INTRODUCTION

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NEUROTENSIN (NT), a 13-amino acid peptide, was first discovered and isolated from bovine hypothalami by Carraway and Leeman (3), who noticed that injections of purified fractions of hypothalamic extracts produced cutaneous vasodilation, hypotension, and cyanosis in anesthetized rats. It was then found that NT is widely expressed in the central nervous system and in peripheral tissues, where it may act as an endocrine or paracrine modulator of neural and smooth muscle function (11). Structure activity studies of NT have shown that much of the biological actions on smooth muscle and on binding to brain membrane fragments are conserved in the COOH-terminal hexapeptide of NT. For example, Granier et al. (9) showed that N-acetyl NT(8-13) [AcNT(8-13)] retained full binding property and pharmacological activity as tested by contraction of the guinea pig ileum. NT and some NT(8-13) analogs also have the unusual property of inhibiting the vascular leakage that occurs after traumatic injury to tissues. For example, in anesthetized rats, AcNT(8-13) inhibits edema formation after thermal injury to skin, after a knife cut to the abdominal muscle, and after freeze injury to the brain cortex (4, 5). AcNT(8-13) also reduced the fluid accumulation in the rat lung after intravenous injection of epinephrine (5).

Transcapillary fluid flux (J_v) is the product of the capillary filtration coefficient (CFC), the hydrostatic pressure (P), and the colloid osmotic pressure (COP) acting across the capillary wall (1). These parameters are interrelated in Eq. 1

(1)

where the subscripts "c" and "if" denote capillary and interstitial fluid pressures (P_if), respectively.  is the capillary reflection coefficient for proteins, and P is the net filtration pressure across the capillary wall. P_if is one of the pressures that controls transcapillary fluid transport. Transcapillary fluid flux is normally kept within narrow limits because of "autoregulation" by P_if and COP_if. Increased fluid filtration and thereby increased interstitial volume will raise P_if and reduce COP_if. These changes will in turn normalize net filtration pressure and hence counteract further filtration. This balance is maintained in normal conditions but undergoes rapid changes in acute inflammation. For example, thermal injury to human skin is accompanied within minutes by increased transvascular fluid flux and blister formation, yet the underlying processes for this phenomenon are not well understood. In earlier studies (13, 15), the egress of fluids into tissues during inflammation was solely attributed to the release of mediators that induced endothelial gap formation, thus increasing capillary permeability. Another mechanism identified in our laboratory is the lowering of P_if. P_if is one of the pressures participating in the control of transcapillary flux according to the Starling hypothesis. P_if normally counteracts accumulation of fluid in the tissue interstitium, but in the early phase of inflammation it becomes a driving pressure for the increased transport of fluid across the transcapillary wall and into the intestititum. Therefore, in our experiments, P_if was measured after the induction of circulatory arrest to avoid the accumulation of fluid in the interstitial space that occurs after tissue injury with intact circulation and thus allows registration of the lowering of P_if as observed in the initial phase after an inflammatory challenge. By using inflammation of tracheal tissue as experimental model, it was shown that increased fluid flux can double interstitial fluid volume within 10 min after initiation of the stimulus when the circulation is intact (12).

Studies from our laboratory (8, 24, 25) have demonstrated that electrical stimulation (ES) of the vagal nerve will lower P_if in the tracheal tissue. The normal slightly subatmospheric P_if becomes additionally negative after the onset of nerve stimulation, and this negativity accompanies the other signs of neurogenic inflammation, namely vasodilation, increased vascular permeability, and leakage of plasma protein, followed by accumulation of extracellular fluid, thus increasing the total tissue volume (14, 15). The lowering of P_if is the major hydrostatic pressure driving the rapid formation of edema that develops in this condition.

