Impaired Responsiveness to Angiotensin I1 in Experimental Cirrhosis: Role of Nitric Oxide

ANNA CASTRO,' WLADIMIRO JIMENEZ,~ JOAN CLhIA,' JOSEFA ROS,l JOSEP MARIA hbRTfNEZ,3 MARTA BOSCH,] VICENTE ARROYO,2 JAUME PIULATS,3 FRANCISCA RIVERA' AND JOAN RODES~

lHormona1 Laboratory and 'Liver Unit, Hospital Clinic i Provincial, University of Barcelona, Barcelona OS036; and 3Merck-IGODA Research Laboratory, Barcelona 0801 0, Spain

lmpaired vascular responsiveness to angiotensin I1 is a common feature in human cirrhosis with ascites. The aim of this study was to investigate whether vascular reactivity to angiotensin I1 is also decreased in rats with carbon tetrachloride-induced cirrhosis and ascites and to assess the role of endogenous nitric oxide in this abnormality. Increasing doses of angio- tensin I1 (from 31 to 600 ng-kg-l hmin-l) induced significantly smaller increases in total peripheral re- sistance in conscious cirrhotic rats with ascites (n = 8) than in control animals (n = 9) at each dose tested. A reduced response to angiotensin I1 was also observed in vitro in aortic rings of rats with cirrhosis and ascites compared with that in control aortic rings (masimal response: 104 -t 16 mgvs. 204 r?: 18 mg; p < 0.001). This in nitro hyporesponsiveness to angiotensin I1 in aortic rings of cirrhotic rats with ascites was reversed on endothelium denudation or nitric oxide synthesis in- hibition with No-nitro-L-arginine but was not influ- enced by cyclooxygenase inhibition with indo- methacin. In conclusion, this study shows reduced vascular reactivity to angiotensin I1 in carbon tetra- chloride-induced cirrhosis with ascites and indicates that this abnormality is mediated by nitric oxide. ( HEPATOLOGY 1993; 18367-372.)

Impairment of systemic hemodynamics is a common finding in advanced liver disease. Patients and experi- mental animals with cirrhosis and ascites have low arterial pressure, hypervolemia, high cardiac output and low peripheral vascular resistance. This hyperdynamic circulation is due to systemic arteriolar vasodilation and occurs despite marked stimulation of the renin-angio-

Received September 8, 1992; accepted March 9, 1993. Portions of this investigation were presented at the 27th Annual Meeting of

the European Association for the Study of the Liver, Vienna, August 26-29,1992. Address reprint requests to: Wladimiro Jimenez, Ph.D., Hormonal Labo-

ratory, Hospital Clinic i Provincial, Villarroel 170, Barcelona 08036, Spain. This work was supported by grants from Fondo Investigaciones Sanitarias

(91/0015) and Direccidn general Investigacion Cientifica y Tecnica (91-0216). J. Ros received a grant from the Fundaei6 Privada Clinic per a la Recerea Biomi?dica.

Copyright 0 1993 by the American Association for the Study of Liver Diseases.

0270-9139/93 $1.00 + .lo 31/1/47363

tensin and sympathetic nervous systems and nonos- motic hypersecretion of vasopressin (1-6).

Decreased reactivity to the vascular effect of angio- tensin I1 (MI), norepinephrine and vasopressin is another remarkable feature in human and experimental cirrhosis with ascites that has been proposed to play a major role in the impairment of circulatory function (7-13). Several mechanisms - including increased circu- lating levels of endogenous substances with vasodilator properties (141, receptor down-regulation (15) and post- receptor signaling defect in the vascular smooth muscle cells (12) -have been suggested for this phenomenon. Recent studies (16-18) in rats with CC1,-induced cir- rhosis suggest that activity of nitric oxide (NO), a powerful local vasodilator of endothelial origin (19,20), is augmented in cirrhosis. This study addresses the question of whether the reduction in vascular reactivity to MI in cirrhosis with ascites is linked to increased NO production.

MATERIAL AND METHODS The study included two protocols performed in male adult

Wistar rats with cirrhosis and ascites and in control Wistar rats. Both groups were fed ad libitum with standard chow and distilled water containing phenobarbital (0.3 gm * L-'). Cir- rhosis was induced with CCl,, which was administered by inhalation twice weekly (Monday and Friday) following a method described elsewhere (4,5). Cirrhotic rats were studied after ascites developed. Because previous investigations from our laboratory have shown that rats treated with CC1, exhibit ascites 9 to 12 wk after the start of the cirrhosis-induction program (5, 21), control rats were studied after 9 to 12 wk of phenobarbital administration.

