20-HETE Requires Increased Vascular Tone to Constrict Rabbit Afferent Arterioles
Abstract Renal production of 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450–dependent arachidonate metabolite, increases during development of hypertension in spontaneously hypertensive rats, and inhibition of its production prevents hypertension. Since 20-HETE is a potent vasoconstrictor, these findings suggest that 20-HETE may contribute to the development of hypertension by elevating renal vascular resistance. In this study we examined the direct action of 20-HETE on the afferent arteriole, a vascular segment crucial to the control of renal vascular resistance. Rabbit afferent arterioles were microperfused at 60 mm Hg in vitro, and 20-HETE was added to the lumen. Although 20-HETE (10−10 to 10−6 mol/L) had no effect on the diameter of nontreated afferent arterioles (n=6), it caused dose-dependent constriction when vascular tone was increased with norepinephrine (0.3 μmol/L); 20-HETE at 10−6 mol/L decreased diameter by 43±4% (n=6, P<.001). This constriction was abolished by disrupting the endothelium (n=5). Moreover, pretreatment with the cyclooxygenase inhibitor indomethacin (50 μmol/L) or the thromboxane/endoperoxide receptor antagonist SQ29548 (1 μmol/L) significantly (P<.03) attenuated 20-HETE–induced constriction: 20-HETE at 10−6 mol/L constricted norepinephrine-treated afferent arterioles by 28±3% (n=6) and 25±4% (n=5), respectively. These results demonstrate that an increase in afferent arteriolar tone is required for the vasoconstrictor action of 20-HETE, which is partly mediated by the endothelial cyclooxygenase pathway. Thus, increased production of 20-HETE in the kidney and increase in afferent arteriolar tone, both of which often precede the development of hypertension, may synergistically contribute to the development of hypertension by elevating renal vascular resistance.
Renal cytochrome P-450–dependent AA metabolism to 20-HETE increases concomitantly with the development of hypertension in SHR,1 2 3 the animal model for human essential hypertension; whereas depletion of renal cytochrome P-450 prevents the development of hypertension in SHR.2 4 Since 20-HETE is a potent vasoconstrictor5 6 7 and is the primary metabolite of AA in the renal cortex,1 8 9 10 including preglomerular microvessels,11 these findings suggest that 20-HETE produced in the kidney may play an important role in the pathogenesis of hypertension by elevating RVR. Thus, it becomes important to study the effect of 20-HETE on RVR to understand the pathophysiology of hypertension. Although 20-HETE constricts dog renal arcuate arteries6 and indirectly affects RVR through tubuloglomerular feedback12 and the myogenic response of renal arcuate arteries,13 its direct action on the preglomerular Af-Art, a crucial vascular segment to the control of RVR, is not well understood.
The purpose of the present study was to examine the direct action of 20-HETE on the Af-Art in the absence of confounding systemic hemodynamic and neurohormonal influences. We microdissected and perfused rabbit Af-Arts in vitro and studied the effect of 20-HETE on the luminal diameter of Af-Arts. Since the vascular tone of Af-Arts increases early in the development of hypertension,14 we examined the action of 20-HETE in Af-Arts with and without increased basal vascular tone. In addition, since the vasoconstrictor response to 20-HETE in rat aortic rings is endothelium and cyclooxygenase dependent,5 we examined the effects of endothelium disruption and indomethacin (a cyclooxygenase inhibitor) or SQ29548 (a thromboxane/endoperoxide receptor antagonist) on the action of 20-HETE in Af-Arts.
Drugs and Chemicals
We obtained medium 199 from GIBCO; BSA, NE, L-NAME, guinea pig complement, acetylcholine, indomethacin, and SQ29548 from Sigma Chemical Co; 20-HETE and U46619 from Cayman Chemical Co; and antibody against human factor VIII–related antigen from Atlantic Antibodies.
