298
Interactions among Inflammatory Mediators
on Edema Formation in the Canine Forelimb
ERIC AMELANG, CM. PRASAD, RICHARD M. RAYMOND, AND GEORGE J. GREGA
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SUMMARY Prostaglandin E,, 4 >ig/min, infused locally intra-arterially (ia) for 80 minutes into
forelimbs perfused at constant pump controlled inflow produced decreases in perfusion pressure,
increases in lymph total protein concentration ( s i g/100 ml), and small increases in weight (23 g) owing
to edema formation. Histamine, 16 /ig base/min, or bradykinin, 10 >tg base/min, infused locally ia for 30
minutes produced large increases in lymph flow, lymph total protein concentration, total protein
transport, and weight (70 g and 130 g, respectively). However, the local ia infusion of prostaglandin Ei,
4 fig/min, together with histamine, 16 /ig base/min, or bradykinin, 10 fig base/min, produced weight
increases of 180 g and 236 g, respectively, and the rate of weight gain during the combination infusions
greatly exceeded that produced by infusions of histamine or bradykinin alone. Moreover, the increases
in lymph flow and in total protein transport far exceeded those produced by infusions of prostaglandin
E, and histamine or bradykinin alone or additively. The edema produced by the local ia infusion of
prostaglandin Ei, 4 fig/min, together with bradykinin, 10 pg base/min, was even more marked in
naturally perfused forelimbs. Similarly, the local ia infusion of histamine, 4 /ig base/min, or bradykinin,
0.8 fig base/min, for 60 minutes into forelimbs perfused at constant inflow produced increases In lymph
flow, lymph total protein concentration, total protein transport, and weight (38 g and 14 g, respectively).
In contrast, histamine, 4 fig base/min, and bradykinin, 0.8 fig base/min, infused together locally ia for
60 minutes produced increases in weight of 118 g. The increase in lymph flow and total protein
transport was considerably more marked during the combined infusions than during the infusions of
histamine or bradykinin alone or additively. Circ Res 49: 298-306, 1S81
TISSUE swelling (edema) is one of the cardinal
signs of inflammation. The edema is largely attributable to the direct action of released histamine,
bradykinin, and possibly other substances on the
microvascular membrane which produce an increase in microvascular permeability to macromolecules. This increases the efflux rate of plasma
proteins from blood to tissue which decreases the
transmural colloid osmotic pressure gradient promoting increased net fluid filtration. The increased
fluid filtration is further enhanced by the arteriolar
vasodilator action of these inflammatory mediators
which increases blood flow, perfused surface area,
and capillary hydrostatic pressure.
In more recent years, the prostaglandins have
been added to the list of inflammatory mediators
(Ferreira and Vane, 1974; Vane, 1976). Prostaglandin E type compounds have been identified in inflammatory exudates (Willis, 1969; Hamberg and
Jonsson, 1973; Plummer et al., 1977; Arturson,
1979), and inhibitors of prostaglandin synthesis
such as indomethacin and aspirin are potent antiinflammatory agents both experimentally and clinically (Ferreira and Vane, 1974; Chahl and Chahl,
From the Department of Pharmacology and Institute for Cardiovascular Studies, University of Houston, Houston, Texas.
Supported, in part, by Grant HL25257 from the Nation*] Heart, Lung,
and Blood Institute.
Address for reprints. George J. Grega. Department of Pharmacology
(SR2). University of Houston, 4800 Calhoun Boulevard, Houston, Texas
77004
Received September 5, 1980: accepted for publication February 2,
1981.
1976; Vane, 1976; Plummer et al., 1977; Arturson,
1979).
Surprisingly, the direct actions of prostaglandin
E type compounds on the microvascular membrane
are very weak compared to those of histamine and
bradykinin (Williams and Morley, 1973; Vane, 1976;
Svensjo, 1978). Prostaglandin E, is reported to exert
the most potent inflammatory action of the E type
prostaglandins (Crunkhorn and Willis, 1971; Williams and Morley, 1973; Ikeda et al., 1975; Flower
et al., 1976; Svensjo, 1978). Based on this observation, it could be questioned whether the prostaglandins play an important role in the inflammatory
process, yet the fact remains that the inhibitors of
prostaglandin synthesis are potent anti-inflammatory agents. Other observations, however, suggest
a way out of this dilemma. Several investigators
have reported that prostaglandin Ei potentiates the
inflammatory action of histamine and bradykinin
(Williams and Morley, 1973; Ferreira et al., 1973;
Lewis et al., 1974; Thomas and West, 1974; Vane,
1976; Williams and Peck, 1977; Svensjo, 1978).
Most of the experimental data providing evidence for a role of the prostaglandins in inflammatory processes are from studies in rats or hamsters.
Studies in the dog, though few in number, fail to
provide evidence for a prostaglandin-induced increase in permeability to macromolecules owing to
a direct action on the microvascular membrane
(Greenberg and Sparks, 1969; Daugherty, 1971; Joyner, 1977). Moreover, the potential interactions
among the prostaglandins and other inflammatory
PROSTAGLANDIN E, POTENTIATION OF INFLAMMATION/zlme/an^ et al.
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mediators apparently have not been studied in the
dog. The mechanism by which prostaglandin Ei
potentiates the inflammatory actions of histamine
and bradykinin is not clear. The interpretation of
the data by Williams and Morley (1973) and Williams and Peck (1977) implies that the potentiation
results simply from the potent arteriolar vasodilator
action of prostaglandin Ei which further increases
blood flow, perfused surface area, and microvascular pressure. In contrast, data presented by Svensjo
(1978) and Joyner et al. (1979) demonstrate that
the potentiation is independent of the arteriolar
vasodilator actions of prostaglandin Ei. Instead, the
potentiation results from a further increase in the
number of large venular pores produced by the
simultaneous infusion of prostaglandin Ei with histamine or bradykinin and produced by an unknown
mechanism.