To characterize the factors that affect P_if, we examined pharmacological agents that reduce inflammatory edema in experimental models of acute tissue injury. These agents include the corticotropin-releasing hormone -trinositol, mystixins, NT, and dynorphin fragments (4, 7, 8, 21, 25). The aim of this study was to determine whether the mechanism responsible for the anti-inflammatory effect of AcNT(8-13) is linked to changes in P_if. The effect of AcNT(8-13) on the lowering of P_if in the rat trachea after ES of the vagal nerve was measured, as well as the effect of pretreatment with AcNT(8-13) on tissue imbibition of water in vitro. Part of the present study has been presented briefly elsewhere (6).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Female Wistar rats (200-250 g) were obtained from M & B (Ry, Denmark) or from Harland (Oxon, UK). Food was available ad libitum. Animals were anesthetized with pentobarbital sodium [initial dose of 50 mg/kg body wt (bw) ip and supplemental doses were administered when required]. A branch of the femoral vein was cannulated for injection of AcNT(8-13) (0.1, 1, 3.1, 10, or 31 µg/kg bw), sodium nitroprusside, or saline (0.9% NaCl), or for the injection of the radiolabeled tracers. Circulatory arrest was induced with 0.5 ml of saturated KCl injected intravenously. All experimental protocols were approved by, and performed in accordance with, the recommendations of the Norwegian State Commission for Laboratory Animals.

Measurements

Interstitial fluid pressure. After the induction of circulatory arrest with saturated KCl, an extrathoracic portion of the trachea was exposed and the muscle lying over the trachea was split longitudinally. The left vagal nerve was dissected free from the carotid artery, at the same level as the exposed trachea, and placed on a stimulating electrode. The tracheal surface and the vagal nerve were covered with mineral oil to avoid desiccation. The time required for the experimental preparation was 3-5 min. Measurement of P_if was initiated thereafter and continued until 60 min after cardiac arrest. P_if was measured with a sharpened glass pipette (4-10 µm) penetrating the frontal portion of the tracheal tissue between the cartilage rings. The pipette tip was inserted into the tissue from the external layer of the adventitia via the submucosa and extended as deep as to the mucosal layer, with registration of pressures in the submucosa, which is the largest layer in the frontal portion of the trachea. We performed the measurements without opening the trachea. The glass capillary was connected to a servocontrolled counterpressure system (22, 23), and the counterpressure created by the servocontrolled pump (model 201, Ling Dynamic Systems; Royston, UK) was recorded with a pressure transducer (model 1280C, Hewlett-Packard) connected to an amplifier and recorder (Gould Instruments; Ballainvilliers, France). The glass pipettes were filled with 0.5 M NaCl colored with Evans blue dye to visualize the tip of the pipette. Micropuncture was performed under visual guidance with a microscope (model M3C, Wild; Heerbrugg, Switzerland), and care was taken to minimize stretch or compression at the site of puncture (23). The measurements were accepted when the following criteria were met: 1) no change of recorded pressure when increasing feedback gain; 2) suction applied by the servocontrolled pump gave increased electrical resistance in the pipette; verifying an open communication to interstitial fluid because of the lower tonicity of the fluid entering the pipette; and 3) the baseline measurements before and after P_if registration were unchanged. The last baseline measurement was performed in a plastic cup filled with saline placed at the level of the site of puncture. All surgical manipulations on or near the trachea were performed postmortem to avoid local inflammation that may increase interstitial fluid volume and confound accurate estimates of P_if.

Arterial pressure. Arterial blood pressure (P_a) was measured before and during administration of the test substance and until circulatory arrest was induced with saturated KCl as part of the experimental protocol for the measurement of P_if. Blood pressure was monitored by a cannula placed in the femoral artery and connected to a pressure transducer (model 840-22, Sensonor) and a recorder (Gould).

Electrical stimulation. ES was performed with a stimulator (model S60, Grass; Quincy, MA) with these parameters: 20 V, 20 Hz, and 0.5 ms for a total of 5 min.

