Contrasting pharmacological ETB receptor blockade with genetic ETB deficiency in renal responses to big ET-1

DAVID M. POLLOCK

Vascular Biology Center, Departments of Surgery, Physiology, and Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2500


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal clearance studies were conducted to determine the role of ETB receptors in the renal response to big endothelin-1 (big ET-1). Two series of experiments were conducted on Inactin-anesthetized rats to contrast acute pharmacological blockade of ETB receptors vs. genetic ETB receptor deficiency. In the first series, Sprague-Dawley rats were given either ETB-selective antagonist, A-192621, or vehicle (0.9% NaCl) prior to infusion of big ET-1 (10 pmol·kg-1·min-1) for 60 min. A-192621 significantly increased baseline mean arterial pressure (MAP; 102 ± 4 vs. 141 ± 6 mmHg, P < 0.05) and urine flow rate (0.5 ± 0.1 vs. 1.3 ± 0.2 µl/min, P < 0.05) without any effect on glomerular filtration rate (GFR) or effective renal plasma flow (ERPF). Big ET-1 significantly increased MAP in both groups but to a higher level in rats given antagonist (120 ± 6 vs. 169 ± 6 mmHg, P < 0.05). Big ET-1 increased urine flow in control rats but decreased in rats given antagonist. GFR and ERPF were decreased in rats given big ET-1, an effect that was exaggerated by ETB blockade. Another series of experiments examined the response to big ET-1 in rats lacking functional renal ETB receptors, known as spotting lethal (sl) rats. Surprisingly, rats heterozygous (sl/+) for ETB receptor deficiency had a significantly higher baseline MAP compared with homozygous (sl/sl) rats (134 ± 6 vs. 112 ± 7 mmHg, P < 0.05), although other variables were similar. Big ET-1 produced no significant change in MAP in either group. Urine flow, GFR, and ERPF were significantly decreased in both groups, although these changes were much larger in sl/sl rats. These experiments indicate that the ETB receptor plays an important role in limiting the renal hemodynamic response to big ET-1. Furthermore, the diuretic actions of big ET-1 require a functional ETB receptor.

endothelin; blood pressure; renal hemodynamics; water-electrolyte balance


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ENDOTHELIN ETB receptors have a multiplicity of functions due, in part, to their location on a variety of cell types and tissue-specific expression. Originally, ETB receptors identified on the vascular endothelium were shown to produce vasodilation through release of nitric oxide and prostaglandins (17). ETB receptors are also located on prostatic stroma, melanocytes, astrocytes, and enteric neurons (10). However, the highest concentration of ETB receptors is on tubular epithelium of the renal medulla (6, 8). In vitro studies have shown that the ETB receptor mediates the effect of endothelin-1 (ET-1) to inhibit ion and water transport in various tubular segments found within the renal medulla (7, 9). In addition, it has been proposed that ETB receptor activation may produce vasodilation in the renal medullary circulation (4). Hoffman and colleagues (5) recently observed that the diuretic and natriuretic response to the ET-1 precursor, big ET-1, can be inhibited by ETB receptor blockade.

The spotting lethal (sl) rat carries a naturally occurring deletion of the ETB receptor gene that renders it nonfunctional (1, 3). Gariepy and colleagues (2) have recently developed an animal model in which tissue-specific ETB receptor expression prevents the lethal aganglionic megacolon that develops, yet for the most part, the animals remain ETB deficient.

The purpose of the present investigation was to further elucidate the role of ETB receptors in the renal response to the ET-1 precursor, big ET-1. Two series of renal clearance experiments were conducted to compare the renal response to big ET-1 during acute pharmacological blockade of ETB receptors with the ETB-selective antagonist, A-192621 (18), compared with the response in rats genetically deficient of ETB receptor expression. We have previously observed that the diuretic and natriuretic response to big ET-1 was greater than ET-1 in Sprague-Dawley rats (14, 15). In addition, we have shown that all of the cardiovascular and renal effects of big ET-1 require conversion to ET-1 (11, 13). One can reason that since ET-1 functions as an autocrine or paracrine fashion, the sites of conversion are more likely to represent sites of biologically relevant effects. Therefore, the present studies examined the response to big ET-1 rather than ET-1.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two series of experiments were conducted. The first series used male Sprague-Dawley rats (200–250 g) from Harlan Laboratories (Indianapolis, IN). In the second series, male rats (200–250 g) that are either homozygous (sl/sl) or heterozygous (sl/+) for a deficiency of the ETB receptor were obtained from our local breeding colony.