These observations prompted the present study
to characterize the effects of prostaglandin Ei on
macromolecular permeability and edema alone and
in combination with other mediators of inflammation in the canine forelimb. Experiments were also
designed to assess the role of increases in blood flow
and macromolecular permeability in the potentiation of the inflammatory actions of histamine and
bradykinin produced by prostaglandin Ei.
Methods
Mongrel dogs of either sex having an average
weight of 23 kg (16-28 kg range) were anesthetized
with sodium pentobarbital (30-35 mg/kg, iv) and
ventilated using a Harvard respirator.
Lymph Flow Rate and Total Protein
Concentration Measurements
Small incisions were made over the brachial artery, cephalic vein (above the elbow), and second
superficial dorsal metacarpal vein (paw) in the right
forelimb. A side branch of the brachial artery, a
lymph vessel, and the vein were isolated. After
intravenous administration of heparin (300 U/kg),
these vessels were cannulated in an upstream direction with polyethylene tubing. The cannulas were
used for drug administration, lymph collection, and
pressure measurement, respectively. The lymph
vessels in the area of the cephalic vein at the elbow
drain forelimb skin and paw. Two or three lymph
vessels were usually tied centrally, and one of them
was cannulated distally with a 10-cm length of PE10 tubing that had been beveled at the cannulating
end.
Lymph was collected in miniature 0.5-ml graduated cylinders constructed from plastic pipettes.
Drugs were infused into the brachial artery with a
Harvard infusion pump. The skin small vein pressure and aortic pressure (via a femoral artery) were
measured with low-volume displacement Statham
pressure transducers (P23Gb). Blood was collected
periodically from the brachial artery for plasma
299
protein determinations. Lymph collections were
made for 10-minute periods, and pressures were
measured at the end of each period. Total protein
concentration in lymph and plasma was measured
by the spectrophotometric (Beckman DB spectrophotometer) method of Waddell (1956).
Prostaglandin Ei, 4 /xg/min, or bradykinin, 10 fig
base/min, was infused locally intra-arterially for 60
minutes. In other experiments, these agents were
infused together ia for 60 minutes under similar
conditions except that the prostaglandin Ei infusion
was initiated 10 minutes prior to the bradykinin
infusion.
In other dogs, a Masterflex roller pump was
interposed in the brachial arterial circulation to
maintain constant pump controlled blood inflow
throughout the experiment. Initially, flow was set
at a rate that produced a perfusion pressure similar
to aortic pressure. Lymph and blood collections,
pressure measurements, and drug infusions were as
described above except that other doses or drugs
were also studied.
Prostaglandin Ei, 4 /tg/min, histamine, 4 or 16
fig base/min, or bradykinin, 0.8 or 10 fig base/min,
was infused locally intra-arterially for 60 minutes.
In other experiments, prostaglandin Ei was infused
together with histamine, 16 /ig base/min, or bradykinin, 10 jug base/min, ia for 60 minutes under
similar conditions except that the prostaglandin Ei
infusion was initiated 10 minutes prior to the histamine or bradykinin infusion. In still other experiments, histamine, 4 \ig base/min, and bradykinin,
0.8 or 10 fig base/min, were infused together ia for
60 minutes.
All drugs were administered locally intra-arterially at a delivery rate of 0.2 ml/min using a Harvard
infusion/withdrawal pump.
At the conclusion of the experiments the animals
were killed. Immediately, the right and left forelimbs were severed approximately 2 cm above the
humeral condyle. The brachialis, biceps, and triceps
muscles were dissected down to their tendons of
insertions on the ulnar and radial tuberosities. The
humerus was cut and forelimbs were then exsanguinated and weighed. The mean weight differences
between the experimental and contralateral control
forelimbs were compared using the £-test. All other
data were statistically analyzed using the StudentNewman-Keuls procedure (Sokal and Rohlf, 1969).
