Inhibition of vasoactive agents by perfluorochemical emulsion

Life Sciences, Vol. 40, pp. 1971-1979 Printed in the U.S.A.

Pergamon Journal

INHIBITION OF VASOACTIVE AGENTS BY PERFLUOROCHEMICAL EMULSION Maythem Saeed, Andreas Hartmann and Richard J. Bing Huntington Medical Research Institutes Huntington Memorial Hospital i00 Congress Street Pasadena, CA 91105 (Received in final form February 24, 1987) Summary Perfluorochemicals are widely used in clinical and experimental studies as volume expanders with high oxygen carrying capacity. We noticed that contractions of rabbit aortic strips, induced by norepinephrine, were inhibited in FC-43 emulsion as compared to Krebs-Henseleit solution (KH). To study this inhibition, a comparative evaluation was made of the contractile responses of norepinephrine, serotonin and histamine in FC-43 emulsion and KH. The effect of these agents was significantly diminished in FC-43 emulsion; concentration-response curves were shifted to the right and the maximum response was diminished. A search was made for the individual constituents of FC-43 responsible for diminution of contraction of vascular smooth muscle. After centrifugation of FC-43 emulsion, the supernatant caused a reduction of contractility and reduced EC~^-values of norepinephrine to the same degree as the fullyD~onstituted emulsion. This excluded perfluorotributylamine, the oxygen carrying particles, as being responsible for this inhibition. When the detergent Pluronic F-68 was added to KH in concentrations equal to that in FC-43 emulsion, inhibition of contraction occurred and EC50-values increased. Contractions induced by norepinephrine were equally inhibited by the addition to KH of a volume expander, hydroxyethylstarch, in a concentration equal to that in FC-43 emulsion. It is concluded that FC-43 emulsion inhibits vasoactive agents and attenuates its pharmacologic effects on vascular smooth muscle. The fractions of FC-43 emulsion responsible for inhibiting the effect of vasoactive agents are Pluronic F-68 and hydroxyethylstarch. Perfluorochemicals are blood substitutes widely used in patients with severe anemia, when blood transfusion cannot be performed on religious grounds (1-3). Recently perfluorochemical emulsions have been used during transluminal coronary angioplasty (2). In perfluorochemicals, oxygen is not bound but dissolved and the amount of oxygen transported depends on its partial pressure. The small particle size of FC-43 particle (0.09~) permits flow through ischemic areas of microcirculation which have become inaccessible to erythrocytes. Reduction of the cardiac infarct size with perfluorochemicals has been shown by a series of investigators (4-7). Improvement of cardiac function with FC-43 emulsion as perfusate has been observed by nuclear magnetic resonance (8,9) and by a study of cardiac function in a supported working heart preparation (i0,Ii). In the course of our experiments with FC-43 emulsion (12-14) it became apparent that certain compounds added to FC-43 emulsion lost some of the Copyright

0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals Ltd.

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pharmacological activity as compared to their activity in crystalloid solution (15). It is the purpose of this report to furnish evidence that FC-43 emulsion diminishes th~ effect of norepinephrine, serotonin and histamine. Pluronic F-68, a detergent, and hydroxyethylstarch a volume expander, are likely to be responsible for these effects. Methods Preparation of the strips: Male albino rabbits weighing 2.5 3.0 kg were anesthetized with intravenous injection of sodium pentobarbital (30 mg/kg). The rabbits were ventilated with 95% 02 and 5% CO 2. After thoracotomy, a segment of thoracic aorta (4 cm length) was excised. Transverse strips (3 mm width) were prepared according to the method of Furchgott and Zawadzki (16). They were mounted in an organ chamber of 20 ml capacity at 37 ° C, containing either Krebs-Henseleit (KH) or perfluorochemical (FC-43 emulsion). KH was oxygenated with 95% 02 and 5% CO^,z and FC-43 emulsion was oxygenated with 90% 02 and 10% CO 2 because of its higher binding O2-capacity (Table I). TABLE I Composition of Krebs Henseleit (KH) and perfluorotributylamine emulsion (FC-43 emulsion) (at 37 ° C, 760 mm Hg). Solubility, oxygen capacity and ionic concentrations (m.M).