All rats were subjected to a similar surgical preparation. Rats were anesthetized with pentobarbital sodium (65 mg/kg ip; Abbott Laboratories, North Chicago, IL) and placed on a servo-controlled heating table to maintain rectal temperature constant at 37°C. The airway was maintained clear by placing a catheter (PE-205) in the trachea. A catheter (PE-50) was placed in the right jugular vein for infusion of A-192621 and big ET-1. The right femoral vein was catheterized (PE-50) for infusion of saline containing [3H]inulin (4 µCi·h-1·100 g-1; Amersham Pharmacia Biotech, Arlington Heights, IL) and 14C-labeled p-aminohippurate ([14C]PAH; 10 µCi·h-1·100 g-1; New England Nuclear, Boston, MA). The right femoral artery was catheterized (PE-50) for monitoring mean arterial pressure (MAP) using a MacLab data acquisition system (ADInstruments, Milford, MA). Another catheter (PE-90) was also placed into the bladder to allow urine collection. Immediately following surgery, intravenous infusion of saline with isotopes was initiated at a rate of 10 µl/min and was continued throughout the experiment.

In Sprague-Dawley rats, a 60-min equilibration period was followed by administration of either the ETB receptor antagonist, A-192621 (30 mg/kg; n = 9), or vehicle (0.9% NaCl; n = 8), given as a bolus (iv). A-192621 has been previously shown to be potent and selective for the ETB receptor compared with the ETA receptor (IC50 = 8.2 µM for ETA and 6.4 nM for ETB) with a functional half-life of over 6 h (18). Fifteen minutes after A-192621 injection, a baseline clearance period (30 min) was begun that included a timed urine collection with a blood sample taken at the midpoint of the period. This was followed immediately by a continuous infusion of big ET-1 at 10 pmol·kg-1·min-1 iv that continued for 1 h. Two additional clearance periods were obtained during big ET-1 infusion. At the end of the experiment, the kidneys were removed, decapsulated, and weighed. In the sl/sl and sl/+ rats, the renal clearance periods were identical, but without any A-192621 treatment (n = 10 and 9, respectively).

Glomerular filtration rate (GFR) was determined from the clearance of [3H]inulin, and effective renal plasma flow (ERPF) was equated with the clearance of [14C]PAH. Urine sodium concentrations were measured using ion-selective electrodes (EL-ISE; Beckman Instruments, Brea, CA). ANOVA for repeated measures was used along with means comparison contrasts to compare individual means between groups during the difference time periods (SuperANOVA; Abacus Concepts, Berkeley, CA). Values are means ± SE, with P < 0.05 being considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Sprague-Dawley rats, the ETB receptor antagonist, A-192621, given prior to big ET-1 significantly increased MAP compared with rats treated with vehicle (Fig. 1). Subsequent infusion of big ET-1 significantly increased MAP in both groups, although the increase during the first 30-min period was significant only in the A-192621 group. Consistent with a pressure diuresis, urine flow rate was also significantly greater in the A-192621 group compared with controls. In rats treated with vehicle, big ET-1 significantly increased urine flow, whereas the opposite occurred in rats pretreated with A-192621. Sodium excretion changed in response to big ET-1 in a pattern similar to urine flow rate. Big ET-1 produced a significant increase in sodium excretion in rats treated with vehicle from 0.39 ± 0.22 to 1.44 ± 0.54 meq/min (P < 0.001). In contrast, sodium excretion was not significantly changed in response to big ET-1 in rats treated with A-192621 (0.41 ± 0.13 meq/min before and 0.32 ± 0.11 meq/min during big ET-1).



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Fig. 1. Effect of big endothelin-1 (big ET-1) on mean arterial pressure (MAP) and urine flow rate in Sprague-Dawley rats treated with the ETB receptor antagonist, A-192621 (n = 9), or vehicle (n = 8). Following a control period (C), big ET-1 was infused during two 30-min clearance periods (E1 and E2). *P < 0.05 compared with the control period. {dagger}P < 0.05 compared with vehicle rats.

 
GFR and ERPF were similar between Sprague-Dawley rats treated with A-192621 and vehicle (Fig. 2). Big ET-1 significantly decreased GFR and ERPF in both groups, but the decrease was significantly greater in rats given A-192621. Hematocrit was not significantly different between the two groups during the control period (Fig. 3). Big ET-1 significantly increased hematocrit in the group treated with A-192621. Big ET-1 infusion tended to increase hematocrit in the vehicle-treated group, but this was not statistically significant.