Protocol 1: Hedynamic Studies. This protocol assessed the cardiovascular response to increasing doses of AII in conscious control rats and rats with CC1,-induced cirrhosis. Eight rats with cirrhosis and ascites and nine control rats were studied. Animals were anesthetized with ketamine (50 mg. kg-l) and fitted with PE-50 polyvinyl catheters (Becton Dickinson, Palo Alto, CA) in the left femoral artery and vein. The right jugular vein was also isolated, and a PE-50 catheter was placed in the right atrium. A thermocouple (Columbus Instruments, Columbus, OH) was advanced to the aortic arch by a left carotid approach to monitor intraarterial temperature during cardiac output (CO) measurement. Catheters were tunneled subcutaneously, brought out at the nape of the neck,

367

368 CASTRO ET AL. HEPATOLOGY August 1993

run through a flexible stainless steel sheath and connected to a swivel (Harvard Apparatus, South Patrick, MA). The sheath was attached to a harness made of polystyrene worn by the animal. The femoral artery catheter was connected to a highly sensitive transducer (Hewlett-Packard, Avondale, PA) and continuously perfused with Ringer's solution (0.1 ml - hr- ') by means of a continuous-flow system (Critiflo; Gould Inc, Oxnard, Ca) to maintain patency. Mean arterial pressure (MAP), heart rate (HR), CO and stroke volume (SV) were determined in a microcomputer system for CO determination (Cardiomax IIR; Columbus Instruments, Columbus, OH) and recorded in a multichannel system (MX4P and MT4; Lec- tromed Ltd., Jersey, Channel Islands, UK). CO was measured on thermodilution after administration of a 200-pl bolus of Ringer's solution (20" to 23" C) into the right atrium. This is a very accurate technique (coefficient of variability: 4.3% t 1.5%); its results closely correlate with results of electromagnetic flowmetry (22). In addition, it has the ad- vantage of allowing repeated measurements of CO in con- scious, unrestrained animals. A spring-loaded syringe was used (model CR-700-200; Hamilton Syringe, Reno, NV) to ensure a constant injection rate and volume. CO and SV indexes (CI and SVI) were calculated by dividing the original values by animal weight. Total peripheral resistance (TPR) was obtained with the formula TPR = MAP/CO. The total volume infused for AII administration and CO measurements never exceeded 1 m1/100 gm body wt during the 45-min study period. Right atrial pressure was not considered in the calculation of TPR. However, in a separate experiment no significant changes in right atrial pressure were observed in control rats (n = 5 ) or cirrhotic rats with ascites (n = 5 ) after AII administration at the doses used in this protocol (see below). Each value is the average of three measurements.

Animals were placed in rectangular cages, unrestrained, and allowed to recover from surgery and anesthesia for 24 hr. A blood sample (1 ml) was taken to measure plasma aldosterone concentration, plasma electrolytes and osmolality. Packed RBCs were reconstituted to an equal volume with Ringer's solution and reinfused over 3 min. After we obtained baseline MAP, HR, CO, SV and TPR measurements, animals received 5-min intravenous infusions of increasing doses of AII (31,62, 125, 250 and 500 ng * kg-' min-l). At each dose rats were allowed to equilibrate for 3 min; then hemodynamic measure- ments were repeated. This equilibration period is sufficient to achieve a new stable pressor response (23). Because rats were not allowed to recover after each dose, results represent responses to cumulative doses. At the end of the study a blood sample (2 ml) was obtained for measurement of plasma AII Concentration.

