Thromboxane synthase and TP receptor mRNA in rat kidney andbrain: effects of sal - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Christopher S.Wilcox and William J.Welch作者单位：Division of Nephrology and Hypertension and Center for Hypertensionand Renal Disease Research, Georgetown University Medical Center,Washington, DC 20007 " I6 O' `) S, q8 u0 m. \7 E
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      【摘要】- H5 Q- o2 E" C0 |" |% K  F
   A TP receptor (TP-R) mimetic causessalt-sensitive hypertension and renal afferent arteriolarvasoconstriction. TP-Rs mediate effects of ANG II on renal vascularresistance and drinking. Therefore, we investigated the hypothesis thatthromboxane A 2 synthase (TxA 2 -S) and/or TP-Rexpression is regulated by salt and/or ANG II. Rats ( n = 6) received high-salt (HS) or low-salt (LS) diets. Additional HS-dietrats received ANG II while other HS- and LS-diet rats received theAT 1 receptor (AT 1 -R) antagonistlosartan. Excretion of thromboxane B 2 by consciousrats was increased with the HS diet compared with the LS diet (126 ± 10 vs. 48 ± 5 pmol/24 h, respectively; P to -actin) inthe kidney cortex was enhanced 30% by the HS diet ( P P 0.001). However, during losartan administration,the effects of salt were reversed; mRNA more than doubled during the LSdiet ( P stem was reduced by 50% with the addition of ANG II( P and during losartan administration wasalmost doubled by the LS diet ( P 2 -S in the kidney cortex also wasincreased many times with the HS diet ( P Incontrast, the mRNA for TxA 2 -S in the brain was unaffectedby salt. ANG II did not affect TxA 2 -S at either site.During losartan administration, TxA 2 -S increased modestlyin the brain stem with the LS diet. mRNA abundance for TP-Rs in thekidney cortex and brain stem is suppressed by ANG II acting onAT 1 -Rs. In the absence of AT 1 -Rs,expression of TP-Rs at both sites is enhanced by LS intake. Incontrast, ANG II does not affect the mRNA abundance forTxA 2 -S. Expression of TxA 2 -S is enhanced by HSintake in the kidney cortex but by LS intake in the brain stem onlyduring losartan administration. Thus TP-Rs are strongly dependent onANG II acting on AT 1 -Rs, whereas TxA 2 -S isregulated differentially in the kidney cortex and brain stem by salt intake. 4 a, z. t! [' k5 V, F$ j6 |
      【关键词】 thromboxane A prostaglandins angiotensin II losartan angiotensin receptor blocker thromboxane prostanoidreceptor, |( t/ E; t: j" j
   INTRODUCTION% w0 Q5 p* _* i" C# h  f

THROMBOXANE A 2 ( T X A 2 ) IS GENERATED from PGH 2 by TxA 2 synthase(TxA 2 -S). TxA 2 -S is expressed in the kidney( 53 ). TxA 2, PGH 2, andisoprostanes, such as 8-iso-PGF 2, and stable mimetics, such as U-46619, are agonists for the TP receptor (TP-R) ( 14, 17 ). TP-Rs are expressed in the blood vessels and in kidney microvessels, glomeruli, mesangial cells, thick ascending limbs (TALs)of the loops of Henle, and collecting ducts ( 1, 10, 11, 48 ).