Isolation and Microperfusion of the Rabbit Af-Art
This study was performed in accordance with the Guide for Animal Experimentation, Tohoku University School of Medicine. We used methods described previously to isolate and microperfuse Af-Arts.15 16 17 Briefly, young male New Zealand White rabbits (1.5 to 2.0 kg) fed standard rabbit chow and tap water ad libitum were anesthetized with sodium pentobarbital (40 mg/kg IV), and their kidneys were removed. From each rabbit, a single superficial Af-Art with its glomerulus (without macula densa) intact was microdissected under a stereomicroscope (model SZH-10, Olympus). Using a micropipette, we transferred the Af-Art to a temperature-regulated chamber mounted on an inverted microscope (model IMT-2, Olympus). The Af-Art was then cannulated with an array of glass pipettes and perfused at 60 mm Hg throughout the experiments with oxygenated medium 199 containing 5% BSA (M199/5% BSA). The bath (M199/0.1% BSA) was exchanged continuously. Microdissection and cannulation of the Af-Art were completed within 90 minutes at 8°C, after which the bath was gradually warmed to 37°C. Once the temperature was stable, a 30-minute equilibration period was allowed before any measurements were taken. Images of Af-Arts were displayed and recorded with a video system consisting of a camera (model CS520MD, Olympus), monitor (model PVM1445MD, Sony), and videotape recorder (model HR-S101, Victor). The diameter at the most responsive point to 20-HETE was measured with a video micrometer (model VM-30, Olympus).
Protocol 1: Effect of 20-HETE
After the 30-minute equilibration period, increasing doses of 20-HETE (10−10 to 10−6 mol/L) were added to the perfusate. We put 20-HETE only into the lumen; since 20-HETE is produced in renal microvessels11 it may act as an autocrine/paracrine factor. Luminal diameter was measured immediately before addition of 20-HETE and observed for at least 10 minutes at each dose.
Protocol 2: Effect of Increasing Basal Tone on the Action of 20-HETE
After the equilibration period, Af-Arts were preconstricted by about 20% with either NE (0.3 μmol/L) added to the bath or the NO synthesis inhibitor L-NAME18 (100 μmol/L) added to the perfusate. Thirty minutes later, we examined the effect of 20-HETE as in protocol 1 in the presence of NE or L-NAME. We previously showed that L-NAME at this concentration blocks acetylcholine-induced vasodilation in Af-Arts.19 We used NE or L-NAME to preconstrict Af-Arts because unlike angiotensin II (which causes transient and segmental constriction in our preparation19 ), they both cause persistent constriction along the entire arteriole.17 19
Protocol 3: Effect of Endothelial Disruption on the Action of 20-HETE
We found that increased basal tone is required for the vasoconstrictor action of 20-HETE in the Af-Art (see “Results”). To examine the possible contribution of the endothelium to 20-HETE–induced constriction, we studied the effect of endothelial disruption on the action of 20-HETE in NE-treated Af-Arts. After the equilibration period, Af-Arts were perfused for 10 minutes with M199/5% BSA containing both antibodies against human factor VIII–related antigen (14.29 μg/mL) and 2% guinea pig complements. This was followed by a 20-minute washout period during which Af-Arts were perfused with M199/5%BSA containing neither antibodies nor complements. We have previously demonstrated that this treatment selectively disrupts endothelial cells without altering vascular smooth muscle cells.20 After endothelial disruption, Af-Arts were constricted with NE (0.3 μmol/L), and the effect of 20-HETE was examined as in protocol 2. At the end of each experiment, we confirmed that Af-Arts did not dilate in response to acetylcholine (10 μmol/L).
Protocol 4: Effect of Cyclooxygenase Inhibition or Thromboxane/Endoperoxide Receptor Blockade on the Vasoconstrictor Action of 20-HETE
To examine whether the vasoconstrictor action of 20-HETE is dependent on cyclooxygenase activity, we studied the effects of indomethacin or SQ29548 on the vasoconstrictor action of 20-HETE in NE-treated Af-Arts. After the equilibration period, indomethacin (50 μmol/L) or SQ29548 (1 μmol/L) was added to the bath and perfusate. Thirty minutes later, Af-Arts were constricted with NE (0.3 μmol/L), and the effect of 20-HETE was examined as in protocol 2. We have previously shown that this dose of indomethacin blocks the effect of AA (10−4 mol/L) on renin release in rabbit Af-Arts.21 In addition, we confirmed the efficacy of SQ29548 to block the thromboxane/endoperoxide receptor by examining its effect on the vasoconstrictor action of the thromboxane/endoperoxide mimetic U46619 (10 nmol/L); SQ29548 abolished U46619-induced constriction in Af-Arts (n=3; 24±4% without SQ29548 versus 3±2% with SQ29548).
