Effects of arginine vasopressin on the sequestration of albumin in extravascular space in the rat

Gen. Pharmac. Vol. 19, No. 3, pp. 381-386, 1988 Printed in Great Britain. All rights reserved

0306-3623/88 $3.00+0.00 Copyright © 1988 Pergamon Press pie

EFFECTS OF ARGININE VASOPRESSIN ON THE SEQUESTRATION OF ALBUMIN IN EXTRAVASCULAR SPACE IN THE RAT I. WERBER and M. HOROWITZ* Division of Physiology, Hadassah School of Dental Medicine The Hebrew University, POB 1172, Jerusalem 91019, Israel (Received 17 August 1987) Abstract--I. The effect of Arginine-Vasopressin (AVP) and phenoxybenzamine on regional sequestration

of albumin in the extracellular space was studied in anesthetized (Na-Thiopentone) rats using a dual albumin labelling technique. 2. Low AVP dose (400 #U/kg) resulted in increased albumin sequestration, particularly in cutaneous and skeletal muscle extravascular space. In higher AVP doses this effect was not pronounced. 3. Phenoxybenzamin resulted in increased albumin sequestration, mostly in cutaneous and skeletal muscle extravascular space. 4. The effect of AVP, a potent pressoric hormone, on albumin sequestration resembles that of a vasodilator drug and contrasts with its pressoric nature.

INTRODUCTION

During acute heat stress, hyperthermia and excessive loss of body fluids occurs, resulting in dynamic changes in plasma volume (Horowitz, 1984; Horowitz and Borut, 1970; Horowitz et al., 1978; Horowitz and Samu¢loff, 1979). An initial phase of plasma expansion is followed by a phase of plasma volume conservation; as dehydration increases, a pronounced loss of water from the plasma compartment will lead to a failure of heat dissipation. During thermal dehydration, plasma volume conservation, as well as the failure of this mechanism are governed primarily by alteration in transvascular albumin flux (Horowitz et al., 1978; Horowitz and Samueloff, 1979). It is evident, that heat induced redistribution of cardiac output plays an important role in protein flux (Horowitz et al., 1985; Horowitz and Samueloff, 1987). However, other events occurring during thermal dehydration may modulate transvascular protein flux. During both thermal dehydration (Epstein et al., 1984) and hypovolemia, (Dunn et al., 1973; Share, 1973) vasopressin (AVP) is secreted. This hormone, in addition to its role in water reabsorption is a potent vasopressor, leading to an elevation of blood pressure and vascular resistance (Heyndrickx et al., 1976; Montani et al., 1980; Schmid et al., 1974; Szezepanska-Sadowska, 1973), particularly in cutaneous and skeletal muscle vascular beds (Hoffman, 1980; Charocopos et al., 1982; Liard et al., 1982). Recently, the counteraction of AVP on protein flux during histamine induced edema, not by its haemodynamic effects, has been reported (Grega and Svensjo, 1984). In view of the multiple effects of AVP on the vascular bed, this hormone may modulate proteins flux during thermal dehydration. As a first step to investigate this possibility, in the present in*To whom reprint requests should be addressed.

vestigation we have studied the effects of AVP infusions on whole body vascular permeability and on the regional changes in plasma volume and in albumin sequestration in tissue interstitial space. The findings have been compared with those obtained following the administration of a vasodilator drug. MATERIAL AND METHODS