Several lines of evidence suggest that TxA 2 -S andTP-Rs in the kidney are regulated by salt intake or ANG II. Thushypertension in response to a prolonged infusion of U-46619 is enhancedby high-salt (HS) intake ( 55 ). U-46619 enhances thesympathetic nervous system of conscious rats ( 13 ) and thevasoconstrictive tubuloglomerular feedback (TGF) response ofanesthetized rats. Both of these effects are enhanced by an HS diet( 56 ). Moreover, the effects of U-46619 on TGF correlatewith the abundance of the mRNA for TP-Rs in kidney and glomeruli ofrats on different salt intakes. Thus the kidney is sensitive to TP-Ractivation, and this response is salt dependent. However, effects ofsalt on TxA 2 -S expression or on thromboxane B 2 (TxB 2 ) excretion are not clear.# A: ?7 D# D7 I0 `
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The brain also generates TxA 2 ( 6, 8, 44-47 ) and expresses TP-Rs ( 12, 34 ) on glialcells and astrocytes ( 12, 18, 34, 35 ). Theintracerebroventricular administration of U-46619 increases bloodpressure. This pathway also is sensitive to salt. Thus the increase inblood pressure with intracerebroventricular U-46619 is enhanced by HSintake ( 12 ). The drinking response to central ANG II isblunted by intracerebroventricular injection of a TP-R antagonist( 19 ). A TP-R mimetic and ANG II, when given into thebrain, synergize to promote drinking ( 19 ). Thus the brain-TP-R pathway apparently interacts with salt intake and ANG II,but the mechanism is unclear.& ?) u. e, T$ L! _7 ?4 `
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Vasoconstrictor PGs enhance many of the effects of ANG II. Thusblockade of TP-Rs, and in some studies blockade of TxA 2 -S, blunts or prevents the pressor response to infused ANG II ( 32, 33, 62 ), the development of renovascular hypertension in the two-kidney, one-clip Goldblatt or aortic coarctation models of renovascular hypertension in the rat ( 23, 59, 60 ), theincrease in renal vascular resistance during ANG II infusion ( 24, 31, 62 ) and the dipsogenic response to intracerebroventricularANG II infusion ( 19 ). These data implicateTxA 2 -S and/or TP-Rs in the renal and central nervous systemactions of ANG II. Therefore, we investigated the hypothesisthat dietary salt intake or ANG II regulate the mRNA abundance forTP-Rs and TxA 2 -S in the kidney cortex and brain stem.0 f1 x- i* C( V. G
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METHODS5 d8 O, r1 `4 X9 l. {& N
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Studies were undertaken on male Sprague-Dawley rats weighing200-250 g. Animals were habituated in their cages to a standard rat chow (Na content 0.3 g/100 g; Ralston Purina) for 5 days before randomization. Thereafter, all rats were fed a regulateddiet with identical composition except for the NaCl (Teklad, Madison,WI). The Na content of the HS diet was 3 g/100 g,and for the LS diet it was 0.03 g/100 g. The LS diet is sufficient fornormal growth over an 8- to 10-day period. Rats were studied after 6 days on each protocol.9 q. W8 ]2 J0 Y0 c: p

Protocols. Rats were studied according to the following protocols: 1 ) series A; group 1, HS intake ( n = 6); group 2, LS intake ( n = 6); and group3, HS ANG II intake ( n = 6); and 2 ) series B; group 4, HS losartan intake( n = 6); and group 5, LS losartan intake ( n = 6). Rats in series A and B were analyzed separately.( @  W; f! u) R! v% T
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Before starting the 6 days of a regulated diet, animals in group3 were anesthetized with pentobarbital sodium (50 mg/kg ip; AbbottLaboratories, North Chicago, IL) and fitted with a subcutaneous osmoticminipump (Alza, Palo Alto, CA) to deliver ANG II at 200 ng · kg 1 · min 1. In a priorseries, our laboratory found that this protocol increased the meanarterial pressure from 107 to 129 mmHg and reduced the renal blood flowfrom 9.6 to 8.0 ml · min 1 · g 1 ( 9 ). Animals in series B were given losartan (1 mg/ml) in the drinking water.4 q9 D: s9 \1 m5 r% b
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Excretion of TxB 2. Rats were studied on day 6 of LS or HS diets. They wereaccommodated for 1 day to metabolism cages, and for the next 24 h a urine sample was collected from the cage. Urine was collected intocontainers with streptomycin (2,000 IU), penicillin G (2,000 IU), andamphotericin B (5 µg) to prevent microbial growth. It wascentrifuged, separated from sediment, and stored at 70°C untilanalyzed for TxB 2. These rats were separate from those used for mRNA studies.

The details of the assay, including dual system purification by organicexcretion and thin-layer chromatography, individual sample recovery,limits of detection, intra- and interassay coefficients of variation,cross-reactivity, and validation against gas chromatography massspectroscopy, have been published ( 57 ).