In vitro swelling of tracheal tissues. The animals were treated as before, with cannulation of the femoral vein and intravenous injection of AcNT(8-13) (10 or 31 µg/kg bw) or saline. After cardiac arrest, the tracheas were dissected out, split longitudinally, and placed in preweighed vials that were then sealed and reweighed. Each vial, containing one trachea, was then filled with 5 ml of phosphate-buffered saline (PBS; 0.15 M adjusted to pH 7.4). After being soaked for 2, 4, 8, and 24 h, the tracheas were removed from the vials, the surface fluid was wiped gently with a paper towel, and they were then weighed. After being weighed, the sample was returned to the vial. After the last weighing (24 h), samples were dried at 65°C to obtain tissue dry weight. Total tissue water (TTW) was then calculated for each time interval assuming a constant dry weight during the 24 h of immersion.

Albumin extravasation and TTW. Albumin extravasation (E_alb) and TTW were measured as the 25-min distribution volume of ¹²⁵I-labeled human serum albumin (¹²⁵I-HSA) and the accumulation of tissue water during the same period of time, respectively. The radiolabeled ¹²⁵I-HSA (0.05 MBq) (Institute for Energy Technique; Kjeller, Norway) was injected intravenously in 0.2 ml, followed immediately by AcNT(8-13) (3.1, 10, or 31 µg/kg bw) or intravenous saline. The injection volume was 0.1 ml/100 g bw plus 0.2 ml saline to flush the cannula. In some experiments, the vagus nerve was dissected free and stimulated 10 min after intravenous injection of AcNT(8-13) or vehicle (see Experimental Protocol). Five minutes before cardiac arrest and 20 min after AcNT(8-13) injection, the rats received an intravenous injection of 0.2 ml ¹³¹I-HSA (0.10 MBq) (Institute for Energy Technique; Kjeller, Norway). Blood samples (0.5-1.0 ml) were taken by cardiac puncture just before cardiac arrest. Samples of trachea were placed in preweighed vials, which were sealed immediately and reweighed. The radioactivity in the plasma and tissue samples was determined with a gamma counter (Wallac 1282, LKB; Turku, Finland) with automatic spillover and background subtraction. The samples were then dried at 65°C until a constant weight was attained and water content [TTW, in ml/g dry weight (dw)] was calculated as fresh weight (fw)  dw/dw

(2)

E_alb (in ml/g dw) was calculated as plasma equivalent space, i.e., counts per minute (cpm) of tracer per gram dry tissue weight divided by cpm per milliliter of plasma. The difference between the 25-min plasma equivalent space for ¹²⁵I-HSA and plasma volume is shown. Plasma volume (PV) corresponds to the plasma equivalent space for ¹³¹I-HSA

(3)

All calculations were referenced per gram dry tissue weight.

Solutions. AcNT(8-13) (mol mass 859), provided by Phoenix Pharmaceuticals (Belmont, CA) or by Bachem (Bubendorf, Switzerland), was made up to stock solution of 100 µg/ml prepared by dissolving AcNT(8-13) in 100 µl of 0.1 M acetic acid subsequently diluted to the required concentration with 0.9% NaCl. This solution or further dilutions with 0.9% NaCl were used for intravenous injections of AcNT(8-13) at 0.1 ml/100 g bw. Sodium nitroprusside (Merck) was made up to a solution of 100 µg/ml by dissolving the drug in 0.9% NaCl. The solution was used for injection at 0.01 ml · min¹ · 100 g bw¹ for a period of 10 min.