02 capacity (mM) SSlubility constant (ml 0o/ml/mm Hg) Viscosity (Pa/s) N

K ~ Ca2+

KH i.i 3.1 x 10-2 1.05 x 10 -5 143

2+

5.94 2.54

Mg _ H2PO 4

1.19 1.19

CI-

HCO-3 SO2- 4

128

25

FC-43 Emulsion 2.5 8.4 x 10 -5 1.91 x 10 -3 128 4.56 2.52 2.1 116.5 25

1.2

Perfluorotributylamine

(g/lO0 ml) Pluronic F-68 (g/lO0 ml) Hydroxyethylstarch (g/100 ml) Glucose (mM)

i0

20 2.56 3.0 10

pH was maintained between 7.35 - 7.45, pO 2 between 510 - 550 mm Hg and pCO^ between 35 - 45 mm Hg by adjusting the gas mixture in the chamber, pH, pO. z and pCO^ were measured by Radiometer (ABL 2, Copenhagen, Denmark) L ~ Isometric transducer (Type UL-20 GR, rated capacity 20 g, Shikoh, Minoba Co., Tokyo, Japan) recorded changes in tension. Strips were stretched to a passive tension of 1.5 g and allowed to equilibrate for 90 minutes during which time the solution was changed every 30 minutes. Endothelial integrity was assessed qualitatively by the degree of relaxation caused by acetyl= -7 -6 -~ cholinel' (ACh) from i0 to i0 M after precontraction with histamine (i0 or 10-"M). To induce the same degree of p~contraction, t~e histamine concentration used in KH was maintained at i0- M, and at 10- M in FC-43 emulsion. Only strips which relaxed with ACh were included in this study (17). Vasoactive agents (20 HI) were added to the aortic strips suspended in either KH or FC-43 emulsion, and the contractile response to various vasocontracting agents was compared. Cumulative concentration response curves were

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Vasoactive Agents in Perfluorochemical

obtained separatel_~ for. serotonin norepinephrine (10u-10-4M).

(I0-8-I0-3M),

histamine

(I0-7-I0-3M)

1973

or

In the first set of experiments the supernatant of FC-43 emulsion (FC-43 emulsion without perfluorotributylamine) was compared to the whole FC-43 emulsion. The supernatant was obtained after centrifugation of perfluorochemical emulsion (6000 rpm for 30 minutes at 4 ° C). Strips were equilibrated for 90 minutes in the supernatant. In the second set of experiments the effect of hydroxyethylstarch as well as pluronic F-68 on norepinephrine (NE) induced contractions was tested. Aortic strips were equilibrated either in KH or in KH solution containing hydroxyethylstarch (20 g/100 ml), or of Pluronic F-68 (2.56 gll00ml). Data are shown as mean ± S.E. Statistical analysis was performed with paired and unpaired Student's t-test. Differences were considered to be significant when p < 0.05. EC~n values (the molar concentration of drug producing 50% of the maximal respon%%) in concentration response curves were determined according to the method of Fleming et al; (18) and Patil et al; (19). Results Effect of vasoactive~gent~ on aortic strips: - / . ^ - ~ -j. -j ~Sero~onin (i0 -i0 M), histamine (i0 -I0 M) and norepinephrine (10-~-I0-"M) caused concentration dependent force-development of isolated aortic strips in both KH and FC-43 emulsio~ as shown in^Figs. I, 2 and 3. At lOWvConcent~ations of serotonin (from i0 -v t~ 3 x i0 -~ ~), histamine (from i0-" to i0 -v M) and norepinephrine (from I0 -U to 3 x I0 -° M) in FC-43 emulsion, no contractions were observed; contractions were present with KH (Figs. i, 2, 3). 3.SO.

Tension Ig)

I,TS -

o':

-LoS $erolonin (M)

FIG. i Tension of aortic strips induced by cumulative concentrations of serotonin in FC-43 emulsion as compared to KH solution. Significant decline in serotonin-induced tension in FC-43 emulsion is shown at all concentrations. Tensions are expressed as the difference between the initial tension (1.5 g) and the tension developed with serotonin.

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j+J+

3o

Tension igl

~/ g,/;////" ••• i .//~.. ~7"'07;0;.................... U;/ a

7

s

s

4

Log Norepinephrine (M)

FIG. 2 Tension of aortic strips induced by cumulative concentrations of histamine in FC-43 emulsion as compared to KH solution. A significant decline in histamine-induced tension in FC-43 emulsion is shown at all concentrations. Tensions are expressed as the difference between the initial tension (1.5 g) and the tension developed with histamine. 3,so

Tension (gl

°

,

0°0

Lo~ Hls~lmine (M~

FIG.

3

Tension of aortic strips induced by cumulative concentrations of norepinephrine in FC-43 emulsion as compared to KH solution. The difference in response to norepinephrine between the two media is statistically significant. Tensions are expressed as the difference between the initial tension (1.5 g) and the tension devloped with norepinephrine.