Protocol 2: Vascular Reactivity in Isolated Aortic Rings. This protocol assessed the in uitro vasoconstrictor effect of AII in aortic rings of control rats and rats with CC1,-induced cirrhosis and ascites. Experiments were performed in intact and endothelium-denuded aortic rings and in intact rings under conditions inhibiting NO synthesis with No-nitro-L- arginine (NNA). Animals were killed by decapitation, the thoracic aorta was dissected, placed in a Petri dish containing prewarmed (37" C) modified Krebs-Henseleit salt solution (in mmol/L: NaC1, 118; glucose, 11.1; HNaCO,, 24; KC1, 4.7; MgSO, 2.4; KH,PO,, 1.2; CaCl,, 1.6), cleansed of surrounding fatty tissue and cut into 5-mm rings. The rings were attached to metal frames and suspended in individual organ-bath chambers filled with 6 ml of modified Krebs-Henseleit solution. The salt solution was continuously bubbled with a mixture of 95% 0,-5% CO, and maintained at 37" C with an outer water jacket and circulating heat pump. Contractions were measured

with an isometric transducer (Biegestab k 30 type 351; Hugo Sachs Elektronik, March-Hugstetten, Germany), recorded in a multichannel polygraph (R-511; Beckman, Philadelphia, PA) and expressed as absolute values (milligrams). The rings were allowed to equilibrate for 45 min, with several adjustments, until a baseline force of 1.5 g was obtained. The integrity of the vessel was then assessed on examination of the maximal force developed to 6 x l o p 6 mol/L serotonin and by the determi- nation of the vasodilator effect of acetylcholine (lo-, mol/L). In preliminary experiments we found that 1.5 g of resting tension is optimal for expression of serotonin (6 x mol/L)-induced contraction of thoracic aortic rings obtained from control and cirrhotic rats.

Noncumulative dose-response curves to AII (lo-' to 3 x mol/L) were constructed in aortic rings from rats with cirrhosis and ascites and from control rats. MI was prepared fresh daily in distilled water and added to the organ bath in 60-pl volumes. After 5 min of exposure to the vasoconstrictor, the preparation was rinsed for 15 min or until the aortic ring regained its initial resting tension (this was done to avoid tachyphylaxis). The rings were then exposed to another dose of AII. Intact or endothelium-denuded aortic rings were studied. We removed the endothelium by gently rubbing the intimal surface with a small pair of forceps. In addition, we studied intact aortic rings of cirrhotic rats with ascites and control rats in the presence of the specific NO biosynthesis inhibitor NNA (3 x mom) or the cyclooxy- genase inhibitor indomethacin ( lop5 mol/L). In preliminary dose-response experiments this NNA concentration maximally inhibited the vasodilator effect of acetylcholine (3 x lo-, mol/L) in serotonin (6 x mol/L)-precontracted aortic rings. On the other hand, previous studies have shown that 10 -' mol/L indomethacin blocks prostaglandin synthesis in most vascular tissue (24) and in the aortas of normal rats (25). Aortic rings from at least six rats were studied in each experimental condition. From each dose-response curve we calculated the maximal response and the concentration of MI required for the 50% of the maximal contractile response using a curve-fitting program (INPLOT4; Graphpad, San Diego, CA). Values of R,, and EC,, were averaged; the results were used for statistical analysis. At the end of the experiment, we tested the functional integrity of the vessel by comparing the vasoconstrictor effect of serotonin (6 x mol/L) with the observed before the dose-response study.

Drugs. Acetylcholine, serotonin, NNA and human synthetic AII were purchased from Sigma Chemical Co. (St. Louis, MO). Indomethacin was supplied by Impex Quimica (Barcelona, Spain). In protocol 1, AII was dissolved at 1 and 10 pg * ml- in Ringer's solution. The 1 pg - ml-' solution was used for the three lowest infusion doses. The 10 p,g*ml-l solution was used for the two highest infusion doses. All drugs used in protocol 2 were dissolved in distilled water except indo- methacin, which was dissolved in Na,CO, (100 mrnom). Concentrations are expressed as final molar concentration in organ chambers.

Measurements and Statistical Analysis. Plasma osmolality was measured from osmometric depression of the freezing point (Osmometer 3MO; Advanced Instruments, Needham Heights, MA), and plasma sodium and potassium concentra- tions were determined on flame photometry (IL 943; Instm- mentation Laboratory, Lexington, MA). Plasma concentration of aldosterone was measured on RIA (Coat-A-Count Aldo- sterone; Diagnostic Products Corp., Los Angeles, CA). AII was extracted from plasma with cold ethanol and measured on RIA (Nichols Institut, Wijchen, The Netherlands).