Values are expressed as mean±SEM, and all statistical analyses were performed using absolute values. A paired t test was used to examine whether the diameter at a given concentration differed from the control value or preconstricted value within each group. When more than one comparison was made, Bonferroni’s multiple comparison adjustment was used to reduce the significance level from .05 to .01 (.05/5; Bonferroni’s adjustment for five doses). Repeated measures ANOVA was used to examine whether the change in diameter at a given concentration differed between groups. For this, a value of P<.05 was considered significant.
Protocol 1: Effect of 20-HETE
Basal luminal diameter of Af-Arts was 17.4±0.3 μm (n=6), which was not altered by 20-HETE at 10−10 to 10−6 mol/L (16.6±0.4 μm at 10−6 mol/L; Fig 1⇓).
Protocol 2: Effect of Increasing Basal Tone on the Vasoconstrictor Action of 20-HETE
NE at 0.3 μmol/L decreased the diameter of the Af-Arts by 20±2%, from 16.7±0.4 to 13.4±0.5 μm (n=6). In contrast to Af-Arts without preconstriction, 20-HETE caused dose-dependent constriction in NE-pretreated Af-Arts, with the decrease in diameter becoming 2.5±0.6 μm (18±5%) at 10−9 mol/L (P<.01) and 5.8±0.5 μm (43±4%) at 10−6 mol/L (Fig 1⇑). To exclude the possibility that the vasoconstrictor action of 20-HETE was unmasked due to its specific interaction with NE rather than increased basal vascular tone, we next examined the effect of L-NAME, which increases vascular tone by inhibiting basal NO synthesis.15 Pretreatment with L-NAME at 100 μmol/L decreased the diameter of the Af-Arts by 20±3%, from 17.3±0.5 to 13.8±0.5 μm (n=5), which was not different from the decrease in diameter induced by NE. In L-NAME–pretreated Af-Arts, 20-HETE caused similar dose-dependent constriction, with the decrease in diameter becoming 2.3±0.4 μm (17±2%) at 10−9 mol/L (P<.01) and 5.6±0.6 μm (40±4%) at 10−6 mol/L (Fig 1⇑). There was no difference in the 20-HETE–induced constriction between NE- and L-NAME–pretreated Af-Arts, demonstrating that an increase in basal vascular tone is required for the vasoconstrictor action of 20-HETE in the Af-Art. In addition, these results demonstrate that NO does not modulate the vasoconstrictor action of 20-HETE, since there was no augmentation in 20-HETE–induced constriction in L-NAME–pretreated compared with NE-pretreated Af-Arts.
Protocol 3: Effect of Endothelial Disruption on the Vasoconstrictor Action of 20-HETE
Treatment with antibodies against factor VIII–related antigen and complements did not alter luminal diameter of Af-Arts (Fig 2⇓); diameters before and after the treatment were 16.3±0.2 and 16.2±0.3 μm, respectively (n=5). In these Af-Arts, NE at 0.3 μmol/L similarly decreased the diameter by 20±2% to 13.0±0.2 μm; however, 20-HETE had no effect on the diameter (12.9±0.4 μm at 10−6 mol/L). These results demonstrate that the vasoconstrictor action of 20-HETE on Af-Arts is completely dependent on the presence of intact endothelium.