Extraction and analysis of mRNA. For preparation of the kidney ( 56 ) and brain( 12 ), groups of rats were anesthetized on day 6 of the protocol with thiobarbital (Inactin, 100 mg/kg ip; ResearchBiochemicals International, Natick, MA). The chest was opened, and theleft ventricle was punctured to flush the organs with ice-cold 0.154 NaCl. The entire brain stem (dissected free from the cerebellum,midbrain, and cortex) and one kidney were removed, cleared ofconnective tissue, and placed in ice-cold saline solution. The kidneywas cut longitudinally and a segment of cortex was removed. Total RNAwas extracted by using RNA ATAT-60 (Tel-test B, Friendswood, TX). ThemRNA was reverse transcribed with oligo(dT) 16 as the primerand murine leukemia virus RT by using an RNA PCR kit (PerkinElmer,Branchburg, NJ). The primers used for PCR for the TP-R gene productwere selected from the published cDNA sequences of the rat TP-R(GenBank accession no. D32080 ) ( 1 ). ForTxA 2 -S, the sense was 5'-TTCCTGCAGATGGTGCTGGATG-3' and theantisense was 5'-AGGTATGTGATGAAGGAGAGCG-3' (235 bp) and TxA 2 -S ( 64 ) (GenBank accession no. D31798 )( 49 ) as described previously ( 12 ). For theTP-R, the sense was 5'- TGGACTGGCGTGCCACTGAT-3' and the antisense was5'-AGCAAGGGCATCCAACACACCGTG-3' (502 bp). The primers for the TP-R wereselected to amplify the - and -isoforms of the receptor( 30 ). -Actin was selected as the "housekeeper gene"for comparison, because -actin mRNA abundance in the rat kidney isindependent of salt intake ( 56 ). The sense primer was5'-GATCAAGATCATTGCTCCTC-3', and the antisense was5'-TGTACAATCAAAGTCCTCAG-3' (426 bp). We detected no differences in -actin expression in the kidneys or brain stem in the differentgroups used in these protocols. Therefore, the amounts of TP-R andTxA 2 -S cDNAs were normalized by the amounts of -actincDNA. The reaction mixture contained 50 pmol of each primer, 1.25 mMdeoxynucleotide mixture, 2.5 µl Taq DNA polymerase, 10 mMTris (pH 10), 50 mM KCl, and 1.5 mM MgCl 2 and was carriedout by the following protocol. After an initial melting temperature of94°C for 4 min, there were 30 s of denaturation at 94°C,45 s of annealing at 60°C, and 45 s of extension at 72°Cfor 7 min. The PCR product was analyzed on a 1.5% agarose gel stainedwith ethidium bromide and visualized under ultraviolet light. The sizeof the products was compared with rat kidney cDNA probes for TP-Rs( 1 ) and TxA 2 -S ( 49 ), kindlyprovided by Dr. Kazu Takeuchi (Tokohu University, Sendai, Japan). Toverify authenticity of the PCR products, amplified TP-R andTxA 2 -S cDNAs from the rat kidney cortex and brain stem were purified with Microcon (Amicon, Beverly, MA) and sequenced with an Ampli Taq cycle sequencing kit (PerkinElmer).& }$ _- ~3 c" [& ?* y  e" |
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Care was taken to optimize conditions for the RT-PCR. For all products,pilot studies were undertaken with graded amounts of cDNA to ensurethat product (as assessed by densitometry) increased log-linearly withthe cDNA amount in the ranges tested. Negative controls were undertakenby PCR without prior RT and by RT-PCR of the buffer used.

Statistical analysis. The data were subjected to ANOVA to assess the effects of salt intake( group 1 vs. 2; series A ) and ANG II( group 1 vs. 3; series A ) and of saltintake during losartan administration ( group 4 vs. 5; series B ). Data are presented as means ± SE.

The studies were designed to test two a priori hypotheses. First, theexpression of mRNA for TxA 2 -S or TP-Rs could be stimulated by ANG II independently of salt intake. This was refuted if there wasno significant difference in mRNA abundance in a comparison of HSintake with HS ANG II intake. Second, the expression of mRNA forTxA 2 -S or TP-Rs could be stimulated by salt intakeindependently of AT 1 receptors (AT 1 -Rs). Thiswas refuted if there was no significant difference between LS losartanintake and HS losartan intake.

RESULTS6 w3 [) v. ~0 r( F- @1 c
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The 24-h excretion of TxB 2 by conscious ratsis shown in Fig. 1. It is apparent thatexcretion is increased significantly by 6 days of HS intake comparedwith LS intake.