Experimental Protocol

Interstitial fluid pressure. AcNT(8-13) was administered intravenously at 0.1, 1, 3.1, 10, or 31 µg/kg bw, with the number of experiments equal to 8, 3, 4, 8, or 10 per dose, respectively. Cardiac arrest was induced 10 min after injection with intravenous saturated KCl at 0.5 to 1 ml, and measurement of P_if on the exposed trachea was commenced after the left vagal nerve was surgically isolated and placed in the electric stimulator. The time of circulation of the agent until induction of cardiac arrest is based on previous experiments (4). One or two recordings of P_if were obtained as control measurements, before sensory nerve stimulation. After nerve stimulation, repeated measurements of P_if were performed until 60 min after cardiac arrest. Two groups were used as controls, both groups consisting of vehicle-treated animals (0.1 ml/100 g bw 0.9% saline iv). The protocol for electrical nerve stimulation was used in the first group (n = 9) but not in the second group (n = 6). Finally, P_if was measured in sodium nitroprusside-treated rats (n = 4). Rats were given intravenous injections of sodium nitroprusside every 0.5 to 1 min for 10 min before cardiac arrest was induced. Measurement of P_if for this group and for the second control group was started immediately after exposure of the trachea and continued until 60 min after cardiac arrest.

In vitro swelling of tracheal tissues. The anesthetized animals were cannulated and pretreated with vehicle (n = 10) or AcNT(8-13) (10 or 31 µg/kg bw, n = 10), at an injection volume of 0.1 ml/100 g bw + 0.20 ml saline to flush the catheter. Cardiac arrest was induced 10 min later.

E_alb and TTW. The experiments were performed in two series with separate control groups and variable doses of AcNT(8-13). The first series consisted of three groups (n = 7 in each) pretreated either with AcNT(8-13) (3.1 or 10 µg/kg bw) or vehicle. The timing for the injection of the tracers was according to the following schedule: 0 min, ¹²⁵I-HSA and AcNT(8-13)/vehicle; 20 min, ¹³¹I-HSA; 25 min, blood sample and KCl. This series was performed to investigate whether 3.1 µg/kg and 10 µg/kg had an effect on TTW and E_alb. The second series consisted of four groups pretreated with intravenous AcNT(8-13) (31 µg/kg bw) or vehicle and further subdivided by absence or presence of ES: vehicle-ES (n = 7); AcNT(8-13)-ES (n = 7); vehicle + ES (n = 8); AcNT(8-13) + ES (n = 8). The timing for the injection of the tracers and ES was according to the following schedule: 0 min, ¹²⁵I-HSA and AcNT(8-13)/vehicle; 10 min, ES/-ES; 20 min, ¹³¹I-HSA; 25 min, blood sample and KCl. This series was performed to investigate whether the high TTW observed when tracheal tissue was allowed to swell was due to the high dose of AcNT(8-13) (31 µg/kg) and whether it had an effect on TTW and E_alb. Furthermore, we investigated whether this high dose was affecting the response to electrical nerve stimulation.

Statistical Analysis

	RESULTS

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Interstitial Fluid Pressure

View larger version (16K): [in this window] [in a new window]   Fig. 1.   N-acetyl neurotensin(8-13) [AcNT(8-13)] inhibits development of lowering interstitial fluid pressure (Pif) in the trachea of anesthetized rats after electrical stimulation of the vagus nerve. Values are means ± SD; n, no. of animals. * P < 0.05, using one-way analysis of variance (ANOVA) with subsequent Bonferroni's comparison and t-tests of Pif from before to after stimulation. ** P < 0.05, significant inhibition of fall in Pif looking at the changes in Pif [change in Pif = (Pif before stimulation)  (Pif after stimulation)] for the control group compared with the experimental groups using one-way ANOVA as described. bw, Body weight.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Pif for vehicle-treated animals (A) and AcNT(8-13) at 10 µg/kg bw (B) (n = 9 and n = 8, respectively). Each solid circle represents mean values for Pif from one animal, measured before and after electrical stimulation (stim) of the vagus nerve. The large standard deviation shown in Fig. 1 is due to 1 of the 8 animals in the AcNT(8-13)-treated group that did not respond to the treatment.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Pif in trachea (means ± SD) in control animals and animals treated with sodium nitroprusside. There is no statistical difference between the two groups, showing that the lowering of arterial pressure (Pa) induced during injection with sodium nitroprusside did not affect the Pif. The figure also shows that the levels of Pif are kept constant throughout the 60-min recording period.