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Fig. 2. Effect of big ET-1 on glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) in Sprague-Dawley rats treated with the ETB receptor antagonist, A-192621, or vehicle. Following a control period (C), big ET-1 was infused during two 30-min clearance periods (E1 and E2). *P < 0.05 compared with the control period. {dagger}P < 0.05 compared with vehicle rats.

 


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Fig. 3. Effect of big ET-1 on hematocrit in Sprague-Dawley rats treated with the ETB receptor antagonist, A-192621, or vehicle (A) and in rats homozygous and heterozygous for ETB receptor deficiency (B). Following a control period (C), big ET-1 was infused during two 30-min clearance periods (E1 and E2). *P < 0.05 compared with the control period. {dagger}P < 0.05 compared with vehicle rats.

 
The responses of rats genetically deficient of ETB receptors to big ET-1 were similar in most but not all respects compared with normal rats with pharmacological blockade of the ETB receptor. MAP was greater in heterozygous ETB-deficient rats (sl/+) compared with those with homozygous deficiency (sl/sl) (Fig. 4). The reason for this difference is unclear. Big ET-1 increased MAP in sl/+, but this increase was of borderline significance (P = 0.059). There were no significant changes in MAP in sl/sl rats although there was a tendency to increase in the first 30-min period. Urine flow rate was similar between the two groups during the control period. Big ET-1 significantly decreased urine flow rate in both groups, but the decrease was significantly greater in sl/sl rats. For reasons that are not known, sodium concentrations in the urine from sl/sl and sl/+ rats were often below the detection level of our instrumentation. Therefore, sodium excretion rates could not be determined in a sufficient number of rats to obtain statistically relevant data. In sl/+ rats, sodium excretion was 0.11 ± 0.01 before and 0.32 ± 0.13 meq/min during big ET-1 infusion (n = 3) and was 0.09 ± 0.02 before and 0.08 ± 0.03 meq/min during big ET-1 in sl/sl rats (n = 4).



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Fig. 4. Effect of big ET-1 on MAP and urine flow rate in rats homozygous (n = 10) and heterozygous (n = 9) for ETB receptor deficiency. Following a control period (C), big ET-1 was infused during two 30-min clearance periods (E1 and E2). *P < 0.05 compared with the control period. §P = 0.059 compared with the control period. {dagger}P < 0.05 compared with heterozygous rats.

 
There was no significant difference between sl/sl and sl/+ rats during the control period for either GFR or ERPF (Fig. 5). Big ET-1 significantly decreased both GFR and ERPF in both groups of rats, with the decrease being significantly greater in rats with the complete deficiency. Similar to findings with pharmacological blockade, hematocrit was increased in rats without a functional ETB receptor, sl/sl, but not in sl/+ rats (Fig. 3).



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Fig. 5. Effect of big ET-1 on GFR and ERPF in rats homozygous and heterozygous for ETB receptor deficiency. Following a control period (C), big ET-1 was infused during two 30-min clearance periods (E1 and E2). *P < 0.05 compared with the control period. {dagger}P < 0.05 compared with heterozygous rats.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These studies demonstrate that the renal response to big ET-1 in rats with a genetic deficiency in a functional ETB receptor were qualitatively similar to the response in normal rats with pharmacological ETB receptor blockade. The similarities include an enhanced renal vasoconstrictor response to big ET-1 and prevention of the diuretic response. These studies provide further support for the hypothesis that the ETB receptor mediates the diuretic response following big ET-1 administration and that the ETB receptor functions to oppose the vasoconstrictor actions of ET-1 mediated via the ETA receptor. It is important to note that all of the effects of big ET-1 require conversion of big ET-1 to ET-1, since the renal vasoconstrictor, diuretic, and natriuretic effects of big ET-1 are blocked by the endothelin converting enzyme inhibitor, phosphoramidon (11, 13). Although this has not been confirmed in the ETB-deficient model, there is no compelling evidence to indicate actions of big ET-1 independent of conversion.

Our findings in Sprague-Dawley rats examining the effects of A-192621 on the response to big ET-1 confirm those from similar experiments by Hoffman et al. (5) in Wistar rats. In both studies, acute treatment with A-192621 allowed for a greater decrease in GFR and ERPF produced by big ET-1. The enhanced vasoconstriction is most likely due to enhanced ETA receptor activation, since blockade of ETB receptors increases plasma ET-1 levels, thus allowing more ET-1 to be available for the ETA receptor (18). Also, we have previously demonstrated that the constrictor actions of big ET-1 are mediated by ETA receptors in the kidney (15). In the current study, rats treated with the ETB receptor antagonist had significantly higher basal arterial pressure compared with vehicle-treated controls, whereas Hoffman et al. (5) did not observe any effect of A-192621 on arterial pressure. The reason for this discrepancy is not clear, but our findings suggest that ETB receptors may be important in maintaining basal arterial pressure in this strain of rats under the conditions of our experiments. Other investigators have reported that A-192621 has no acute effect on basal arterial pressure when given orally to conscious rats (18).