Liver specimens were obtained from the middle lobe of each

HEPATOLOGY Vol. 18, No. 2, 1993 CASTRO ET AL. 369

TABLE 1. Baseline hernodynamic values in cirrhotic and control rats

Parameters Cirrhotic Control p Value" ~~~~~~

MAP (mm Hg) 109 -c 1 122 i 2 < 0.001 HR (beats * min- l) 391 c 20 404 * 13 NS CO (ml . min - 268 i 1 175 c 15 <0.001 CI (ml*min-'-100 gm-l) 59 * 4 39 -c 2 < 0.001 SVI (ml . lo3 1 100 gm-') 172 ? 16 97 c 4 <0.01 TPR (mm Hg . min ml- l) 0.41 -c 0.01 0.74 i 0.06 c 0.005

"Data calculated with Student's t test for unpaired data.

animal, fixed in 10% buffered formalin and stained with hematoxylin and eosin, reticulin and Masson's trichrome for histological examination.

Statistical analysis of results was performed with one-way ANOVA and paired and unpaired Student's t tests when appropriated. In accordance with the criteria of Meddings, Scott and Fick (261, R,, and EC,, in protocol 2 were calculated with a nonlinear regression method (27) with the software package previously indicated. Results are expressed as mean * S.E.M. and considered significant at a p value of 0.05 or less.

The protocols were performed according to the criteria of the Committee for the Care and Use of Laboratory Animals of the Hospital Clinic i Provincial.

RESULTS Histological examination of livers from all animals

treated with CC1, showed a finely granulated surface and the characteristic features of cirrhosis. All these animals also had ascites at the time of the study; volume ranged from 3 to 40 ml. Control rats had no appreciable alterations in hepatic histological appearance.

Protocol 1: Hemodynamic Studies. At the time of study, mean body weights were 457 & 15 gm and 447 t 19 gm in cirrhotic and control rats, respectively. Cirrhotic animals showed marked hyponatremia (135 & 3 vs. 144 & 2 mEq * L-l; p < 0.001), hypoosmo- lality (273 k 4 mOsm * kg-I vs. 286 2 3 mOsm * kg-l; p < 0.001) and hyperaldosteronism (118 2 21 ng * dl-I vs. 41 L 4 ng - d1-l; p < 0.001). Baseline MAP, HR, CO, CI, SVI and TPR values in cirrhotic and control rats included in this protocol are shown in Table 1. Compared with control animals, cirrhotic rats had a significantly reduced NIAP and TPR values and a significantly increased CO, CI and SVI values. No significant differ- ences were observed between cirrhotic and control animals with respect to HR.

Figure 1 shows the hemodynamic responses to AII in cirrhotic and control rats. Similar dose-dependent in- creases in MAP were observed in the two groups of animals (one-way ANOVA for both groups, p < 0.001). CI decreased significantly from preinfusion values only during the highest dose of MI. At this dose, CI fell to 56 2 4 ml * min-l. 100 gm-l in cirrhotic rats and to 36 & 2 ml - min-l- 100 g m - l in control rats (p < 0.05 and p < 0.025 vs. baseline values, respectively). The reduction in CI during administration of the highest dose of AII was associated with decreased HR during this period (344 +- 13 beats-min-l in cirrhotic rats and

361 & 10 beats-min-' in control rats). SVI did not change during the study. We saw no significant changes in TPR during administration of the two lowest doses of AII in the two groups of animals. However, a dose- dependent increase in TPR was observed with higher doses. Figure 2 shows that the absolute increase in TPR was significantly lower in cirrhotic rats than in control animals at each dose of AII given.

At the end of the study, plasma MI levels were not significantly different in cirrhotic (3.2 5 0.4 pg. m1-l) and control (3.0 2 0.3 p,g m1-l) rats.

Protocol 2: Vascular Reactivity in Isolated Aortic Rings. The contractile effect of increasing doses of AII on isolated aortic rings of cirrhotic and control rats is illustrated in Figure 3. AII administration was asso- ciated with an initial concentration-dependent increase in tension followed by a decline at the highest dose.

Marked differences in constrictor response between cirrhotic and control rings with intact endothelium were observed (Fig. 3A). The aortic rings of cirrhotic rats showed impaired reactivity to the vasoconstrictor effect of AII, as indicated by the approximate 50% reduction in response to the four highest concentrations of AIL Accordingly, the R,, was significantly lower in cirrhotic than in control aortic rings (Table 2). This difference cannot be attributed to distinct sensitivity to AII because aortic rings of cirrhotic and control rats showed similar EC,, values (Table 2).

