stimulated with at 8 Hz (60 V, 1 msec)

leaving

20-min

intertrain

intervals.

of perivasof 50 V, and 3-min trains

Parameters

of the stimu-

lating pulses are calibrated and monitored with a Beckman Circuitmate 9020 oscilloscope. Under these experimental conditions, tissue contractions are tetrodotoxin (10e6 M) sensitive, confirming their neurogenic origin.

Estimation of r3HINorepinephrine After

a 60-min

equilibration

fused/superfused cific activity

Release

period,

at a rate of 2 mL/min

14.2 Ci/mmol

(New

segments

of the rabbit

with 0.5 FM (-)-

England

Nuclear,

ear artery were

[3Hlnorepinephrine,

Boston,

MA,

USA)

perspe-

for 60 min.

To conserve labeled norepinephrine and avoid radioactive contamination of the entire perfusion system, [3H]norepinephrine in a volume of 3 mL was recirculated between with

a 5-mL reservoir radioactive

and the tissue chamber

norepinephrine

was

using separate

followed

by

a

tubing.

60-min

Incubation

perfusion

with

[3H]norepinephrine-free Krebs’ solution containing 10 FM cocaine. Cocaine was present for the rest of the experiment. After this washing-out period, 2-min fractions of the perfusate/superfusate were collected. Radioactivity in a I-mL aliquot of each fraction sample

was determined Beckman

with

Ready-Solv

a Beckman CP premixed

liquid

scintillation

counter

using a 6 mL/

liquid scintillation

cocktail.

Tritium

efflux

is expressed in dpm/sample and plotted against the fraction number, i.e., perfusion time. Each tissue is stimulated five times (S, to S,). The L3H]norepinephrine overflow evoked

by electrical

taneous

tritium

the total tritium

stimulation

is calculated

efflux determined

just before

efflux for each fraction

by subtraction

of the averaged

and after each stimulation

during

stimulation

(Farnebo

spon-

period

from

and Hamberger,

1971).

Endogenous

Norepinephrine

Release

Segments of rat tail artery were perfused and superfused at a rate of 1.5 mL/min in the presence of deoxycorticosterone and cocaine (both 1O-5 M) to inhibit extraneuronal

and neuronal

norepinephrine

uptake,

respectively.

The perfusate

was

collected at 30 set after initial stimulation for a total of 6 min and a total volume of 9 mL. Stimulations were repeated three times (Sl, S2, S3) with a 20-min equilibration period

between

Collected 1.5 M acetic buffer

each stimulation.

perfusate acid.

was placed

Subsequently

on ice and immediately pH was adjusted

acidified

to 8.6 with

(1.5 M tris, 54 mM EDTA), and 50 mg acid washed

alumina

with 240 FL of

2.5 mL of tris-EDTA was added

to each

tube along with 1 ng of dihydroxybenzylamine hydrobromide (DHBA) internal standard. If the amount of alumina was increased to 75 mg or 100 mg, the extraction efficiency of norepinephrine was reduced and larger volumes of perchloric acid (1.0-1.5 mL) were 50 mg of alumina

required to elute the norepinephrine bound to the alumina. Thus, was found to be the optimal amount for the extraction of nor-

epinephrine from a perfusate volume of 9 mL. Tubes were shaken alumina was allowed to settle, and the supernatant was decanted pipette.

The alumina

was washed

three

times

with

5 mL of double

for 15 min, the with a Pasteur distilled

water

45

A. 60

8 r---I.-.,--‘,‘

8500

_

8

8

Time

B.

40

.I..

I..

120 Putses I’--’

(min)

7500 6500

4500 3500

2500 1500

8

8

!

I

0 12000 _

5

8

10000

t2.0

I

8

I

10

15

20

25

Fraction

c.

40

I

Pulses I

30

1

35

Number

y= -1854+164x R-0.987

8000

10

s

20

I

30

Peak

t

40

I

I

50

60

height

I

I

70

80

(mmtig)

FfGURE 3. Stimulation-evoked changes of the perfusion pressure (panel A) and [3H]norepinephrine overflow (panel 6) in a perfused-superfused segment of rabbit ear artery. Tissue was stimulated for 8, 40, and 120 pulses at 8 Hz. Correlation between the mechanical response and the release of neurotransmitter is shown in panel C. The uppermost panel is an actual experimental recording taken by the computerized data acquisition system.