Male Rattus norvegieus (Zabar strain, albino vat) weighing 250-300 g were used. The animals were divided into six groups: two control groups, three experimental groups receiving infusions of Arginine-Vasopressin (AVP) and one experimental group in which alpha adrenergic receptors were blocked with phenoxybenzamine. The control groups comprised animals receiving saline infusion for either 30 ((230) or 120 (C120) rain respectively. AVP groups were maintained on steady AVP levels of 400 # U/kg (AVP400), 800/~U/kg (AVP800) and 4000pU/kg (AVP4000) respectively, by constants AVP (Sigma) infusion. For calculation of AVP concentration in the infused solution an average hi2 of 20 rain used (Lauson, 1974). Alpha-adrenoceptor blockade was obtained by i.v. administration of phenoxybenzamine 2mg/kg (Smith Kline). Complete blockade of the receptors was validated by blood pressure measurement following i.v. injection of Phenylephrine, 0.I mg/kg (Sigma). Experiments were carried out under anaesthesia (Nathiopentone 5 mg/100 g body wt). Following cannulation of the jugular vein and the right carotid artery, the accumulation of labelled plasma albumin in tissue extravaseular space was measured using a dual albumin tracer technique modified from Studer and Potchen (1971) and Dewey (1959). One albumin tracer (nSI-RISA) was used to trace albumin sequestration while the second (99Tc-albumin) served for plasma volume determinations. The animals were placed in a temperature controlled chamber at 31°C, the jugular vein cannula was connected to an infusion pump (Harvard) and either AVP or saline were infused at a rate o f 10#I/rain. Concomitantly, t25I-RISA was administered i.v. as a bolus and was allowed to circulate in the blood for the entire experiment. Peripheral blood samples were taken from the tail at 30 rain intervals. Five rain before termination of the experiment 99Tc-Albuminwas

381

I. WERBERand M. HOROWITZ

382

administered. An additional plasma sample was then taken. The animal was then sacrificed by an overdose of Nathiopentone and tissue samples were dissected and transferred into pre-weighed counting vials. The following organs or tissues were sampled: tail, scrotum, leg muscle, chest skin, liver lobules, spleen, heart, gut, stomach, salivary gland and mesentary. Tracer activity in plasma and tissue samples was counted on Kontron Gammamatic II. To avoid counting errors the height of each sample was limited to 1 cm above the bottom of the vial. Extravascular activity of nSI-RISA (I-EXV) was calculated as follows: Organ PV = 99Tc activity (cpm)/99Tc/#l plasma

(I)

PV = Plasma volume Using this data, intravascular ~25I-RISA activity in each tissue

Table 1. 125I-RISA tl/2 values (min) of control (C-120), AVP

perfused and phenoxybenzamine treated rats Vasopressin Control

Phenoxybenzamine

C-120 AVP400 AVP800 AVP4000 371+33 265+33* 254+15" 259_+13" *Significant to control (P < 0.025), n = 12-15.

(2 mg/kg) 246+__22*

almost unchanged these findings suggest an increase in total vascular permeability due to the hormonal action. However, no difference between various doses of the hormone was observed. Phenoxybenzamine, similarly to AVP, resulted in an increased total vascular permeability (Table l).

Sequestration o f albumin in interstitial space

Statistics

Data of the control groups showed the basic nature of albumin sequestration of extravascular space in our experimental setup. In skeletal muscle and cutaneous tissue albumin accumulation was low, whereas in splanchnic organs such as intestine, stomach and mesentary, larger quantities of the tracer accumulated. The various organs differed in tracer sequestration dynamics with time. The PI values in the scrotum, salivary glands and gastrointestinal organs in the C l 2 0 group were significantly higher than in the C30 group. Organ tissues such as skeletal muscle, heart, tail and skin had almost the same PI values, in both the C30 and C l 2 0 groups (Fig. 1). In contrast, organs having blood sinusoids such as the liver and the spleen, showed a significantly higher PI values in the C30 group than in the matched organs in C120 group (Fig. 2).

The unpaired Student t-test with 0.05 level of significance was employed.

A VP action

(12sI-IVS) was calculated (equation 2). t25I-IVS = (12~I/,ul plasma) x (organ PV)

(2)

with equation (3) the sequestered albumin in tissue extra vascular space (12SI-EXV) was calculated: 125I-EXV = [total t25I-activity (cpm)] - (I25I-IVS)

(3)

From the obtained value a permeability index (PI) was defined: PI = (I-EXV.g/Inj. count.g) x 1000 Inj. count, g = injected I2~I-RISA (cpm)/g.body wt (4) Circulating plasma 12SI-RISA half-life (tl/2) values for each ' experimental group was obtained from the 125 I-RISA whashout curves.