Results
Responses in Forelimbs Perfused at Natural
Flow
Effects of Prostaglandin E\ and Bradykinin
Alone and in Combination on Vascular Pressure,
Lymph, and Weight (Table 1)
Mean aortic pressure and plasma total protein
concentration were not significantly affected during
the local ia infusion of prostaglandin Ei, 4 /ig/min,
CIRCULATION RESEARCH
300
VOL. 49, No. 2, AUGUST
1981
TABLE 1 Effects of 60-Minute Local Intra-arterial Infusions of Prostaglandm Eh Bradykinin, or Bradykinin and
Prostaglandm E, on Vascular Pressure, Lymph Flow, Lymph Total Protein Concentration, Total Protein Transport,
and Weight in Naturally Perfused Canine Forelimbs
Control periot
Infusion period
-30 to-20
-20 to -10
-10 to 0
Oto 10
min
min
min
min
10 lo 20
min
20 to 30
min
30 to 40
40 to 50
50 to 60
min
min
min
124
±6
122
±5
126
±5
127
±7
123
±5
123
±5
129
±5
123
±5
123
±5
131
±5
122
±4
122
±4
20
±2*
22
±2*
44
±4*
19
±1*
21
±2*
51
±5*
19
±1*
20
±2*
57
±5*
21
±2*
20
Mean aortic pressure (mm Hg)
PGE,
Bradykinin
PGEi + bradykinin
122
±5
120
±3
127
±4
121
±6
120
±3
127
±4
123
122
±6
±5
120
±3
126
±4
119
±3
127
±4
123
±5
121
±4
128
±4
Shin small vein pressure (mm Hg)
PGE,
Bradykinin
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PGE, + bradykinin
PGE,
Bradykinin
PGE, + bradykinin
10
±1
11
±1
14
±1
10
±1
10
±1
14
±1
0.02
±0.01
0.01
±0.02
0.02
±0.01
0.01
±0.01
0.01
±0 01
001
±0.01
2.3
±€2
1.9
±0.2
2.2
±0.3
2.3
±0.2
2.1
±0.2
2.3
±0.3
0.42
±0.06
0.63
±0.07
0.52
±0.04
0.33
±0.06
0.71
±0.06
0.61
±0.06
10
±1
10
±1
27
±3'
19
±1*
26
±3*
33
±4*
20
±2*
24
±3*
38
±5*
Lymph : flow (mil 10 min)
0.03
0.04
0 01
±0.01
±0.01
±0.01
0.01
0.01
0 01
±0 01
±0.08*
±0.1*
008
1.91
2.51
±0.03
±0.3*
±0.01
0.05
±0.01
0.01
±0 2*
1.81
±0.01
±2'
61
±6'
0.08
0.07
0.07
±0.02f
0.01
±0.2*
±0.03f
±0.03t
0.78
±0.2*
0.93
±0.3*
100
±0.01
0.78
±0.2*
0.93
±0.3*
Lymph total protein concentration (g/100 ml)
PGE,
Bradykinin
PGE, + bradykinin
PGE,
Bradykinin
PGE.+ bradykinin
23
2.8
±0.2
±0.2
Total protein
0.34
±0.06
0.71
±0.06
1.94
±0.05f
3.5
±0.3*
3.2
5.7
62
6.3
6.2
±0.3
±0.3
±0.3
±0.3
1.40
±0.3
41.4
1 64
±0.3t
2.35
230
±0.6t
±0.5f
40.3
±5*
65
±9*
37.4
±6*
53
±7*
3.7
4.3
4.9
5.3
±0.4*
±0.5*
±0.2
±0.2
2.8
±0.3
3.2
±0.2f
3.4
±0.3*
4.9
±0.4*
6.3
±0.4*
2.1
±0.2
3.1
±0.2f
transport (mg/19 min)
0.65
1.15
±0 2
±0.3
20.3
33.7
±4*
118
±9*
±6*
156
±14*
±6*
123
±9*
38.6
±6*
67
±8*
±03*
4.6
±0 4*
6.3
±0.4*
Plasma total protein concentration (g/100 ml)
PGE,
Bradykinin
PGE, + bradykinin
5.8
5.7
5.7
±0.2
±0.2
±0.2
5.6
5.6
5.7
±0.3
±0.3
±0.3
6.7
6.6
6.5
±0.4
±0.4
±0.4
PGE, — prostaglandin E,. 4 fig/min, ia (n — 6); bradykinin — 10 jig base/min, la (n = 6); PGE, + bridykinin — (n — 8). The mean weight of the
experimental forelimba exceeded the mean weight of the contxalateral control forelimb* by 35 ± 6 g', 158 ± 13 g*T and 293 ± 27 g* following the cessation
of the prostaglandin E,r bradykinin, and procuglandin E,-bradykinin infusions, respectively.
• P 5 0.01 relative to -20 to —10 minute time.
t P 3 0.06 relative to -20 to -10 minute time.
and bradykinin, 10 /ig base/min, alone or in combination. Skin small vein pressure was increased
throughout the infusion period in all three experimental groups, especially during the combined prostaglandin Ei-bradykinin infusion. There was a small
increase in forelimb weight during the prostaglandin Ei infusion, a large increase in forelimb weight
during the bradykinin infusion, and a massive in-
crease in forelimb weight during the combined prostaglandin Ei-bradykinin infusion. The increases in
lymph flow, lymph total protein concentration, and
total protein transport produced by prostaglandm
E[ were small, by bradykinin very large, and those
produced by the combined prostaglandin Ej-bradykinin infusion (except for total protein concentration) were exceptionally marked.