Protocol 4: Effect of Cyclooxygenase Inhibition or Thromboxane/Endoperoxide Receptor Blockade on the Vasoconstrictor Action of 20-HETE
Pretreatment with indomethacin did not alter basal diameter of Af-Arts; diameters before and after the treatment were 17.1±0.5 and 17.0±0.5 μm, respectively (n=6). NE at 0.3 μmol/L decreased the diameter of indomethacin-treated Af-Arts by 19±2%, to 13.6±0.4 μm. Treatment with indomethacin significantly (P<.03) attenuated the 20-HETE–induced decrease in diameter at 10−8 to 10−6 mol/L; in indomethacin-treated Af-Arts, 20-HETE began to cause constriction from 10−7 mol/L (by 2.2±0.5 μm or 16±4%), and at 10−6 mol/L diameter decreased by only 3.9±0.5 μm (28±3%), to 9.7±0.4 μm (Fig 3⇓). Pretreatment with SQ29548 did not alter basal diameter of Af-Arts; diameters before and after treatment were 17.0±0.4 and 16.7±0.6 μm, respectively (n=5). NE at 0.3 μmol/L decreased the diameter by 25±2%, to 12.5±0.5 μm. SQ29548 also significantly (P<.01) attenuated the 20-HETE–induced decrease in diameter at 10−8 to 10−6 mol/L; 20-HETE began to cause constriction from 10−7 mol/L (by 2.1±0.4 μm or 16±3%), and at 10−6 mol/L diameter decreased by only 3.2±0.6 μm (25±4%), to 9.2±0.3 μm (Fig 3⇓). There was no difference in the vasoconstrictor action of 20-HETE between indomethacin- and SQ29548-treated Af-Arts, suggesting that 20-HETE–induced constriction in Af-Arts is mediated in part by vasoconstrictor endoperoxide(s) produced by cyclooxygenase.
There is considerable evidence that the kidney plays an important role in the pathogenesis of hypertension.22 Abnormalities in renal function (such as reduced sodium excretion and decreased renal blood flow due to elevated RVR) precede, and their resetting may be necessary for, the development of hypertension.23 Although the factor or factors responsible for these early abnormalities are unclear, Sacerdoti et al1 2 have suggested that in young SHR these abnormalities may be a functional expression of an alteration in renal cytochrome P-450–dependent AA metabolism. They have demonstrated that renal cytochrome P-450 content and its related metabolizing enzymes are increased in young SHR1 and that its inhibition markedly decreases blood pressure and improves sodium excretory function in 7-week-old SHR.2 20-HETE, one of several cytochrome P-450–dependent metabolites of AA, is produced in renal preglomerular microvessels (between 10 and 50 μm in diameter)11 and exerts vasoconstrictor action in some vascular beds,5 6 7 suggesting that endogenous 20-HETE may participate in the regulation of RVR. These possibilities prompted us to examine the direct action of 20-HETE on the Af-Art, a crucial vascular segment in the control of RVR.
In the present study, we found that although 20-HETE had no effect on Af-Arts without preconstriction, it caused dose-dependent constriction in Af-Arts with increased basal tone. Such characteristics may be unique to 20-HETE, since a number of vasoconstrictors we have tested thus far in the Af-Art (eg, angiotensin II, NE, adenosine, endothelin, and phenylephrine) do not require elevated basal tone for their action. We also found that 20-HETE–induced constriction was abolished by disruption of the endothelium and was significantly attenuated by either cyclooxygenase inhibition or thromboxane/endoperoxide receptor blockade, suggesting that the vasoconstrictor action of 20-HETE is mediated in part through the endothelial cyclooxygenase pathway.
Although the exact mechanism by which 20-HETE constricts Af-Arts is not clear from the present study, our results suggest that 20-HETE may stimulate endothelium to release vasoconstrictor endoperoxide(s). It is also possible that 20-HETE constricts Af-Arts through its conversion to endoperoxide(s) by endothelial cyclooxygenase because 20-HETE can be used as a substrate for endothelial cyclooxygenase, since its structure is identical to native AA except for a terminal hydroxyl group. Indeed, Schwartzman et al24 have shown that the constrictor response to 20-HETE in rat aortic rings is mediated by 20-hydroxy-prostaglandin G2 and 20-hydroxyprostaglandin H2, labile endoperoxides of 20-HETE produced by the endothelium.