RESULTS

Total vascular permeability Infusions of all AVP doses resulted in a significant reduction in 125I-RISA half-life. Since plasma volume of the animal throughout the measurement was

In non sinusoidal vascular beds A V P raised PI values in all the organs studied. Figure 3 depicts a typical PI AVP dose-response curve. The most pronounced effect was found in the low AVP dose (AVP400). In the higher doses (AVPS00, AVP4000) the AVP effect was less pronounced. Skeletal muscle, skin, heart, tail gut, stomach and mesentary shared

1400 CONTROL 30 min (C-30)

12OO

x >.

[ - - ' - I CONTROL 120 rain (C-120)

1000

i

800

_1 '~

600

400 200

TAIL

SCROT,

CHEST LEG SKIN MUSCLE

HEART

GUT

STOMACH MESENT.

SALIV. GLAND

Fig. i. Regional t:SI-RISA sequestration, expressed in PI values, 30 and 120min following a bolus injection of the tracer. Vertical lines denote SD, Asterisks indicate statistical significance (P < 0.05) between the two groups, n = 12-15.

Vasopressin and albumin tissue sequestration

383

I000 CONTROL C-30

I

I

320

CONTROL C-120 280

800

240

x z°

600

200

//

>-

J < uJ

160 400 120 0

2O0

i

I

I

I

I

I

'

i

CON 400 800

i

4000 AVP pU/kg

LIVER

Fig. 3. Effects of vasopressin on skin PI values--a typical AVP dose response curve. Vertical lines denote SD; Asterisks indicate statistical significance (P < 0.025) from control values, n = 12-15.

SPLEEN

Fig. 2. '25I-RISA s e q u e s t r a t i o n in sinusoidal o r g a n s , expressed in PI values, 30 and 120 min following a bolus injection of the tracer. Vertical lines denote SD Asterisks indicate statistical significance (P < 0.05) between the two groups, n = 12-15.

600

o-..o

x

400

z

this pattern (Table 2). In contrast, sinusoidal organs did not show any significant change in PI values in the AVP400 group. However, 4000 p U / k g AVP resulted in a significant rise in PI in both spleen and liver (P < 0.05 and P < 0.005 respectively) as demonstrated in Fig. 4. In Table 3 tissue plasma volumes are presented. In non-sinusoidal organs a physiological AVP dose, resulted in a reduction in PV in all tissues except for the gut. In higher AVP doses no clear changes were observed. N o clear correlation was found between changes in PI and PV values.

Experiments with phenoxybenzamine In alpha-blocked rats a significant increase (P < 0.05) in PI values was recorded in most organs. The most pronounced effect was found in the skin ( + 350%) and leg muscle ( + 130%), a moderate effect was found in the tail and salivary glands ( + 6 7 and + 6 5 % respectively) and the smallest effect was observed in the heart and splanchnic organs (from +30-+36%).

>I.3

200 Q.

I

I

I

I

I

I

I

i

I

CON 400 800

C- 120 (X + SEM)

Chest SK Tail Scrotum Leg muscle Heart Salivary gland Stomach Gut Mescnt.

95.0 _+ 15 213.0 + 29 473.0 _+ 57 42.7 _+ 12 400.0 _+44 182.0 _+43 1166.0 + 174 736.0 + 70 224 _+ 26

AVP pU/kg

Fig. 4. Effects of vasopressin on PI values of sinusoidal

organs---a dose-response curve. Vertical lines denote SD; Asterisks indicate a statistical significance (P < 0.05) from spleen control; A--indicates a statistical significance (P < 0.05) from liver control, n = 12-15. DISCUSSION

In this investigation a dual albumin labelling technique was used to characterize the effect of AVP on albumin shifts. This technique allowed us to measure the net amount of albumin sequestered in tissue interstitial space: [transvascular albumin flux