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1975

Inhibition of the three vasoactive agents was in a noncompetitive manner as indicated by the attenuation of the maximum response (Fig. 1,2,3). Table 2 shows the EC.^-values of serotonin, histamine and norepinephrine in KH solution as c o m ~ r e d to FC-43 emulsion. A significant shift to the right of the concentration response curves in FC-43 emulsion was seen, denoting inactivation of the vasoactive agents. TABLE II AGENTS KH (M) FC-43 (M) Emul~ion Serotonin 3.4 x i0-~ ± i.i (n=6) 3.7 x i0-~ ± 1.8 ** (n=ll) Histamine 5.6 x I0-~ ± 0.8 (n=6) 1.5 x I0-~ ± 2 I* (n=12) Norepinephrine 1.6 x I0 -/ ± 0.2 (n=19) 6.9 x I0 -/ ± 0.8** (n=16) *p < 0.02, **p < 0.001 FC-43 emulsion compared with KH The inhibitory effects of FC-43 emulsion were reversible since repeated washing with KH of the strip restored the normal response to norepinephrine when the strip was contracting in KH solution. For instance, strips incubated ~n FC-43 emulsion showed a mean of 18.9 ± 4.7% (n=16) of contraction with 10--M norepinephrine as compared to KH 100% (n=lg). After repeated washing, contractile response to norepinephrine returned to 118.9 ± 13.4%. The effect of Perfluorotributylamine, Pluronic F-68 and hydroxyethylstarch on aortic strips: To detect the fraction of FC-43 emulsion responsible for inhibiting contractility, the emulsion was separated by centrifugation. The supernatant contained Pluronic F-68, an emulsifying, dispersing and defoaming agent, as well as hydroxyethylstarch, a volume expander. The precipitate contained the particles of FC-43 emulsion, perfluorotributylamine, which is responsible for O~ carrying. The supernatant fraction diminished the contractility and r~duced EC~0 of norepinephrine to the same degree as the fully constituted emulsion (~Ig. 4). 3,00-

Tension (g)

sS••

•~

~ ~.~.~s~

SLrlpseQulltbraLed in FC 43(h16)

J l'-"'~

~

~

l

,

-Log Not'epinephrine (M)

FIG. 4 The effect of supernatant as compared to FC-43 emulsion. No significant difference in contractile response to norephinephrine was observed in both media. See figure for explanation of symbols. Tensions are expressed as the difference between the initial tension (1.5 g) and the tension developed with norepinephrine.

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When Pluronic F-68 was added to KH in concentrations equal to that contained in FC-43 emulsion (2.56 g/100 ml), inhibition of contraction occurred (Fig. 5)° The concentration response curve of NE was shifted to the right (Fig. 5).

Tension (g) 3.00-

l/

I/"

,/'1

I j .,,,~o+t

, B

/

.."!i

I

.,,."

..... o-.,.,..

"-! !!!!!::::::= :: .. P(.02

• ..

| 7

p~O01

, 6

, 5

, 4

-Log Norepinephrine (M) FIG. 5 Shows the increase in EC_ 0 and the shift to the right with norepineD phrine in KH solution containing Pluronic F-68. Tensions are expressed as the difference between the initial tension (1.5 g) and the tension developed with norepinephrine. Contractions were equally inhibited by the addition of hydroxyethylstarch to KH (Fig. 6). The maximum response to norepinephrine was significantly reduced but no significant changes in ECN0-value were noticed (Fig. 6). Therefore both Pluronic F-68 and hydroxyethyIstarch were responsible for diminished contractile response to NE. Discussion In the course of previous experiments with FC-43 emulsion we noticed that the contractile effect of norepinephrine on rabbit aortic strip was diminished as compared with KH solution. Later it was discovered that the contractile effects of histamine and serotonin were also reduced in perfluorochemical emulsion. These observations prompted us to perform an evaluation of FC-43 emulsion on the pharmacological activity of these compounds. This was accomplished by studying the effect of the supernatant or particulate fraction of FC-43 emulsion on the contractile response to norepinephrine. In addition, hydroxyethylstarch and pluronic F-68, constituents of the supernatant fraction, were added to KH solution and their effect on the contractile response to NE was observed. e

The diminished contractile effect of serotonin, histamine and norepinephrine in perfluorochemical emulsion is ~llustrated i~ Figures 1,2 and 3. A~ low~concentrations of serotonin from i0-_ to 3 x I0 -° M, norepinephrine from i0 -~ to 3 x i0 -~ M or histamine from 1 0 - / to i0 - b M, no tension development

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Vasoactive Agents in Perfluorochemical

1977

Tension (g} 3,00-

Ec

I "0 / , I.---" 0 , :--. ,0

~/J,[,,,,.'f""

/...... I

EC~o-: ~~0 "-,~-

'::,i::::7,:;::: ',::':,'.";:...... ,,,

Log Norepinephrine {M)