Our finding that 20-HETE had the same effects on Af-Arts preconstricted with either NE or L-NAME suggests that the vasoconstrictor action of 20-HETE requires an increase in Af-Art tone, which is independent of the method used. The reason why 20-HETE requires increased Af-Art tone to exert its vasoconstrictor action is not clear. It may be that increased basal tone of vascular smooth muscle cells of the Af-Arts augments action of vasoconstrictors stimulated by 20-HETE (that is, an increase in the vascular tone may increase the affinity of 20-hydroxyendoperoxides for the thromboxane/endoperoxide receptor). Another possibility is that increased vascular tone (or maneuvers that elicit it) may elevate the levels of 20-HETE–induced vasoconstrictors or decrease the metabolic rate of 20-hydroxyendoperoxides to prostaglandin E2 or I2, which would offset the vasoconstriction. However, since 20-HETE caused significant constriction in indomethacin- or SQ29548-treated Af-Arts, 20-HETE seems to have another endothelium-dependent but not cyclooxygenase-dependent vasoconstrictor effect on the Af-Art. Further studies are apparently required to characterize the mechanism by which 20-HETE causes constriction in the Af-Art.
In contrast to our observations, a preliminary study by Imig et al25 has reported that 20-HETE added to the bath constricts rat juxtamedullary Af-Arts and that this response is not inhibited by indomethacin. Although the reason for this discrepancy is unclear, there are several possibilities other than species differences (rabbit versus rat). First, it may be related to the difference in the preparation used (particularly the presence or absence of structures such as collecting tubules and medullary interstitial cells, which are major sites of vasodilator prostaglandin production). Since the activity of vasodilator prostaglandins is much higher in the juxtamedullary Af-Art than the cortical Af-Art,26 vasodilator prostaglandins may modulate the 20-HETE–induced constriction, as stated by McGiff,27 in juxtamedullary but not cortical Af-Arts. Indeed, 20-HETE at 1 μmol/L decreased the diameter of juxtamedullary Af-Arts by only 15%, which is much less than that observed in our preparation (40% or 43%). If this difference is mediated by vasodilator prostaglandins, indomethacin would have little effect on the vasoconstrictor action of 20-HETE in the juxtamedullary Af-Art because it inhibits the formation of both vasoconstrictor and vasodilator cyclooxygenase metabolites. Second, it may be the difference in administration route (bath versus perfusate). Imig and Navar28 have recently demonstrated that AA added to either the bath or perfusate similarly constricts rat Af-Art and that indomethacin causes much stronger inhibition of AA-induced constriction when AA is added to the perfusate than to the bath. Thus, it is conceivable that AA and its related products would be metabolized through different enzymatic pathways when they are added to the bath or the perfusate. Third, since we used isolated Af-Arts, we could not observe the effect of tubuloglomerular feedback that contributes to the control of Af-Art resistance in juxtamedullary nephron preparations in which tubular and vascular relations are preserved. 20-HETE is known to inhibit Na+-K+-2Cl− cotransport at the thick ascending limb of Henle29 and to affect the Af-Art resistance through tubuloglomerular feedback.12 Therefore, the presence or absence of an intact tubuloglomerular feedback system may account for the difference between our results and those of Imig et al.
In summary, we provide evidence that an increase in vascular tone triggers endothelium-dependent and cyclooxygenase-dependent vasoconstrictor action of 20-HETE in in vitro microperfused rabbit Af-Arts. We have shown that 20-HETE constricted Af-Arts only when basal tone was increased, that this constriction was abolished by endothelium disruption, and that this constriction was significantly attenuated by indomethacin or SQ29548. Thus, increased production of 20-HETE in the kidney and an increase in Af-Art tone, both of which often precede the development of hypertension, may synergistically contribute to the development of hypertension by elevating RVR.
Selected Abbreviations and Acronyms
|BSA||=||bovine serum albumin|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|RVR||=||renal vascular resistance|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported in part by grants from the Ministry of Science, Education, and Culture of Japan (No. 07457237) and from the Ministry of Health and Welfare of Japan (for Renal Diseases; Kurokawa-han). The authors gratefully thank Hiromi Takahashi and Akiko Kohinata for their secretarial assistance.
Sacerdoti D, Escalante B, Abraham NG, McGiff JC, Schwartzman ML. Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats. Science. 1989;243:388-390.
Omata K, Abraham NG, Escalante B, Schwartzman ML. Age-related changes in renal cytochrome P-450 arachidonic acid metabolism in spontaneously hypertensive rats. Am J Physiol. 1992;262:F8-F16.
Levere RD, Martasek P, Escalante B, Schwartzman ML, Abraham NG. Effect of heme arginate administration on blood pressure in spontaneously hypertensive rats. J Clin Invest. 1990;86:213-219.