AVP400 (.( _+ SEM) 292.0 409 932.7 80.2 603.0 251.0 1542.0 958.0 305.0

I

4000

Table 2. Permeability index (PI) in organs and tissues of control rats (C-120) and its changes following AVP perfusion Organ

I

+ 30.3*** + 45.8*** _+ 107.0"** + 9.2** _+46.0*** _+ 50.0 _+ 192.0 _+ 53.0* + 29.0t

***P < 0.005; **P <0.025; *P <0.01; t P < 0,05; n = 12-15.

AVP800 (,~ _+ SEM) 198.0 + 42.0** 374.0 + 45.0*** 10)36.0 _+ 102.0"** 60.0 _+ 12.5 407.0 + 46.0 172.0 + 27.0 1523.0 + 229.0 809.2 + 52.0 284.0 _+42.0

AVP4000 (,~ + SEM) 206.0 + 40.0** 296.0 + 33.0* 524.0 _ 62.4 69.0 _+ 19.1 442.0 _+ 30.0 149.0 _+ 22.5 1672.0 + 262.0 739.0 + 37.0 268.0 + 40.0

384

I. WERBERand M. HOROWITZ Table 3. Tissue plasma volume in control (C-120) and AVP infused rats Organ Chest SK Tail Scrotum Leg muscle Heart Salivary gland Stomach Gut Mesent.

C- 120 ( ~ +_ SEM)

AVP400 (~' + SEM)

AVPS00 (.~' + SEM)

AVP4000 (.~ +_ SEM)

18.7_+1.1 22.7 _+ 1.7 34.9 _+ 2.3 10.6 +_0.6 79.3 +_ 3.0 22.8 _+ 1.3 23.4 _+ l.l 28.6 _+ 1.4 30.6 __ 3.9

17.86_+0.6 17.1 +0.9*** 26.0 _+ 0.7*** 8.32 _+ 0.5*** 66.9 -+ 2.4*** 18.0 _+ 0.6*** 19.5 _+ 1.4"* 32.5 _+ 1.3t 33.3 _+ 2.9

20.5_+0.8 24.1 +_ 1.4 30.9 _+ 1.8 10,6 _+ 0.6 78.6 -+ 2.9 20.0 _+ 0.7t 23.8 _+ 1.2 36.3 +_ 1.9"** 36.6 _+ 3.7

18.0 -+ 0.9 17.1 _+0.5*** 23.1 _+ 1.6"** 8.9 _+ 0.4** 69.6 -+ 2.5** 17.6 _+ 0.7*** 19.2 _+ 1.0*** 35.0 _+ 0.9*** 40.9 _+ 3.6~

***P <0.005; **P <0.025; I"P <0.05; n = 12-15.

- a l b u m i n drained with lymphatic flow] in many vascular beds simultaneously. It has previously been reported that in non-sinusoidal tissues such as muscle or skin, 2-72 hr after bolus injection of albumin tracer, the tracer sequestered in the interstitial space is hardly affected by lymphatic flow (Bill, 1977, 1979). Furthermore, in the gut, where lymph production is higher than in other internal organs, 5 hr are required to clear 35% of the albumin tracer from the extracellular space (Sheppard and Sterns, 1975). In view of these findings and in view of the fact that vasopressin reduces lymph flow (Quillen et al., 1977) we hypothesized that PI values, measured in this investigation 2 hr following bolus injection of albumin tracer may reflect albumin sequestration, mostly due to transvascular protein flux. Our findings on interstitial 99Tc-albumin sequestration in various tissues in the control group agree with those of other authors. Sequestration of albumin in skin and muscle extravascular space was the lowest whereas that of splanchnic organs was the highest. Tracer sequestration with time agree with previous reports as well (Studcr and Potchen, 1971). The variation in PI values obtained for the various vascular beds fits well with the variations in permeability between these tissues. Thus skin and muscle capillary have the lowest PI and very low permeability whereas