PROSTAGLANDIN Ej POTENTIATION OF
Responses in Forelimbs Perfused at Constant
Pump-Controlled Inflow
Effects of Prostaglandin £\, Histamine, and
Bradykinin on Vascular Pressure, Lymph, and
Weight (Table 2)
Mean aortic pressure, skin small vein pressure,
and plasma total protein concentration were not
INFLAMMATION/AmelangetaL
301
significantly affected during the infusions of prostaglandin Ei, histamine, or bradykinin. All agents
studied produced decreases in perfusion pressure
which were sustained (prostaglandin Ei and histamine) or transient (bradykinin). The infusions of
histamine or bradykinin produced large dose-related increases in lymph flow, lymph total protein
concentration, total protein transport, and forelimb
TABLE 2 Effects of Prostaglandin E\, Histamine, or Bradykinin Infused Locally Intra-arterially in Canine
Forelimbs Perfused at Constant Inflow
Control period
-20 to -10
min
Infusion period
-10 toO
min
0to 10
10 to 20
min
min
30 to 40
min
20 to 30
min
40 to 50
50 to 60
min
min
Mean aortic pressure (mm Hg)
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PGE,-4
H-t
H-16
B -.8
B-10
124 ±
130 ±
128 ±
115 ±
PGE.-4
H-4
H-16
B-8
B-10
118 ± 8
127 ± 7
132 ± 5
PGE.-4
H-4
H-16
B-.8
B-10
11 ± 1
13 ± 1
8
5
7
5
142 ± 7
124 ±
128 ±
128 7
117 6
142 +
7
4
118 ±
124 ±
132 5
111 ±
140 5
8
5
5
7
11 ±
13 ±
13 ±
10 1
14 ±
1
1
1
1
1
2.4 ±
2.8 ±
3.0 ±
1.8 ±
3.3 ±
0.3
0.2
03
0.2
0.3
7
6
7
127
129
128
118
140
±7
±3
±8
±6
±7
56
86
86
86
107
± 4*
± 9*
± 10*
± 5*
± 10*
138 ± 11
128 ± 5
124 ± 9
121 ± 5
137 ± 8
141 ± 12
129 ± 5
123 ± 9
121 ± 5
140 ± 5
138 ± 12
129 ± 5
122 ± 8
123 ± 5
ia5 ± 8
141 ± 11
130 ± 4
120 ± 9
123 ± 5
136 ± 6
142
130
122
123
138
64 ± 3 *
88 ± 9 *
88 ± 9 *
120 ± 13
133 ± 7
69 ± 5*
88 ± 7*
80 ± 4*
127 ± 13*
133 + 6
± 138
± 4
± 10
± 5
± 7
Perfusion pressure (mm Hg)
110 ± 6
140
±
5
5
58 ± 3*
88 ± 9*
81 ± 10*
95 ± 7*
106 ± 8 *
60 ± 3*
88 ± 10*
82 ± 10*
105 ± 8
110 ± 8*
62 ± 3*
87
82
115
123
±8*
±8*
± 11
±5f
Skin small vein pressure (nun Hg)
13 ± 1
10 ± 1
14
±
1
13 ± 2
13 ± 1
16 ± 1
11 ± 2
14 ± 1
13 ± 2
14 ± 2
15 ± 1
11 ± 2
15 ± 1
13 ± 2
14 ± 1
15 ± 2
10 ± 1
15 ± 1
Lymph total protein concentration
PGE,-4
H-4
H-16
B-.8
B-10
2.4 ±
2.8 ±
3.0 ±
1.8 ±
33 ±
03
0.2
0.3
02
0.3
2.7
3.5
3.8
2.1
4.6
± 0.3
± 0.3*
± 0.5
± 0.2
± 0.4t
3.0 ± 4 |
3.9 ± 0.2*
5.1 ± 0.4*
2.4 ± 0.3t
5.4 ± 0.5*
13 ± 2
14 ± 2
15 ± 2
10 ± 1
15 ± 2
13 ± 2
14 ± 2
14 ± 2
10 ± 1
15 ± 2
13 ±
14 ±
14 ±
10 ±
15 ±
2
2
2
1
2
(g/100 ml)
34 ± 0.3*
4.7 ±0.4*
5.2 ± 0.5
2.9 ± 0.4*
59 ± 0.3*
3.5
4.8
5.3
2.9
5.9
± 0.2*
±0.3*
±04*
± 0.3*
±0.3*
3.4 ±0.3*
4.3 ±0.3*
5.2 ±0.4*
2.8 ±0.3*
5.2 ±0.2*
3.5 ± 0.2*
3.8 ± 0 2 *
4.9 ± 0.4*
2.8 ± 0.3*
5.2 ± 5.2
Lymph flow (ml/10 min)
PGE.-4
H-4
H-16
B-.8
B-10
0.02
0.02
0.02
0.01
0.01
± 0.01
± 0.01
± 0.01
± 0.01
± 0.01
0.02
0.02
0.02
0.01
0.02
±
±
±
±
±
0.01
0.01
0.01
0.01
0.01
0.03 ± 0.01
0.07 ± 0.02f
0.29 ± 0.1*
0.04 ± 0 02
0 17 ± 0.07*
PGE.-4
0.3
0.3
0.5
0.2
0.6
± 0.04
± 0.06
± 0.1
± 0.04
± 0.1
0.3
0.4
0.5
0.3
0.7
±
±
±
±
±
0.04
0.08
0.1
0.04
0.2
0.9 ± 0.04
2.5 ± 0.7f
10 ± 5f
0.5 ± 0.03
7.4 ± 2j-
0.05 ± 0.0If
0.16 ±0.04*
0.52 ± 0.3*
0.08 ± 0.04t
0.38 ± 0.1*
0.04 ± 0.02
0.18 ± 0 04*
0.44 ± 0.3*
0.14 ± 0.05*
0.49 ± 0.1*
0.03 ±0.01
0.15 ±0.03*
0.40 ±0.2*
0.14 ±0.5*
0.32 ±0.1*
0.03
0.12
0 42
0.12
0.36
±0.01
±0.03*
±0.2'
± 0.05*
±0.04*
0.03
0.02f
0.1*
0.12
0.28
± 0.01
± 0.03f
± 0.1*
± 0.05'
± 0.04*
Total protein transport (mg/10 min)
H-4
H-16
B-.8
B-10
1.5 ± 0.6*
6.3 ± 1.6*
26 ± 6 *
2.7 ± 0.04*
20.0 ± 6*
1.6 ±0.6*
7.8 ± 1.3*
25 ± 5 *
3.7 ± 0.08'
25.6 ± 4*
1.2 ±0.3f
6.7
22
5.9
20.4
± 1*
±4*
± 0.07*
±3*
1.0 ± 0.2
4.9 ± 1*
23 ± 5 *
5.3 ±0.06*
17.4 ± 2 *
0.7 ±
3.4 ±
19 ±
5.1 ±
13.1 ±
0.2
1*
4*
0.04'
2*
Plasma total protein concentration (g/100 ml)
PGE.-4
H-4
H-16
B-.8
B-10
6.0 ± 0.3
6.6 ± 0.1
66 ±0.2
3.9 ± 0.2
6.8 ±0.1
6.1 ±0.3
6.6 ±0.1
6.5 ±0.2
4.1 ±0.2
6.7 ±0.2
6.0
6.6
6.5
4.1
6.9
± 0.2
± 0.1
± 0.2
± 0.2
± 02
PGEi-4 — prostaglandin Ei, 4 itg/min, la, from 0 to GO minutes (n — 6), H-4 — huurnine, 4 fig baae/min, ia, from 0 to 60 minutes (n — 6), H-16 hutamine, 16 fif ba*e/min, m, from 0 to 60 minutes (n - 6), B-.8 - bradykinin, 0.8 jig bose/min ia, from 0 toflOminutes (n " 8), B-10 = bradykinin, 10/ig