Fig. 1. Values are means ± SE and represent excretion ofthromboxine B 2 (TxB 2 ) by conscious rats after 6 days of accommodation to a high-salt (HS) or low-salt (LS) diet.

The mRNA abundance for TP-Rs (relative to -actin) extracted from thekidney cortex and brain stem during HS, LS, and HS ANG II intake areshown in Fig. 2. For the kidney but notthe brain, HS intake enhanced the mRNA for TP-Rs by 30%( P both the kidney and the brain, theabundance of TP-Rs during HS intake was reduced by ~50% by ANG IIinfusion ( P conclude that ANG II infusioninhibits mRNA for TP-Rs in the kidney cortex and brain stemindependently of salt intake.! X1 ^" Y: H: E# y
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Fig. 2. Values are means ± SE and represent mRNA abundancefor TP receptors expressed as density relative to -actin comparingkidney cortex ( A ) with brain stem ( B ) in ratsadapted to HS, LS, or HS ANG II intake. ns, Not significant.# J8 O( M3 G% M; q" U) G
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During losartan administration, LS intake led to a large increase inthe mRNA for TP-Rs in the kidney cortex (2-fold) and brain stem (60%)(Fig. 3 ). Thus salt restrictionstimulates TP-R expression in the kidney and brain independently ofAT 1 -Rs.
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Fig. 3. Values are means ± SE and represent mRNA abundancefor TP receptors expressed as density relative to -actin comparingkidney cortex ( A ) with brain stem ( B ) in ratsadapted to HS and LS intake during losartan administration.2 }& g+ Q. E0 B/ `% P5 B

The mRNA abundance for TxA 2 -S (relative to -actin) inthe kidney cortex and brain stem during HS, LS, and HS ANG II intake isshown in Fig. 4. The TxA 2 -Sabundance in the kidney was increased 20-fold during HS intake,whereas the abundance in the brain was not changed significantly.Unlike TP-Rs, there were no effects of ANG II during HS intake.. s) W1 }: N$ J7 w3 q

Fig. 4. Values are means ± SE and represent mRNA abundancefor thromboxine A 2 synthase by conscious rats after 6 daysof accommodation to an HS or LS diet expressed as density relative to -actin comparing kidney cortex ( A ) with brain stem( B ) in rats adapted to HS, LS, or HS ANG II intake.% Z, _3 F( a* c0 x8 E0 A1 H. Q0 {

During losartan administration, there was a similar effect of saltintake on TxA 2 -S in the kidney (Fig. 5 ) as seen in the absence of losartan(Fig. 4 ). However, during losartan administration there was an increasein mRNA for TxA 2 -S in the brain stem during LS intake of60%. We conclude that salt intake has divergent effects onTxA 2 -S expression in the kidney and brain. Salt loadingstimulates expression in the kidney, and salt restriction stimulatesexpression in the brain. These effects are largely independent of ANGII and AT 1 -Rs.& \5 o5 [: S% P# h5 `1 e8 V- |
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Fig. 5. Values are means ± SE and represent mRNA abundancefor thromboxine A 2 synthase expressed as density relativeto -actin comparing kidney cortex ( A ) with brain stem( B ) in rats adapted to HS and LS during losartanadministration.
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DISCUSSION
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Our laboratory found previously that HS intake enhances mRNAabundance for TP-Rs in the kidney cortex and glomeruli( 56 ). This was confirmed in the present study. The mainnew findings are that this effect of HS intake to increase mRNAabundance for TP-Rs is specific for the kidney because it is not foundin the brain stem and is dependent on ANG II and AT 1 -Rs atboth sites. Remarkably, during losartan administration, the increasedTP-R expression in the kidneys seen during HS intake is reversed, and in both the kidney and the brain, TP-R expression is increased substantially by salt restriction when the effects ofAT 1 -Rs are prevented by losartan. These data demonstratethat the prominent effect on TP-R expression in these protocols issuppression by ANG II acting on AT 1 -Rs. This action of ANGII apparently conceals an effect of salt restriction to enhanceexpression that is seen only during AT 1 -R blockade. BecauseANG II levels diminish during HS intake and ANG II suppresses theexpression of TP-Rs, the increase in TP-Rs in the kidneys with HSintake may be ascribed to a loss of the inhibitory effect of ANG IIacting on AT 1 -Rs. Similar to TP-Rs, the mRNA abundance forTxA 2 -S also increases with HS intake in the kidney,although it is not changed in the brain. However, ANG II does notchange the expression of TxA 2 -S in the kidney or brain.Moreover, the increase in TxA 2 -S with HS intake in the kidney and with LS intake in the brain persists during losartan administration. We conclude that the major determinant ofTxA 2 -S expression is salt intake independent of ANG II andAT 1 -Rs. These data indicate the quite distinct effects ofANG II on the regulation of the mRNAs for TP-Rs and TxA 2 -S.. F& c. x( C+ ^+ F( V$ E  B