splanchnic capillaries with their fenestrations are very permeable (Rhodin, 1980) and their PI value is high. The data of the present investigation demonstrate an increased sequestration of 99Tc-albumin in nonsinusoidal organs in response to AVP infusion. The most pronounced response was observed for the lowest AVP dose (AVP400), while the effect of higher AVP doses was not significant. The tissues with the highest response to vasopressin were the skin and the skeletal muscle. These vascular beds are known to be the most sensitive to vasopressin pressoric action as well (Liard et al., 1982; Hoffman, 1980; Charocopos et al., 1982). In contrast to most organs, in the sinusoidal organs, where endothelium is discontinued (Wisse, 1970) and lymphatic flow is high, PI values are not indicative of permeability changes even a short time following bolus injection of a tracer. The high lymphatic flow causes rapid loss of any accumulated tracer within the tissue space (Mayerson, 1963) and thus produces low PI values. A further reduction in PI may reflect changes in lymphatic washout rate rather than permeability changes. In both spleen and liver PI values increased with the increment in vasopressin doses. In these organs hydrostatic pressure is a major factor in transcapillary exchange (Brauer et al., 1959) and lymph production. Since vasopressin has been found to decrease portal vein flow

14OO ~] IL:)OO

CONTROL C-120

['-'-'l PHNOXYBENZAMINE 2 mg/kg

IOOO ¢3 _z >I-.% 65 <[ tkl :Z ¢¢:

800

600

400

200

O

TAIL

SCRO'E Ct'IIEST LEG HEART GUT SKIN MUSCLE

STOM- MES- SALIV. LIVER SPLEEN ACH ENT. GLAND

Fig. 5. Regional =25I-RISAsequestration in controls and phenoxybenzamine treated rats, expressed in PI values, 120 rain following injection of the tracer. Vertical lines denote SD; Asterisks indicate a statistical significance (P < 0.05) between the two groups, n = 9-11.

Vasopressin and albumin tissue sequestration (Charocopos et al., 1982), AVP induced reduction in lymphatic clearance may explain our findings of elevated PI values. In addition to these findings AVP infusion resulted in shortening of 125I-RISA 11/2 by 30% in the three groups studied. Since no change in plasma volume except in AVP4000, was observed, the reduction in 125I-RISA ti/2 can be accounted for by an increased total vascular permeability in response to AVP infusion. The regional changes observed in albumin sequestration in AVP400 agree with these findings. However, data on albumin washout to extraceilular space of the other experimental groups do not. The additive effect of both increased PI and increased plasma volume, even though not statistically different from that of the control group may account for the observed changes. A positive correlation between flow and permeability changes has been repeatedly documented (Renkin, 1984). In the present investigation, administration of phenoxybenzamine, an alpha-blocker drug, resulted in increased PI values in many organs. Unexpectedly, AVP, a potent vasopressor, and known for its antiinflammatory drug by antagonizing histamine induced increased permeability (Grega and Svensjo, 1984) brought about an increase in the albumin sequestered in the interstitial space, A vasopressin dose which induces 64.5% reduction of skin blood flow in rats (Charocopos et aL, 1982) increased PI value by 205%. Similarly, in skeletal muscle, 45% reduction in blood flow coincided with an 85% increase in albumin sequestration. These findings suggest a dissociation between AVP pressoric action and its effect on the endothelial wall. A combination of pressoric effect, together with increased permeability of the capillary bed, possibly by formating venular gaps by partial detachment of endothelial cell junctions was reported for 5-hydroxytryptamine (Rippe and Folkow, 1980). In the present investigations, the effect of AVP on PI value at high AVP dose may have been masked by AVP pressoric action, resulting in a decrease in capillary surface area, available for the escape of macromolecules. Our results differ from those reported by Grega and Svensjo (1984). The discrepancy may be due to differences in the organ studied, doses used by various authors or by species variation. In conclusion, the data of this investigation suggest two modes of action of AVP on transvascular protein etflux. The data suggest that the hormone is not involved in the regulation of transvascular protein flux during moderate thermal dehydration.

385

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