baae/min, u, from 0 to 60 minutes (n - 6). The mean weight of the experimental forelimbs eiceeded the mean weight of the contralateral forelimbs by 23
± 4 g", 38 ± 4 g", 70 ± 7 g*, 14 ± 3 g*, and 130 ± 7 g' after the cessation of the prostajlanrlin E,, but*muie-4, hKtflmine-16, brmdykinin-.8, and bradyltinin10 infiifiionB, respectively
' P < 0.01 relative to -10 to 0 minute time.
t P < 0.05 relative to -10 to 0 minute time.
CIRCULATION RESEARCH
302
weight. The infusion of prostaglandin Ei produced
only small increases in these parameters.
Interaction between Prostaglandin Ei and
Histamine or Bradykinin (Table 3)
Prostaglandin Ei, 4 ng/min, infused together with
histamine, 16 jug base/min, or bradykinin, 10 fig
base/min, locally ia for 60 minutes failed to alter
mean aortic pressure or plasma total protein concentration significantly. Perfusion pressure was decreased, whereas skin small vein pressure was gradually increased, especially during the prostaglandin
Ei-bradykinin combined infusion. Lymph flow,
lymph total protein concentration, total protein
VOL. 49, No. 2, AUGUST 1981
transport, and forelimb weight were increased
markedly during both combined infusions. The increases in lymph flow, total protein transport, and
forelimb weight were far greater than those produced by prostaglandin E,, histamine, or bradykinin, alone or additively.
Interaction between Histamine and Bradykinin
(Table 4)
The infusion of histamine, 4 fig baae/min, together with bradykinin, 0.8 jug/min or 10 \i% base/
min, locally ia for 60 minutes failed to alter mean
aortic pressure or plasma total protein concentration significantly. Lymph total protein concentra-
TABLE 3 Effects of Prostaglandin E\ Infused Locally Intru-arttrially with Histamine or Bradykinin in Canine
Forelimbs Perfused at Constant Inflow
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PGE.-4 + B-10 or H-16 infusion period
ConLrol period
-.30 to -20
min
- 2 0 to - 1 0
min
-lOtoO
min
0 t o 10
min
10 to 20
min
20 to 30
min
30 to 40
40 to 50
50 to 60
min
min
min
122
±8
123
±10
121
±10
121
117
±11
117
120
±11
120
±11
Me an aortic pressure (mm Hg)
PGE,-4 + B-10
PGE.-4 + H-16
142
±9
134
131
±8
±9
123
121
116
±6
±7
±6
133
±7
124
±7
134
132
±7
132
±8
70
±8'
115
±9
116
±8
±11*
14
14
16
±0.8
±0.8
±1.5
±8
123
±7
±12
±9
Perfusion pressure (mm Hg)
PGE,-4 + B-10
PGE.-4 + H-16
78
82
±11'
81
±10*
91
95
100
±11'
±11*
±11*
92
97
106
±6f
±8f
±10
100
±11*
102
±15
109
±10*
105
±12
Skin small vein pressure (mm Hg)
PGE.-4 + B-10
PGE i -4 + H-16
14
13
13
±0.7
±0.7
3.3
±0.3
3.0
±0.3
3.3
±0.3
3.0
±3.3
3.6
±0.3
3.5
±0.3
0.01
±0.01
0.02
±0.01
0.01
±0.01
0.02
±0.01
0.03
±0.01
003
±0.01
±0.8
22
±3'
16
±1
28
±4"
17
±1
33
±3*
36
±3*
20
±1*
37
±3*
±1*
38
±4*
17
±lf
6.0
±0.4*
5.5
±0.3*
5.9
±4'
5.7
±0 3*
5.7
±0.4*
5.6
±0.3*
5.4
±0.5*
5.1
±0.2*
1.5
±0.4*
0.87
±0.2*
1.4
±0.4*
0.70
±0.2*
L.O
±0.3*
0.51
±0.1*
0.92
±0.2*
0.44
±0.1*
19
±1
18
Lymph total protein concentration (g/100 ml)
PGE.-4 + B-10
PGE.-4 + H-16
4.4
±0.3t
5.0
±3*
5.8
±0.4'
5.6
±0.3*
Lymph flow (mil 10 mm)
PGE.-4 + B-10
PGE.-4 + H-16
0.70
±0.3*
0.30
±0.1
1.4
±0.5*
0 91
±0.2'
Total protein transport (mg/10 mm)
PGE,-4 + B-10
0.4
0.4
PGE.-4 + H-16
±0.03
O.G
±0.2
±0.03
0.7
±0.2
1.3
±0.5
11
±0.5
27
79
74
±llf
18
±21*
54
±10*
±27*
58
±11*
±3t
83
±22*
40
±10*
67
±16*
30
±6*
49
±18*
25
±6*
Plasma total protein concentration (g/100 mi)
PGE.-4 + B-10
68
6.7
6.6
±0.3
6.6
±0.3
±0.2
PGE.-4 + H-16
±0 2
6.9
±0.3
6.4
±0.3
PGEi-l - prostaglandin E,. 4 /ig/min, la. from —10 to 60 minutes. B-10 = bradykinin, 10 mi baae/min, ia, from 0 to 60 minutes, H-16 — rustamine, 16
base/min, i», from 0 to 60 minutes; PGF.,-4 + B-10 - (n - G». PGE,-4 + H-1S - (« - 61. The mean weight of the experimental forelimbs eicetded the
an weight of the contndateraJ fordimba by 236 ± 27 g* and 180 ± 20 g" after the ceeaauon of the PGE,-4 + B-10 and PGE.-4 + H-16 infusions.