Transmitter Release from Small Blood Vessels and transferred

in 1 mL of water to microliter

test tubes (Bioanalytical

West Lafayette, IN, USA) fitted with millipore filters. After RPM for 5 min, the water was discarded. The norepinephrine the alumina

were

eluted

with

300 FL of 0.1 N perchloric

centrifuged at 2,000 RPM for 5 min. One hundred microliter samples of eluted

amines

Systems

centrifugation and DHBA

Inc.

at 2,000 bound to

acid, and the tubes were

were

injected

into an HPLC

system with electrochemical detection (Bioanalytical Systems Inc., West Lafayette, IN, USA) using the following parameters: flow rate, 1.5 mL/min, and detector attenuation,

1 nA for full scale pen deflection.

Reverse

phase

columns

(C-18) were

supplied by Phenomenex (Phenomenex Inc., Palos Verdes, CA, USA). The mobile phase consisted of 0.1 M sodium acetate, 0.02 M citric acid, 0.2 mM sodium EDTA, 150 mg of sodium-octyl-sulfate Norepinephrine FL = Peak height peak

height

and methanol

was quantitated of NE/Peak

DHBA

height

standard/peak

of NE and DHBA standards

volume

for recovery

of DHBA

(recovery

NE standard

height

DHBA

formula:

picogram

x 330 picogram internal

of total tissue

is known, varied

this value is multiplied

of norepinephrine by 3 and corrected

from 85-95%/o).

norepinephrine

of stimulation

FL x

(concentrations

content

for calculation

of fractional

NE release has been described previously (Handa and Duckles, 1987). norepinephrine release is calculated by the formula: pg NE released/pg content/number

NE in 100

DHBA/lOO

standard

were 330 pg/lOO FL). Once the quantity

in the 100 FL injection Quantitation

3% by volume.

with the following

Fractional NE tissue

pulses.

RESULTS Figure

3 shows

stimulation-evoked (panel

B)

in

an

application

contractile

a 5-cm

example

responses

segment

of

of the

(panel

isolated

perfusion

A) together

rabbit

ear

system with tritium

artery

measuring overflow

preloaded

with

5 5 1500 2000

”

*g

z

1000

2

a,

B

600

Lu =

0 60

20

Time (sn) FIGURE 4. Effect of time on release of endogenous norepinephrine from rat tail artery determined by HPLC and electrochemical detection. Three subsequent stimulation periods, Sl, S2, and S3, are shown. Deoxycorticosterone and cocaine, both lo-5M, are present throughout. Values shown are means f SEM, n = 3.

47

48

D. Budai et al.

[3Hlnorepinephrine. Stimulation of the tissue with as few as eight electrical pulses delivered at 8 Hz evoked measurable increase in both parameters. There was an excellent linear correlation (r = 0.98) between the vasoconstriction measured in mmHg and stimulation-evoked [3Hlnorepinephrine overflow (Figure 3C) for stimulation trains of 8, 40, and 120 pulses at a frequency of 8 Hz. Release of endogenous norepinephrine from the rat tail artery as quantitated by HPLC and electrochemical detection is shown in Figure 4. Amount of norepinephrine release is constant for three repeated stimulation trains, each at 8 Hz for 3 min. Stimulation-evoked increases in perfusion pressure also remained constant during these three stimulation trains (data not shown). This quantity of norepinephrine release corresponds to a fractional release of 3.5 x 1O--6 + 0.3. DISCUSSION The results demonstrate that our novel perfusion-superfusion apparatus allows the simultaneous registration of vasoconstriction and quantitative determination of stimulation-evoked transmitter release, which are well correlated when stimulation train length is varied. The low volume and the relatively high perfusion rate limited the dilution of the substances to be assayed. Because of the smooth baseline, i.e., the excellent signal to noise ratio, very small changes in perfusion pressure could be recorded. The computerized data acquisition made the data handling more accurate and reliable. Since each artery was perfused-superfused in an individual closed circulation, changing between drug-containing medium and normal Krebs’ solution was automated by timer-controlled solenoid valves. To provide optimal conditions for preloading the perivascular sympathetic nerve terminals with labeled norepinephrine, a recirculating perfusion-superfusion system was used to expose both the extra- and intraluminal surfaces to [‘Hlnorepinephrine (Avakian and Gillespie, 1968; Bevan et al., 1969; de la Lande et al., 1967; T&ok and Bevan, 1971). After excessive (nonspecifically bound) radioactivity was washed out, cocaine was added to block reuptake of any isotope-labeled norepinephrine liberated from nerve terminals. Use of longer segments instead of strips or ring preparations made it possible to monitor the perfusion pressure and also provided greater surface for norepinephrine release. Since 90% of the radioactivity released by nerve stimulation appears in the superfusate (Allen et al., 19731, the perfusate was allowed to superfuse the adventitial surface. The HPLC quantitation of norepinephrine released by nerves in very small blood vessels represents a difficult problem, since the amount of norepinephrine released is in the picogram range. We have shown that stimulation-evoked endogenous norepinephrine release in small blood vessels can be quantitated with electrochemical detection with longer stimulation times (>I min) and utilization of the optimal amount of alumina (50 mg) for the extraction of norepinephrine. Increasing the amount of alumina to 75 or 100 mg decreased the extraction efficiency for norepinephrine and required larger volumes of perchloric acid to elute the norepinephrine bound to the alumina. This resulted in the dilution of norepinephrine and the reduced ability to detect extracted compounds by electrochemical detection.