FIG. 6 Cumulative concentration response curves to norepinephrine of aortic strips equilibrated in KH solution or in KH solution containing hydroxyethylstarch. The addition of hydroxyethylstarch diminish the maximum response to norepinephrine denoting its inhibitory effect. Tensions are expressed as the difference between the initial tension (1.5 g) and the tension developed with norepinephrine. occurs in FC-43 emulsion (Fig. 1,2,3); when the vasoactive agents are added to FC-43 emulsions, the concentration response curves are shifted to the right (Fig. 1,2,3,). The effective concentration which produces half maximal contraction (ECnn) is significantly higher in FC-43 emulsion as compared to KH solution (Ta%~e 2). The inhibitory effect of serotonin is particularly conspicuous (Fig. I). The reason for this observation is not understood. When either pluronic F-68 or hydroxyethylstarch are added to KH solution in the organ chamber, both compounds inhibit contraction of aortic strips to increasing concentrations of norepinephrine (Fig. 5,6). This suggests that these two constituents may be responsible for the diminution of vascular smooth muscle contraction. When FC-43 emulsion is centrifuged and the effects of rising concentrations of norepinephrine are tested in the supernatant, inhibition of contraction of the same magnitude as observed in whole FC-43 emulsion is noticed (Fig. 4). This provides further proof that the O~ carrying particles in FC-43 emulsion (perfluorotributylamine) are not responsible z for the reduction in_She diminished response to NE. It is also unlikely that differences in 2 Mg concentration between FC-43 emulsion and KH solution play a role in the altered responses to the vascular agents. As shown by Altura and Altura (20), only concentrations 95 Mg ~ above 5mM diminish smooth muscle contra~i tion. Concentration of Mg ~ in KH and FC-43 emulsion are much lower (Mg ~concentration KH: 1.19mM; in FC-43 emulsion: 2.19mM, Table i).

1978

Vasoactive Agents in Perfluorochemical

Vol. 40, No. 20, ].987

It has been suggested that free fluoride ions in perfluorochemical emulsions are toxic. This toxicity occurs particularly after ultrasonication of the emulsions, when the concentration of free fluoride ions is particularly high 60-80 ppm (21). At lower concentrations of fluoride as present in FC-43 emulsions (3.7 ppm) no toxic effects on cardiac performance are noticeable (10,11,22). A possible cause for the effect of pluronic F-68 is its action on cell membranes. Different detergents affect membrane solubility to a different degree. In general, neurotransmitter receptors have resisted solubilization from synaptic membranes with a notable exception of nicotinic receptor in muscle and nerves (23-25). However, a prolonged effect of pluronie F-68 is unlikely, since washing of the strips with KH solution restores tension and tension development. If the detergent were to affect integrity of the plasma membrane, one might also expect changes in intracellular Ca--. However, Tomero and Gayer (22) using FC-43 e m u l s i ~ in a recycling perfusing heart system, found no change in intracellular Ca- concentration. The possibility that the difference in 02 carrying capacity between FC-43 emulsion and KH solution is responsible for the difference in contractility, has been excluded by adjusting the p O ? and pC02 in the organ chamber (by oxygenating KH solution with 95% 0^ and 5% COn, and in FC-43 emulsion with 90% Ov and 10% COy). Thus pH (7.~5-7.45), P~2 (510-550 mm Hg), and pC02 (35-4F mm Hg) are ~qual in both solutions. Our findings with pluronic F-6~ are supported by the results of Lane and Lan~kin (26) and by Vercelotti et al (27). Lane and Lamkin (26) found that inhibition of chemotaxis of polymorphonuclear neutrophiles is not the result of the perfluorocarbons themselves but of Pluronic F-68 and Vercelotti et al (27) described pulmonary dysfunction in dogs after administration of products containing pluronic F-68. The second component of FC-43 emulsion, responsible for diminishing contractility of smooth muscle appears to be hydroxyethylstarch. It is a substituted amylopectin of molecular weight of 69,000, with molar substitution of hydroxyethylgroups of 0.7. It has little toxicity in patients and in animals (28). There are no reports of the toxic effects of hydroxyethylstarch on structure or function of individual cells or cell cultures. It is possible that hydroxyethylstarch, because of its relatively high osmotic pressure, results in a decrease in intracellular water, interfering with smooth muscle relaxation (29). Acknowledgments This work was supported by grants from The Council for Tobacco Research, USA and the Margaret W. and Herbert Hoover, Jr. Foundation. Dr. Hartmann is a Fellow of the Humboldt Foundation, Bonn, West Germany. We appreciate the excellent technical help of Marianne Metz and the secretarial help of Peggy Reiter. References I. 2. 3. 4. 5. 6.

K.R. T.J. K.K. Eng. G.J. Ann. D.H. L.C. R.E. L.C. G.R.

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7.

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Vasoactive Agents in Perfluorochemical

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and

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