mean
respectively.
" P < 0.01 relative to tune —20 to -10 minut* time.
t P < 0.05 relative to time —20 to —10 minute time.
PROSTAGLANDIN E, POTENTIATION OF INFLAMMATION/Amelong
et al.
303
4 Effects of Histamine Infused Locally Intraarterially with Low or High Doses of Bradykinin in Canine
Forelimbs Perfused at Constant Inflow
TABLE
Control period
-20 to -10
-10 toO
H-4 + B- 8 or H-4 + B-10 infusion period
0 to 10
10 to 20
20 to 30
30 to 40
40 to 50
50 to 60
137 ± 4
115 ± 13
138 ± 4
115 ± 16
96± 11*
143 ± 26
94 ± 12
139 ± 19
17 ± 1*
36 ± 12'
17 ± 1'
36± 12'
17 ± 1*
32 ± 10
H-4 + B-8
H-4 + B-10
142 ± 3
135 ± 8
144 ± 4
136 ± 8
Mean aortic pressure (mm Hg)
139 ± 4
138 ± 5
138 ± 5
133 ± 7
125 ±12
120 ± 1 1
139 ± 5
120 ±11
H-4
H-4
141 ± 6
140 + 8
140 ± 5
139 ± 8
Perfusion pressure (mm Hg)
82 ± 8 *
92 ±11*
92 ± 11*
106 ± 12+
105 ± 10+
135 ± 18
165 ± 27
B-.8
B-10
Skin small vein pressure (mm Hg)
16 ±0.7'
16 ±0.7*
17 ± 1 *
23 ±6*
30 ±9*
36 ±13*
94 ± 11*
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
H-4
H-4
B-.8
B-10
13 ±0.7
12 ± 1
13 ±0.7
12 ± 1
H-4
H-4
B-.8
B-10
3 0 ± 0.3
29 ±0.1
3.0 ±0.3
2.9 ±0.1
Lymph total protein concentration (g/100 ml)
4.1 ±0.4*
5.3 ±0.4*
5.4 ± 0.9*
5.1 ±0.2"
5.2 ± 0.2*
4.4 ±0.3*
5.4 ± 0.9
5.2 ± 0.2
5.0 ± 0.3*
5.1 ± 0.2*
5.3 ± 0.3*
5.1 ±0.1*
H-4 I-B-.8
H-4 - B-10
0.01 ± 0.01
0.01 ± 0.01
001 ± 0 0 1
0.01 ± 0.01
Lymph flow (ml/10 min)
0.09±0.03f
0.27 ±0.06''
0.43 ±0.1*
0.38 ± 0.2*
1.3 ± 0.6*
1.1 ±0.2*
0.39 ± 0 . 1 '
0.89 ±0.2
0.34 ± 0 . 1 '
0.86 ± 0.2*
0.36 ± 0 1*
0.72 ± 0.2*
H-4 • B - 8
H-4 hB-10
0 3 ± 0.03
06 ±0.1
0.3 ± 0 03
0.6 ± 0.1
Total protein transport (mg/10 min)
4.1 ± 2+
17.3 ± 6*
24.3 ± 7*
18.4 ± 9-j63.3 ±21*±6*24.355.8± 16*
23.3 ± 6'
46.7 ± 10'
19.5 ± 4*
44.5 ± 10*
19.4 ± 5*
36.7 ± 11*
6.3 ± 0 2
6.3 ± 0.3
Plasma total protein concentration (g/100 ml)
6.4 ±0.1
6.3 ± 0.2
H-4
H-4
B-.8
B-10
6.3 ± 0.3
6.3 ± 0.2
H-4 - histamine, 4 jig baje/min, la, from 0 to 60 minutes; B-.8 - bradykinin, 0.8 n% base/min, la, from 0 to 60 minutes; B-10 - bradykinin, 10/ig base/
min, la, from 0 to 60 minutes; H-4 + B- 8 — (n • 6), H-4 + B-10 ™ {n — 6) The mean weight of the experimental forelimbs exceeded the mean weight of
the contralateral control forelimbs by 118 ± 17 g* and 243 ± 27 g* after the cessation of the H-4 + B-8 and H-4 + B-10 infusions, respectively
' P < 0.01 relative to —10 to 0 minute time
t P < 0 06 relative to -10 to 0 minute time.
tion was increased markedly during both combined
infusions. Perfusion pressure was decreased and
skin small vein pressure was increased, especially
during the combined infusions with the high dose
of bradykinin. Lymph flow, total protein transport,
and forelimb weight were markedly increased during both infusions, and the increase was gTeater
than that produced by histamine or bradykinin
alone or additively.