Transmitter Release from Small Blood Vessels In conclusion,

our novel experimental

design is suitable

for either tritium

labeling

or measurement of endogenous transmitters as well as recording vasoconstrictions. Perfusion of small blood vessels in a closed system gives good control of oxigenation,

temperature,

studying

release

sufficiently

sensitive

This work Heart

perfusate. or other

This

apparatus

substances,

could

be used

for

such as peptides,

with

assay system.

was completed

Association,

supported

and pH of the of norepinephrine

during

California

in part by NIH

the tenure

Affiliate,

grants

of a research

and with funds

#DK

fellowship

contributed

to D. Budai from

by the Central

Valley

the American

Chapter.

It was

36289 and AC 06912.

REFERENCES Allen GS, Rand MJ, Story DF (1973) Techniques studying

adrenergic

lated perfused Avakian

OV,

artery.

Gillespie

drenaline

Cardiovasc

Res 7:423-428.

nerves, with

for

in an iso-

Uptake

tissue in isolated

its correlation

response.

release

JS (1968)

by adrenergic

and connective ies and

transmitter

muscle

perfused

arter-

the vasoconstrictor

of bound

JV, Bevan RD (1969)

norepinephrine

wall. Eur

train length / Pharmacol de la Lande

release

of stimulation inhibition

in the rabbit

of

ear artery.

Exp Ther 247:839-843. IS, Frewin

Influence

of age on

in arteries Aging

AA (1948) Perfusion

and veins of 8:511-516. of rabbit’s

substances.

ear

Methods

H (1927)

Adrenalingehalt

Untersuchungen

des Blutes.

uber

den

Arch Exp Path Phar-

mako/l21:160-203.

SP (1988) Influence

on the opioid-induced

norepinephrine

Pharmacol

Res 1:123-129.

Schlossmann

/ Pharmaco/5:299-301. Budai D, Duckles

SP (1987) content

Fischer 344 rats. Neurobiol Page IH, Green Med

Distribution

in the arterial

RK, Duckles

norepinephrine

for study of vasoconstrictor

BrJPharmacolChemother32:168-184.

Bevan JA, Osher

HJ (1959) Bioassay procedures.

Rev 11:241-249. Handa

of nora-

smooth

Gaddum

of sympathetic

sensitive

to noradrenaline.

OS,

Inhibition

JG (1967) The

innervation

on vascular

Br] Pharmacol

Chem-

Furchgott

RF, Kirpekar

of adrenergic

parasympathomimetics Pharmacol

D, Waterson

influence

Steinsland

SM (1973)

neurotransmission

by

in the rabbit ear artery. /

Exp Ther 184;346-356.

Su C, Bevan JA (1967) Release of L3H]noradrenaline from vasoconstrictor

nerves.

/ Pharm Pharmacol

19:625-626.

other 31:82-93. Su C, de la Lande IS, Harvey JA (1965) A new and sensitive bioassay

for catecholamines.

/ Pharm Pharmacol

17: 589-593. Farnebo

L-O,

changes from

43:96-106.

nique

Hamberger release

stimulated

B (1971) of rat

Drug-induced

J3HJ-noradrenaline iris.

Br /

Pharmacol

JA (1970) in arterial

The strips

of superfusion

ulation.

in the

field

Bevan

nephrine

release

of ‘H-norepi-

studied

by the tech-

and transmural

/ Pharmacol

nerve stim-

Exp Ther 172:62-68.

Torok J, Bevan JA (1971) Entry of 3H-norepinephrine into

arterial

613-620.

wall.

/

Pharmacol

fxp

Ther

177:

49