Figure 3B and C shows the A11 concentration- response curves of control and cirrhotic aortic rings after endothelium removal and NO inhibition with NNA. Neither removal of endothelium nor the presence of the specific NO biosynthesis inhibitor NNA induced signif- icant changes in the constrictor effect of AII in control aortic rings. In contrast, these maneuvers were asso- ciated with significantly increased response to AII in aortic rings of cirrhotic rats (intact endothelium vs. endothelium-denuded; p < 0.001; intact endothelium vs. intact endothelium plus NNA: p < 0.001 [one-way ANOVA]). Under conditions of NO inhibition with NNA (Fig. 3C), the increase in tension induced by AII in cirrhotic rings was almost identical to that observed in control rings. Accordingly, no significant differences were observed in R,, between endothelium-denuded and NNA-treated control and cirrhotic aortic rings (Table 2).

The addition of the cyclooxygenase inhibitor indo- methacin did not modify hyporesponsiveness to AII in

370 CASTRO ET AL. HEPATOLOGY August 1993

MEAN ARTERIAL PRESSURE CARCXAC INDEX

16@l

m I

E

lo?

1.00

0.75 4

I- E

E -$ 0.50

E E

- c

0.25

8

Angiotcnsln I1 (ng.kg-'.min-') Anglotensin I1 (ng.kg-'.mln-')

TOTAL PERIPHERAL RESlSTANCE

T

b 31 62 155 250 560 Anglotensln I1 (ng.kg-'.min-')

FIG. 1. MAP, CI and TPR under basal conditions and after administration of increasing doses of angiotensin I1 in cirrhotic (*I and control (0) rats. a: p < 0.05, b: p < 0.01, c: p < 0.005 and d: p < 0.001 between cirrhotic and control rats (Student's t test for unpaired data).

35 T T

- m - L A

a L E a, E 25 a, 30!

-05' 31 62 125

Angiotensin I1 (ngb

250

-1, In- 1)

500

FIG. 2. Increase in TPR induced by angiotensin 11, with respect to baseline values, in cirrhotic (hatched bars) and control (open bars) rats. a: p < 0.05, b: p < 0.01 and c: p < 0.001 between cirrhotic and control rats (Student's t test for unpaired data).

cirrhotic vessels. In this condition R,, was markedly lower in cirrhotic than in control rings (Table 2).

DISCUSSION Since early investigations by Laragh et al. (7) and

Ames et al. (8) demonstrating impaired pressor response to A11 in patients with cirrhosis and ascites, many studies have been performed to assess vascular respon- siveness to vasoconstrictors in human and experimental cirrhosis. Experimental studies have been made in rats with CC1,-induced cirrhosis without ascites and in bile duct-ligated rats and dogs with cirrhosis with and without ascites, using AII, norepinephrine, serotonin, methoxamine, vasopressin and phenylephrine as vaso- constrictors (28). Vascular reactivity to these agents was assessed on the basis of changes in MAP. Although clear

resistance to the pressor effect of vasoconstrictors occurred in cirrhotic animals in some studies, normal pressor response was observed in other investigations. Differences between the models, vasoconstrictors, study design (conscious vs. anesthetized animals) and animal characteristics (ascitic vs. nonascitic) have been pro- posed to account for this variability in results.

In this study, vascular reactivity to AII was assessed on the basis of changes in MAF' and TPR. Pressor response to increasing doses of AII was similar in control and cirrhotic rats. However, the absolute increase in TPR induced by A11 was significantly lower in cirrhotic than in control rats at any given dose of the vasocon- strictor, indicating reduced vascular response to AII. This failure to demonstrate hyporesponsiveness to vasoconstrictors in terms of changes in MAP but not in TPR was also observed by Lee et al. (29) in a study assessing vascular response to methoxamine in control and portal vein-ligated rats. The absolute changes in MAP induced by methoxamine were similar in the two groups of animals. In contrast, the absolute increase in TPR was significantly lower in portal vein-ligated rats. Therefore TPR is a more sensitive parameter than arterial pressure in the assessment of vascular reactivity to vasoconstrictors in experimental portal hypertension and cirrhosis.