Control Forelimb Weights
The mean weight of the contralateral control
forelimbs of all animals employed in this study was
558 ± 16 g. There was no significant difference
among the mean weights of the contralateral control forelimbs among the various groups used in this
study.
Discussion
Sixty-minute local intra-arterial infusions of histamine or bradykinin into forelimbs pump perfused
at constant inflow produced dose-related increases
in lymph flow, lymph total protein concentration,
total protein transport, and weight similar to that
previously reported (Grega et al., 1972a; Kline et
al., 1973; Marciniak et al., 1978; Maciejko et aL,
1978; Svensjo et al., 1979; Grega et al., 1980; Raymond et al., 1980). The weight increases are attributable to edema formation subsequent to a fall in
the transmural colloid osmotic pressure gradient
owing to an increase in microvascular permeability
to macromolecules. This is evidenced by the
marked rise in lymph total protein concentration
and lymph flow rate. Because the blood pump prevents arterial blood flow from increasing as it does
under natural flow conditions, a rise in capillary
hydrostatic pressure subsequent to arteriolar vasodilation would not be expected in forelimbs pump
perfused at a constant flow rate. In naturally perfused forelimbs, 60-minute local intra-arterial infusions of bradykinin produced even more marked
increases in edema and lymph flow, and accelerated
the rate of weight gain and the rate of protein efflux
compared to that in forelimbs pump perfused at
constant inflow. This reflects the contribution of
the increase in capillary hydrostatic pressure superimposed on the increase in microvascular permeability to macromolecules which further enhances
the rate of net fluid filtration (Kline et al., 1973).
Local intra-arterial infusions of prostaglandin Ei
for 60 minutes produced increases in weight, lymph
total protein concentration, and lymph flow in fore-
304
CIRCULATION RESEARCH
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limbs perfused naturally or at constant pump controlled inflow. The increase in weight is attributable
to edema formation subsequent to an increase in
microvascular permeability to macromolecules. In
the naturally perfused forelimbs, an increase in
microvascular pressure subsequent to arteriolar
vasodilation would also contribute to the edema
formation. Thus, as histamine and bradykinin, prostaglandin Ei produces an increase in macromolecular permeability to plasma proteins. This conclusion is supported by the observation that other
vasodilator drugs which do not alter macromolecular permeability fail to increase lymph total protein concentration, and do not produce edema formation when the forelimb is perfused at constant
pump controlled inflow (Maciejko et al., 1978; Marciniak et al., 1978; Grega et al., 1980; Raymond et
al., 1980). It was also observed that increasing the
prostaglandin E, infusion rate did not produce doserelated increases in protein efflux and edema formation as does histamine and bradykinin. Thus,
these data are in agreement with others suggesting
that the direct action of proataglandin Ei on the
microvascular membrane is weak relative to that of
histamine and bradykinin (Freeman and West,
1972; Williams and Morley, 1973; Vane, 1976;
Svensjo, 1978).
The failure of other investigators (Greenberg and
Sparks, 1969; Daugherty, 1971; Joyner, 1977) to find
evidence of a direct action of prostaglandin Ei on
the microvascular membrane in the dog is probably
related to the small doses studied. For example,
Joyner (1977) reported that, in the dog, intradermal
injections of prostaglandin Ei increased the efflux
rate of labeled albumin as did histamine, evidence
for an increase in microvascular permeability to
plasma proteins. However, in the limb, subcutaneous injections of substantially smaller doses of
prostaglandin Ei did not increase the concentration
of proteins in lymph, thus providing no evidence for
an increase in microvascular permeability to macromolecules. The differences, therefore, are likely
related to dose.
The infusion of prostaglandin Ei together with
either histamine or bradykinin produced massive
increases in forelimb weight owing to edema formation. The edema greatly exceeded that produced
by prostaglandin Ei and histamine or bradykinin
alone or additively. The rate of edema development
was very rapid during the combined infusions. For
example, during the prostaglandin Ei-bradykinin
infusion, the forelimbs were frequently massively
edematous in less than 10 minutes under both natural or constant pump controlled flow conditions.
The edema was visibly evident, and characterized
by a spreading of the digits. During the infusion of
bradykinin alone, it usually took more than 30
minutes to detect visible signs of edema formation.
The edema was associated with exceptionally
marked increases in lymph flow, lymph total protein
concentration, and total protein transport, and is
VOL. 49, No. 2, AUGUST 1981
attributable to a very dramatic increase in microvascular permeability to macromolecules which
greatly increases net protein efflux. The edema was
frequently so massive (especially during the prostaglandin Ei-bradykinin infusion) that venous pressure increased with time even in the forelimba pump
perfused at a constant flow rate. The increased
venous pressure is attributable to extravascular
compression subsequent to marked increases in interstitial fluid volume. This is suggested because,
on cessation of the infusion, perfusion pressure
promptly returned to control, but venous pressure
failed to decrease. In those experiments in which
the edema formation was more modest, there was
no increase in venous pressure. Moreover, neither
bradykinin, histamine, nor prostaglandin Ei infused
alone produced increases in skin small vein pressure
in forelimbs pump perfused at constant inflow. The
potentiation of the bradykinin-induced edema formation produced by prostaglandin Ei is in agreement with that reported by many others in rats,
hamsters, and guinea pigs (Williams and Morley,
1973; Ikeda et al., 1975; Vane, 1976; Williams and
Peck, 1977). However, some investigators have not
been able to demonstrate a potentiation of histamine-induced edema formation by prostaglandin E]
(Lewis et al., 1974; Thomas and West, 1974; Vane,
1976). The reason for this is not clear.