Rat glomeruli and mesangial cells contain high-affinity binding sitesfor TP-R ligands ( 10, 14 ). The distribution of TP-R protein within the kidney has been studied by immunocytochemistry. Receptors are located on arterial walls, microvessels, glomeruli, proximal tubules, TALs, distal tubules, and collecting ducts ( 4, 48 ). In situ hybridization studies have located the mRNA for TP-Rs in glomeruli, afferent and efferent arterioles, the luminal aspects of the TALs, macula densa cells, collecting ducts( 1 ), and the renal medulla ( 27 ).Receptor-mediated contractile responses to TP-R activation have beenshown in isolated glomerular mesangial cells and renal afferentarterioles ( 16, 25, 28, 43 ). A thromboxane mimeticenhances NaCl reabsorption in the loop of Henle and enhances the TGFresponses ( 58 ). These data suggest that TP-Rs aredistributed to renal microvessels, glomeruli, macula densa cells, andtubules of the kidney. TP-Rs also are expressed in the brain( 3 ), where they are distributed to glial cells andastrocytes ( 12, 18, 34 ).# I( \  V6 z) w- a
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We considered two explanations for the reduction of TP-R expression byANG II. First, this could represent ligand-associated receptordownregulation ( 39 ). ANG II stimulates phopholipases thatsubstantially enhance PG formation in the kidney ( 26, 29 ). ANG II infusion increases renal excretion ofTxB 2, implying enhanced renal TxA 2 generation( 26 ). Indeed, blockade of TxA 2 -S or TP-Rs blunts the renal vasoconstriction with prolonged ANG II infusion ( 31, 62 ). An increase in vasoconstriction has beenascribed to PGH 2, which also activates TP-Rs( 22 ). Moreover, ANG II leads to oxidative stress and anenhanced excretion of 8-iso-PGF 2 ( 41, 42 ).Thus all the identified ligands of the TP-R are generated in responseto ANG II infusion. Ligand-associated downregulation of TP-Rs couldaccount for the reduced expression of TP-Rs in the kidney during ANG IIinfusion and the enhancement during AT 1 -R blockade with losartan.- b$ T& C+ A8 X+ A; l2 ]* ?