In protocol 2 of this study the in uitro pressor effect of AII was assessed in aortic rings of control and cirrhotic rats with ascites. This kind of experiment has the advantage over studies in intact animals that the vascular effect of homeostatic changes in endogenous vasoactive substances induced by pressor action of A11 are prevented. AII concentration-response curves in aortic rings of control and cirrhotic rats were qualita- tively similar, with an initial increase in tension followed by a decline at the highest dose. Our results parallel those found in previous studies and confirm that vascular desensitization to A11 occurs on reexposure of

HEPATOLOGY Vol. 18, No. 2, 1993 CASTRO ET AL. 371

.. Anglotensh I1 (H) Anglotensin I1 (M) Angiotensin I1 (M)

FIG. 3. Curves of dose response to angiotensin I1 in (A) intact, (B) endothelium-denuded and (C) intact, NNA pretreated aortic rings from cirrhotic (0) and control (0) rats. a: p < 0.05, b: p < 0.01, c: p < 0.005 and d: p < 0.001 between cirrhotic and control rats (Student's t test for unpaired data).

arterial vessels, with (30) and without endothelium, to high doses of this peptide (31). The decrease in response to the highest dose of AII is attributed to a receptor deactivation resulting from repeated exposure of the arterial vessel to the vasoconstrictor (32). Although the AII concentration-response curves were qualitatively similar, marked differences existed between control and cirrhotic rings with respect to intensity of the response. The force generated by AII was markedly lower in cirrhotic than in control rings. These findings indicate that vascular hyporesponsiveness to A11 in cirrhosis can also be detected in isolated vessels.

Endothelium removal was not associated with significant changes in response to AII in control aortic rings, although we noted a tendency toward a higher response in the vessels without endothelium. In con- trast, in cirrhotic aortic rings endothelium removal was associated with a marked increase in the response to AII such that the maximal response to A11 of endothelium- denuded cirrhotic aortic rings was similar to that of control aortic rings with and without endothelium. These data indicate that vascular hyporesponsiveness to A11 in cirrhotic aortic rings depends on the presence of endothelium and suggest that an endothelial factor is involved in the pathogenesis of this abnormality.

In this study we investigated the roles of prosta- cyclin and NO, the two most important endothelium- derived vasodilators known (20, 331, in the diminished vascular reactivity to AII of cirrhotic aortic rings with the cyclooxygenase inhibitor indomethacin and NNA, a potent inhibitor of NO synthesis. Prostaglandin inhi- bition was not associated with significant change in vascular response to AII in control or cirrhotic aortic rings. Nor did NO synthesis inhibition produce signif- icant changes in control aortic rings. In contrast, NO inhibition normalized the response to AII in cirrhotic aortic rings. These findings indicate that prostacyclin is not involved in the reduced sensitivity to MI in cirrhotic aortic rings and suggest that this abnormality is largely, if not solely, mediated by NO. This contention is in keeping with recent in uiuo studies suggesting increased activity of NO in rats with CC1,-induced cirrhosis with and without ascites (16-18).

TABLE 2. R,, and EC,, in cirrhotic and control aortic rings under the different experimental conditions

Intact rings Cirrhotic Control

Endo thelium- denuded rings Cirrhotic Control

to NNA Cirrhotic Control

Intact rings exposed to indomethacin Cirrhotic Control

Intact rings exposed

104 ? 16". 204 ? 18

261 f 31 251 t 26

238 t 23 276 f 33

144 t 24" 224 ? 26

3.1 t 0.7 5.4 t 0.9

4.5 i 1.4 2.9 t 0.7

6.7 t 0.9 9.4 ? 2.0

6.6 ? 2.0 4.9 t 1.0

"Data expressed as mean t S.E.M. 'p < 0.001 vs. control rings (Student's t test for unpaired data). cp < 0.05 vs. control rings (Student's t test for unpaired data).

In summary, this study demonstrates a reduced vasoconstrictor effect of AII in conscious cirrhotic rats with ascites and in aortic rings obtained from these animals. The in uitro hyporesponsiveness to A11 in this experimental model of cirrhosis was not influenced by indomethacin but was completely reversed after NO synthesis inhibition with NNA. These observations suggest that vascular resistance to AII in cirrhosis is mediated by NO.

Acknowledgment: We are indebted to Rosario Sanchez for skillfull technical assistance.

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