Escalante B, Sessa WC, Falck JR, Yadagiri P, Schwartzman ML. Vasoactivity of 20-hydroxyeicosatetraenoic acid is dependent on metabolism by cyclooxygenase. J Pharmacol Exp Ther. 1989;248:229-232.
Ma YH, Gebremedhin D, Schwartzman ML, Falck JR, Clark JE, Masters BS, Harder DR, Roman RJ. 20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine arcuate arteries. Circ Res. 1993;72:126-136.
Harder DR, Gebremedhin D, Narayanan J, Jefcoat C, Falck JR, Campbell WB, Roman RJ. Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels. Am J Physiol. 1994;266:H2098-H2107.
Morrison AR, Pascoe N. Metabolism of arachidonate through NADPH-dependent oxygenase of renal cortex. Proc Natl Acad Sci U S A. 1981;78:7375-7378.
Oliw EH, Lawson JA, Brash AR, Oates JA. Arachidonic acid metabolism in rabbit renal cortex. J Biol Chem. 1981;256:9924-9931.
Zou AP, Imig JD, Kaldunski M, Ortiz de Montellano PR, Sui Z, Roman RJ. Inhibition of renal vascular 20-HETE production impairs autoregulation of renal blood flow. Am J Physiol. 1994;266:F275-F282.
Zou AP, Imig JD, Ortiz de Montellano PR, Sui Z, Falck JR, Roma RJ. Effect of P-450 ω-hydroxylase metabolites of arachidonic acid on tubuloglomerular feedback. Am J Physiol. 1994;266:F934-F941.
Kauser K, Clark JE, Masters BS, Ortiz de Montellano PR, Ma YH, Harder DR, Roman RJ. Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renal arcuate arteries. Circ Res. 1991;68:1154-1163.
Imig JD, Falck JR, Gebremedhin D, Harder DR, Roman RJ. Elevated renovascular tone in young spontaneously hypertensive rats: role of cytochrome P-450. Hypertension. 1993;22:357-364.
Ito S, Arima S, Ren YL, Juncos LA, Carretero OA. Endothelium-derived relaxing factor/nitric oxide modulates angiotensin II action in the isolated microperfused rabbit afferent but not efferent arteriole. J Clin Invest. 1993;91:2012-2019.
Ito S, Johnson CS, Carretero OA. Modulation of angiotensin II-induced vasoconstriction by endothelium-derived relaxing factor in the isolated microperfused rabbit afferent arteriole. J Clin Invest. 1991;87:1656-1663.
Juncos LA, Ito S, Carretero OA, Garvin JL. Removal of endothelium dependent relaxation by antibody and complement in afferent arterioles. Hypertension. 1994;23(suppl I):I-54-I-59.
Itoh S, Carretero OA. Role of macula densa in renin release. Hypertension. 1985;7(suppl I):I-49-I-54.
Trippodo NC, Frohlich ED. Similarities of genetic (spontaneous) hypertension: man and rat. Circ Res. 1981;48:309-319.
Beierwaltes WH, Arendshorst WJ, Klemmer PJ. Electrolyte and water balance in young spontaneously hypertensive rats. Hypertension. 1982;4:908-915.
Schwartzman ML, Falck JR, Yadagiri P, Escalante B. Metabolism of 20-hydroxyeicosatetraenoic acid by cyclooxygenase: formation and identification of novel endothelium-dependent vasoconstrictor metabolites. J Biol Chem. 1989;264:11658-11662.
Imig JD, Falck JR, Roman RJ. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent endogenous constrictor of renal arterioles. FASEB J. 1993;7:A888. Abstract.
Steinhausen M, Ballantyne D, Fretschner M, Hoffend J, Parekh N. Different responses of cortical and juxtamedullary arterioles to norepinephrine and angiotensin II. Kidney Int. 1990;38(suppl 30):S55-S59.
Imig JD, Navar GL. Differential involvement of enzymatic pathways in the afferent arteriolar response to arachidonic acid. J Am Soc Nephrol. 1994;5:605. Abstract.
Escalante B, Erlij D, Falck JR, McGiff JC. Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science. 1991;251:799-802.