Arterial Pressure

In Vitro Swelling of Tracheal Tissues

View larger version (15K): [in this window] [in a new window]   Fig. 4.   In vitro accumulation of fluid in the tracheal tissues of vehicle-treated animals was significantly increased when comparing the fresh weight data to all other periods in this group. There was no significant accumulation of fluid in the AcNT(8-13) at dose 10 µg/kg bw, indicating an inhibitory effect. For this group, there is a significant inhibitory effect of the swelling at 2 h [total tissue water at 2 h (TTW2h)] compared with the control group at the same time period. ** P > 0.05, using same ANOVA as above. AcNT(8-13) (31 µg/kg bw) induced a significant increase in fresh weight TTW (TTW0h) compared with the fresh weight TTW of the two other groups. dw, Dry weight. * P > 0.05; § P < 0.05, one-way ANOVA with subsequent Bonferroni and t-tests. Values are means ± SE.

E_alb and TTW

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Intravenous pretreatment with AcNT(8-13) at 3.1 and 10 µg/kg bw did not induce significant changes in the plasma equivalent space for 125I-labeled human serum albumin (125I-HSA), TTW, and albumin extravasation (Ealb) compared with the vehicle-treated controls. However, there was a significant increase in the Ealb measurements for 10 µg/kg bw, compared with the 3.1 µg/kg bw. P = 0.042, using Kruskal-Wallis one-way ANOVA on ranks with subsequent pairwise multiple comparison with Dunn's method, * P < 0.05. Values are means ± SD.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Treatment either with AcNT(8-13) at 31 µg/kg bw, electrical stimulation (ES) of the vagus nerve or a combination of both resulted in highly significant increase in plasma equivalent space for 125I-HSA, TTW, and Ealb with exception of 125I-HSA and Ealb after AcNT(8-13) (31 µg/kg bw). P < 0.0001, using one-way ANOVA with subsequent Bonferroni and t-tests. Intravenous pretreatment with AcNT (8-13) at 31 µg/kg bw in combination with ES of the vagus nerve induced a significant increase in all three parameters, compared with all the other treatments. * P < 0.05 using analysis as described above. Treatment with ES induced a significant increase in all three parameters compared with the control group. § P < 0.05 using the analysis as described above. All three treatments (31 µg/kg bw ES, 31 µg/kg bw +ES and vehicle +ES) induced a significant increase in TTW compared with the control group.  P < 0.05 using analysis as described above. Values are means ± SD.

	DISCUSSION

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In a previous study, we showed that AcNT(8-13) administered at 5-8 µg/kg bw intravenous to anesthetized rats 10 min before thermal injury to the paw, inhibited 65-75% of the volume of swelling that occurred 30 min later (5). The results here show that AcNT(8-13) inhibits the lowering of P_if, which develops during neurogenic inflammation in the trachea of rats. Pretreatment of the animals with AcNT(8-13) at 10 µg/kg bw also reduced the fluid accumulation of tissues placed in PBS, but at 31 µg/kg bw, AcNT(8-13) increased TTW and no imbibition of water occurred in vitro. The higher dose of AcNT(8-13) also potentiated TTW accumulation in the trachea both before and after ES of the vagus nerve because the values of water content in the tracheal tissue were markedly increased compared with both the vehicle-treated animals and the animals treated with AcNT(8-13) at 10 µg/kg. The highest dose of AcNT also induced a large increase in plasma albumin leakage when combined with the ES of the vagus nerve. These results provide a complex picture of how a peptide such as AcNT(8-13) modulates fluid dynamics in tissues during inflammation.