These data provide evidence that the potentiation of the histamine and bradykinin edema formation produced by prostaglandin Ei results from
a direct interaction of these agents on the microvascular membrane which further increases microvascular permeability to macromolecules, thus dramatically increasing the efflux rate of plasma proteins from the microcirculation (Svensjo, 1978; Joyner et al., 1979). These data do not support the
conclusions of some other investigators who suggested that the potentiation of the bradykinin
edema formation produced by prostaglandin Ei results simply from the arteriolar vasodilator action
of this agent which further increases blood flow
(Williams and Morley, 1973; Williams and Peck,
1977; Kopaniak et al., 1978). These conclusions are
based on the following observations. The potentiation of the edema formation was equally marked in
naturally perfused forelimbs in which blood flow
markedly increases (Daugherty, 1971), and in
pump-perfused forelimbs in which an increase in
blood flow is mechanically prevented by the pump.
Other vasodilators in doses which produce vasodilation equal to that produced by prostaglandin Ei
do not potentiate the edema produced by histamine
and bradykinin (Maciejko et al., 1978; Marciniak et
al., 1978; Svensjo et al., 1978; Svensjo et al., 1979;
Joyner et al., 1979; Grega et al., 1980; Raymond et
al., 1980). The very potent arteriolar vasodilator
isoproterenol, in fact, prevents the edema formation
produced by histamine or bradykinin, and by combined infusions of prostaglandin Ei and bradykinin
(unpublished observation). Svensjo (1978) and Joy-
PROSTAGLANDIN E! POTENTIATION OF INFLAMMATION/Amelang et al.
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
ner et al. (1979) have demonstrated by intra-vital
light microscopy that prostaglandin Ej and bradykinin increase the number of large venular gaps
through which labeled macromolecules rapidly escape from the microcirculation. The development
of the large venular gaps is due to endothelial cell
separations in the postcapillary venules, and is not
influenced by even major changes in blood flow.
Moreover, the number of large venular gaps formed
by prostaglandin Ei and bradykinin given together
greatly exceeds that produced by either agent administered alone. The data in this study are consistent with the accelerated opening of many large
pores, owing to an interaction among the inflammatory mediators at the level of the microvascular
membrane, despite the fact that in the forelimbs
perfused at constant inflow—the pump mechanically prevents blood flow from increasing. Williams
and Morley (1973), Williams and Peck (1977), and
Kopaniak et al. (1978) did not eliminate the possibility that the potentiation of the bradykirun-induced edema formation by prostaglandin Ei resulted from a direct action on the microvascular
membrane independent of the increase in blood
flow. These investigators simply noted that the
potentiation produced by prostaglandin Ei was
greatest at doses which produced maximal vasodilation. In this study, it was noted that prostaglandin
Ei produced measurable increases in lymph total
protein concentration only in doses exceeding those
which produced maximal vasodilation.
A microvascular interaction between histamine
and bradykinin also was observed in this study. The
edema produced by combined infusions of histamine and bradykinin greatly exceeded that produced by infusions of histamine or bradykinin alone,
and was associated with marked increases in lymph
total protein concentration, lymph flow, and total
protein transport. The increases in lymph flow and
total protein transport exceeded those produced by
histamine or bradykinin alone or additively. During
the combined histamine-bradykinin (low dose) infusion, there was no increase in venous pressure.
However, during the combined infusion with the
high dose of bradykinin, venous pressure increased
with time owing to extravascular compression subsequent to increases in interstitial fluid volume. In
those experiments in which the edema was more
modest, a rise in venous pressure did not occur,
although potentiation of the edema formation was
still evident.
These data demonstrate that, as interactions
among vasoactive agents occur which affect vascular smooth muscle contractility, interactions also
occur at the level of the microvascular membrane
which affect macromolecular permeability. These
microvascular interactions may modulate the increases in microvascular permeability to macromolecules produced by inflammatory mediators
either antagonizing or potentiating the increased
protein efflux and edema formation produced by
305
histamine-like agents. The catecholamines and vasopressin function as antagonists of the direct action of the inflammatory mediators on the microvascular membrane, whereas the E type prostaglandins function as potentiatora of the inflammatory
process. Moreover, the data in this study also demonstrate that a microvascular interaction between
histamine and bradykinin occurs which greatly enhances protein efflux and facilitates edema formation. Thus, the existing physiological state may
profoundly influence the direct response of the inflammatory mediators on the microvascular membrane. For example, perfusion of the forelimb with
blood from animals subject to severe bleeding negates the direct action of histamine-like agents on
the microvascular membrane, even if forelimb blood
flow is held constant with a blood pump (Marciniak
et al., 1977). The microvascular response to intraarterial and intravenous histamine is dramatically
different. Intravenously infused histamine, even in
massive concentrations, produces extravascular
fluid reabsorption rather than edema formation
(Grega et al., 1972b; Marciniak et al. 1977), in part,
subsequent to activation of the sympathoadrenal
system.
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Circ Res. 1981;49:298-306
doi: 10.1161/01.RES.49.2.298
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