Without ETB receptor blockade, big ET-1 produces a diuretic and natriuretic response despite a concomitant and significant decline in GFR in normal Sprague-Dawley rats (15). During administration of an ETB antagonist, however, urine flow rate and sodium excretion decreased in response to big ET-1, and this did not change in a similar study by Hoffman et al. (5). The decline in urine flow and sodium excretion is probably due to blocking the renal tubular effects of big ET-1, but are also exaggerated by the larger decreases in GFR and ERPF. In a previous report, we demonstrated that ETA receptor blockade could completely prevent the increase in MAP and the decrease in GFR and ERPF produced by big ET-1, yet the diuretic actions were unaffected (15). Taken together, these findings provide further evidence that the diuretic responses to big ET-1 are mediated by the ETB receptor.

Our studies in homozygous and heterozygous ETB-deficient rats are the first to examine renal function in this model. A somewhat surprising result is that the heterozygous rats had higher basal MAP compared with homozygous. This difference was not observed in conscious rats when MAP was measured by telemetry (unpublished observations) or by chronically implanted catheters (2), so this would appear to represent a differential response to anesthesia. The mechanism responsible for this observation is not known. Although all other variables were similar between heterozygous and homozygous rats during the control period, GFR and ERPF in both of these groups were lower than in Sprague-Dawley rats. The reason for these clear strain differences is not readily apparent from these studies. Since ETB-deficient rats are derived from a mixture of the spotting lethal and Wistar strains, it is not known whether this apparent difference in renal functional parameters is related to the ETB deficiency or genetic variability between strains.

An unexpected observation, which contrasts with the pharmacological experiments, was that big ET-1 did not significantly increase MAP in the homozygous rats. Although speculative at this point, the lack of a significant pressor response in homozygous rats may be due to intense ETA-mediated vasoconstriction unopposed by ETB-mediated vasodilation such that cardiac output could not be sustained. There was a tendency for MAP to increase during the first 30 min of infusion of big ET-1, but appeared to drop again in the second half of the 60-min infusion period. There were a few individual animals that had large sustained increases in MAP, while others began to increase but then declined precipitously. The latter pattern is not too unlike that observed with extremely high doses of ET-1 or big ET-1, where the vasoconstriction is so intense that cardiac output is severely reduced (12). In a recent study, we observed a dose-dependent pressor response to bolus injections of ET-1 that was exaggerated in homozygous rats (16). At the highest doses, we observed short-lived increases in MAP which are followed by a gradual and persistent decline to the point of vascular collapse. The exaggerated vasoconstrictor response in homozygous rats in the current study is clearly indicated by the severe decreases in ERPF and GFR compared with more modest changes observed in the heterozygous rats.

Another difference between the genetically altered rats and the Sprague-Dawley strain was that the former did not exhibit a diuretic response to big ET-1, and in fact, both heterozygous and homozygous ETB-deficient rats had a significant decrease in urine flow rate. Based on the pharmacological studies, one could predict that big ET-1 would have produced a diuretic response in the heterozygous rats. However, it is important to consider that the heterozygous rats may express significantly less ETB receptor compared with normal strains and that this partial deficiency may negate ETB-dependent diuresis. There is in vitro evidence that ETB receptors antagonize the actions of antidiuretic hormone in the kidney that contribute to the diuretic response to endothelin peptides.

In summary, experiments with both pharmacological ETB receptor blockade and genetic deficiency for the ETB receptor demonstrated that the ETB receptor plays an important role in limiting the renal hemodynamic response to big ET-1. Furthermore, these studies provide additional in vivo evidence that the diuretic actions of big ET-1 require a functional ETB receptor.


    ACKNOWLEDGMENTS
 
I thank Vera Portik-Dobos and Deborah Garner for expert technical assistance. I also thank Drs. Cheryl Gariepy and Masashi Yanagisawa for supplying ETB-deficient breeder rats for our colony. Additional thanks go to Dr. Jerry L. Wessale of Abbott Laboratories for supplying A-192621.

These studies were supported by National Heart, Lung, and Blood Institute Grants HL-60653 and HL-64776 and by an American Heart Association Scientist Development Grant.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. M. Pollock, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500 (E-mail: dpollock{at}mail.mcg.edu).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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