The ability of AcNT(8-13) at 10 µg/kg bw to suppress the development of negative P_if during inflammation provides an intuitive explanation of its actions in vivo and in vitro. AcNT may act on cellular receptors that control the tension of the extracellular matrix. A possible pathway would be the stimulation of the ₁-integrin receptor system, which appears to be a common and final step involved in generating a lowering of P_if because polyclonal and monoclonal antibodies to ₁- and ₂₁-integrins, respectively, decreased P_if (18-20). Thus we suggest that AcNT(8-13) acts via a cellular receptor, which in turn causes strengthening of the cytoskeleton, thus stabilizing the ₁-integrin function, but the exact process by which AcNT(8-13) may affect P_if and swelling via the integrin system and the intracellular pathway is unclear.

AcNT(8-13) at 10 and 31 µg/kg bw produces hypotension. A change in P_a may reduce the transmural driving forces for fluid and protein egress from the microcirculation via an effect on capillary pressure (P_c), as described in Eq. 1, and thereby may also affect P_if. ES of the vagus nerve also changes P_a, but this effect is not of interest in the current experiments because ES was imposed after cardiac arrest. Several facts, however, suggest that the effect of AcNT(8-13) on P_if is not determined by changes in P_a. First, P_if measurements after AcNT(8-13) and before nerve stimulation are the same as in the vehicle-treated animals, i.e., slightly subatmospheric. Second, we showed that lowering of P_a with sodium nitroprusside over a 60-min period did not have effects on P_if. Thus we conclude that the effects produced by AcNT(8-13) on P_if are not secondary to a fall in P_a.

We (8) have previously shown that tracheas from rats pretreated with the synthetic peptide mystixin-7 (20 µg/kg bw) reduced the swelling rate of the tracheal tissue placed in PBS. On the basis of this finding, we performed similar experiments with the two doses of AcNT(8-13) that inhibited the lowering of P_if, namely at doses of 10 and 31 µg/kg. Pretreatment with the lowest dose reduced the swelling, measured as a lower content of TTW, after 2 h of imbibition, compared with the vehicle-treated tissues and the water content in the tissue before the immersion in PBS was not increased. In contrast, the treatment with AcNT(8-13) at a dose of 31 µg/kg induced a large increase in TTW, indicating a proinflammatory effect of AcNT(8-13) at this dose. The increase in water preload was unexpected and so large that when the trachea was placed in the PBS solution the tissues in fact had a reduction in TTW content. There is no evidence that the tissue osmotic pressure was hypotonic compared with the PBS solution because the latter does not contain proteins. The observed preload could be explained by changes in the mechanical properties of the interstitial tissue in balancing the constraining force of the tissue, fibers and connective tissue cells, and the expanding forces (glycosaminoglycans). The water preload of 31 µg/kg bw may be sufficient to attenuate the generation of negative P_if during inflammation. The proinflammatory effect was not seen at lower doses of AcNT(8-13). Furthermore, the experiment with soaking of the tracheal tissue did not include the electrical nerve stimulation. Nevertheless, AcNT(8-13) inhibited the rate of swelling, indicating that this peptide could not exert its inhibitory effect by affecting the release of sensory neuropeptides. In light of this and in combination with the role of the ₁-integrin in controlling the tension of the extracellular matrix, there appears to be indications that the inhibitory effects of AcNT(8-13), both on lowering of P_if and swelling rate, are mediated via mechanisms which improve binding of the structures in the extracellular matrix.

We performed two series of experiments using the radiolabeled tracer technique measuring the ¹²⁵I-HSA, TTW, and E_alb at different doses of AcNT(8-13). The first series simply looked at these parameters following pretreatment with doses of 3.1 and 10 µg/kg bw compared with the vehicle-treated ones, to confirm that AcNT(8-13) at these doses did not increase plasma protein leakage or content of tissue water. The results clearly indicate that none of these treatments led to changes compared with the control group, except for the surprising observation that the plasma protein leakage measured as the 5-min albumin space, E_alb, was significantly reduced in the 3.1 µg/kg bw dose compared with the 10 µg/kg bw dose. The findings suggest that compared with the controls, AcNT(8-13) at the doses used here has no proinflammatory effect.