A second possible explanation derives from the evidence that losartanalso blocks TP-Rs ( 7, 21 ). Therefore, the losartan-induced increase in mRNA for TP-Rs may represent upregulation of the receptor during pharmacological blockade. It is not clear how this could explainthe large effects of salt intake on TP-R expression during losartan administration.5 }- F1 g# I* A7 i( m' W
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The protein for TxA 2 -S is expressed in renal microvessels,interstitial fibroblasts, and macrophages ( 36, 40 ), and in the distal nephron of the kidney ( 37 ). Isolated glomeruliand glomerular mesangial cells and podocytes release TxB 2 and therefore presumably express TxA 2 -S ( 29 ).Microdissected nephron segments also release TxB 2. Thelargest sites for release are glomeruli, TALs, and collecting ducts( 2 ). Thus TxA 2 can be widely synthesized inthe kidney. In the brain, TxA 2 is synthesized predominantly in glia ( 44, 45 ). Very large quantities ofTxB 2 are released from the brain in response tohypoperfusion or trauma ( 6, 8, 46 ). However, we are notaware that the regulation of TxA 2 -S in the kidney or brainhas been studied directly.% S' v, c( I/ Z
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The cause for the salt-dependent, but ANG II- andAT 1 -R-independent, increase in mRNA abundance forTxA 2 -S in the kidney is not clear. This is analogous to thesalt-dependent, but ANG II- and AT 1 -R-independent,expression of mRNA and protein for nitric oxide synthase type I in thekidney cortex and macula densa cells ( 51, 52 ). Itdemonstrates that the kidney contains potentially unique mechanismsthat would provide it with the potential to enhance the generation ofnitric oxide in the juxtaglomerular apparatus during LS intake and ofTxA 2 during HS intake. This could contribute tokidney-specific regulation of vascular tone during changes in saltintake. The large increase in TxA 2 -S mRNA expression in thekidney cortex observed during HS intake may explain the finding thatrenal TxB 2 excretion (and hence TxA 2 generationin the body) increases two- to threefold during HS intake compared withLS intake (Fig. 1 ).
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These results suggest that during LS intake there is normally apowerful stimulus for expression of TP-Rs in kidney cortex and brainstem. However, an associated increase in ANG II, acting onAT 1 -Rs, feeds back to reverse the effect of salt in thekidney and prevent the effect in the brain stem. This is analogous to studies of cyclooxygenase-2 (COX-2) in the kidney cortex. COX-2 expression is enhanced during LS intake; this effect is exaggerated byblockade of ANG II generation with an angiotensin-converting enzymeinhibitor ( 15 ). This has led to the hypothesis that ANG IIfeeds back to blunt salt-induced changes in COX-2 expression. The majordifference in the renal regulation of TP-Rs and COX-2 appears to be inthe relative importance of ANG II, which is predominant for TP-Rs,resulting in an actual reversal of the direct effects of salt on TP-Rexpression in the kidney.  y( D& K, U3 d$ P
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A limitation of our study is the use of mRNA to assess receptor orenzyme function. However, there is a correlation among several findingswith mRNA expression in the present series and functional data. Thusthe 10-fold increase in TxA 2 -S mRNA expression in thekidney with HS intake (Fig. 4 ) is mirrored by a two- to threefoldincrease in TxB 2 excretion (Fig. 1 ). The increase in TP-RmRNA expression in the kidney with HS intake (Fig. 2 ) is mirrored bythe increased responsiveness of the afferent arteriole to U-46619microperfused into the renal interstitium during HS intake( 56 ) and the greater increase in blood pressure( 55 ) and sympathetic nervous system activity( 13 ) in response to prolonged infusion of U-46619 duringHS intake. The finding that the expression of TP-R mRNA in the brainstem is downregulated by ANG II (Fig. 2 ) may be relevant to theinteraction between ANG II and TP-Rs in the brain in the control ofdrinking ( 19 ).' Y( T. Y5 D& ]9 [! @* P
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Perspectives. There are several implications of our study. First, the upregulation ofTP-Rs and TxA 2 -S in the kidney cortex during HS intake maycontribute to the prohypertensive role of vasoconstrictor PGs insalt-dependent forms of hypertension, such as ANG II-salt hypertension( 32 ), DOCA-salt ( 20, 38 ), or the Dahlsalt-sensitive rat ( 50, 54, 63 ). Our laboratory had shownpreviously that hypertonic NaCl infusion selectively increases therelease of TxB 2 into urine and renal lymph( 61 ). An accompanying increase in afferent and efferentarteriolar resistance is blocked by inhibition of COX orTxA 2 -S ( 5 ). The present finding that HS intakeenhances TxB 2 excretion and enhances TP-Rs andTxA 2 -S in the kidney cortex suggests that salt loadingcould increase TxA 2 generation and response to TP-Ractivation. Third, HS intake potentiates the increase in blood pressurethat occurs during a prolonged infusion of a TP-R mimetic( 55 ) and the increase in afferent arteriolar vasoconstriction during local microperfusion of a TP-R mimetic into thekidney ( 56 ). This correlates with an increase in the mRNAfor TP-Rs in the kidney during salt loading. Therefore, these newfindings that salt intake and/or ANG II regulates TP-R and TxA 2 -S mRNA expression in the kidney and brain likely havefunctional significance.
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ACKNOWLEDGEMENTS- y6 d  S* }+ o& `5 M. X$ M

We are grateful to Dr. Bo Peng for technical assistance and SharonClements for the preparation of this manuscript.
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