In the second series of experiments we used the radiolabeled tracer technique measuring the ¹²⁵I-HSA, TTW, and E_alb, and pretreatment with AcNT(8-13) at 31 µg/kg alone or in combination with ES of vagus nerve. The treatment with AcNT(8-13) at 31 µg/kg alone induced a significant increase in TTW as observed in the swelling experiments, but this treatment is not followed by increased protein leakage. The combination of the two treatments, 31 µg/kg AcNT(8-13) and ES of the vagus nerve, also induced an increase in TTW with responses comparable with the two other treatments alone and significantly increased compared with the vehicle-treated group. However, the protein leakage was increased threefold compared with the group with ES of the vagus nerve, thus demonstrating a synergistic response to protein extravasation for the combination of two treatments. The neurogenic inflammation response induced by ES releasing sensory neuropeptides is known to give increased protein leakage mainly created by the formation of gaps between the endothelial cells of the postcapillary venules by the neuropeptide substance P (15). Gully et al. (10) showed that the release of histamine is prevented by a selective NT receptor antagonist. This indicates that activation of this receptor by AcNT(8-13) could induce mast cell activation. It is therefore possible that the highest dose of AcNT(8-13), 31 µg/kg, is able to induce protein extravasation via mast cell activation while the lower doses of the peptide are not able to induce the same response. It is known that NT and NT fragments at higher doses release inflammatory mediators from mast cells (3, 10) and such release may contribute to the proinflammatory effect seen at AcNT(8-13) at 31 µg/kg bw. It should be noted, however, that AcNT(8-13) inhibits vascular leakage from blood vessels of the brain cortex and lung alveoli, tissues insensitive to inflammatory mediators such as histamine and bradykinin (5). Furthermore, we (8) observed a similar dose-dependent response in a recent study with the use of synthetic peptide mystixin-7. Unpublished observations (H. Strukova and E. T. Wei) indicate that the mystixin-7 peptide induced release of histamine from isolated rat peritoneal mast cells in vitro at doses of 0.1 µM. These observations may indicate that variable response to these peptides is based on a dose-dependent response to release of histamine from local mast cells. It is also known that some substances can induce both pro- and anti-inflammatory reactions depending on the route of administration (16), tissue, or species (2, 17). With the radiolabeled tracer technique, the proinflammatory effects were not observed at 10 µg/kg iv, a dose that inhibits lowering of P_if after nerve stimulation. At this lower dose, the ability of AcNT(8-13) to inhibit tissue imbibition of water, as measured by tissue weights, was also demonstrated.

Compliance, the relationship between the changes in the two parameters, interstitial fluid volume (IFV) and P_if, is important for the control of tissue fluid because it determines the level of counterpressure exerted by P_if concomitant with the increase in IFV. A proper determination of compliance requires a set of corresponding P_if and interstitial volume measurements. A change in tissue compliance can be measured if two different data sets have a clearly different relationship between these two parameters. Alternatively, if one parameter is constant whereas the other one changes, which is the case when P_if is lowered after vagal nerve stimulation when circulation is arrested, these must clearly be a change in the interstitial volume-pressure relationship but not necessarily the compliance (i.e., IFV/P_if). Our conclusion is that P_if is dependent on compliance, but more so under the present protocol, the acute changes are not a passive effect due to the relationship between P_if and IFV but rather the effect of AcNT on stabilizing the interstitial matrix. Thus the present data are conclusive in this regard and that determination of compliance after AcNT will not alter this conclusion.

The pathophysiological processes that underlie vascular leakage in acute inflammation remain a relatively unexplored topic in research. The interactions between pharmacological agents that suppress vascular leakage and affect P_if are now, however, better defined and may provide tools for further investigation of the molecular mechanisms in tissue stroma that generate the negative P_if.

In summary, we have characterized the actions of AcNT(8-13) on the physical forces that generate edema in the acute phase of inflammation. The receptive mechanisms of this peptide in the extracellular matrix may allow a more accurate description of the targets and cellular receptors that influences P_if and control edema formation.