228

Intrarenal Dopamine Production and Distribution in the Rat

Physiological Control of Sodium Excretion

Zhl-Qm Wang, Helmy M Srragy, Robin A Felder, Robert M Carey

Abstract Dopamme (DA), produced by the renal proximal tubule, has been demonstrated as an mtrarenal paracnne hormone mediating dmresls and natrmresls The precise mechanism by which DA exerts its cell-to-cell action 1s not fully understood In the present study, renal mterstltlal flmd (RIF) DA (by m VIVO mlcrodlalysls) and urinary DA excretion (U,,V) were compared m anesthetized rats on either normal (0 28% NaCl, NS) or high (4 0% NaCl, HS) sodium balance and m response to acute Y-L- glutamyl-L-dopa (gludopa) admmlstratlon Urme flow (UV) and sodium excretion (U,,V) m HS were greater than m NS rats U,,V was increased m HS compared with NS rats RIF DA was significantly lower m HS than NS rats Gludopa at 3, 5, and 7 5 nmol/kg (IV bolus) produced a larger Increase m UoAV than RIF DA Only the highest dose of gludopa (7 5 nmol/kg), which re- sulted m a 7 3-fold mcrease m UoAV and 1 7-fold increase m RIF

DA, was associated with slgmficant dmresls and natrmresls Cor- tical and medullary blood flow remamed unchanged after gludopa (7 5 nmol/kg) admmlstratlon, while anglotensm II (100 ng kg-’ mm-‘) mduced slgmficant reductton m cortical and medullary blood flow Prior bilateral renal denervatlon did not have a slg- mficant effect on basal DA levels (RIF DA and U,,V) or gludopa- induced DA production or natrmresls and dmresls The\e data demonstrated that both chronic sodium loadmg and acute gludopa admmlstratlon stimulated renal DA production and release pre- dominantly mto the tubule lumen, where DA had a direct tubule action m the control of UN,V Renal DA productlon and its renal effects were not significantly regulated by renal sympathetic nerve activity (Hypertension. 1997;29[part 2]:228-234.)

Key Words l dopamme l extracellular space l gludopa l hdney l mlcrodlalysls l sodium

T he renal dopammergrc system plays an important role m the regulation of blood pressure, sodmm balance, and ktdney functron 1.2 Proximal tubule

cells, rich in aromatic ammo acrd decarboxylase, can take up crrculatmg and/or filtered L-dopa for decarboxylatton to DA 1.3 Substanttal evidence suggests that most of the DA appearing m urine 1s of proximal tubule orrgm How- ever, the extent to which the UDAV reflects the amount of the amme that has been synthesized m tubule cells and the fate and outflow of newly formed DA from the prox- tmal tubules remams unknown.

In the kidney, Dl-like receptors are localized in renal blood vessels, the luxtaglomerular apparatus, the proxt- ma1 tubule (both apical and basolateral), cortrcal collect- mg duct, and medullary thick ascending limb D,-like re- ceptors have been described m the endothehal and adventural layers of renal vasculature, the glomerulus,r and m renal proximal tubules and the renal papilla. More- over, a D4-hke receptor may also be present m the cortical collectmg duct 4 DA generated mtrarenally has a well- documented natrmrettc effect, and mfuston of DA antag- onists decreases UN,V independently of hemodynarmc changes.5.6 Because DA 1s synthesized, stored, and re- leased wrthm the kidney m close proximity to Its target sue(s), rt is believed that mtrarenal DA serves as a para- crme factor, locally modulating renal hemodynamlc and/or excretory function 1.2 The precrse mechanism by which DA exerts its cell-to-cell action, however, IS not understood

From the Departments of Medlcme and Pathology (R A F ), Um- verslty of Virgmla Health Sciences Center, Charlottesvdle

Correspondence to Dr Robert M Carey, Box 395, Umversny of Vlrgmla Health Sciences Center, Charlottesville, VA 22908

0 1997 American Heart Assoclatlon, Inc

The purpose of the present study was to characterize mtrarenal DA production and drstrrbutron m response to chronic sodium loading and gludopa, a DA prodrug, m anesthetrzed rats with and without renal denervatron We used a novel m situ mterstrtral mrcrodtalysrs technique to sample rat RIF DA and to compare the effects of chrome sodmm loading and acute gludopa admmrstra- tron on RIF and UDAV m the rat. Responses of renal Un,V and mtrarenal blood flow distribution to gludopa also were examined

Methods Microdialysis Technique Microdialysis Probe Construction

For the determination of RIF DA, we constructed a mlcrodl- alysls probe as previously described 7-12 Each end of smgle 0 5- cm-long hollow fiber dialysis tubing (0 l-mm inner diameter, transmembrane molecular mass cutoff, 5000 D, Hospal) was m- serted mto a manually dilated end of a 30-cm-long (inflow and outflow) hollow polyethylene tube (0 12-mm inner diameter, 0 65-mm outer diameter, Bloanalytlcal Systems) The distance between the ends of the polyethylene tubes was 3 mm (dialysis area) and the dlalysls fiber was sealed m place within the poly- ethylene tubes with cyanoacryhc glue The dead space volume of the dlalysls and outflow tubes was 3 6 PL

In Vitro Relative Recovery of DA In vitro relative recovery of DA was evaluated by Immersing

dlalysls membranes of mdlvldual probes (n=8) m a beaker con- taming 20 pg/,uL DA The inflow tube of each probe was con- nected to a gas-tight syrmge filled and then perfused with lactated Rmger’s solution at 1, 3, and 5 pL/mm (Harvard Apparatus, pump model 22) each for 90 minutes After 60 minutes of eqml- lbratlon, the effluent was collected from the outflow tube mto a mlcrocentrlfuge tube containing 10 PL 4% acetlc acid

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Wang et al Renal Dopamine Production and Sodium Excretion 229

Selected Abbreviations and Acronyms DA = dopamme

gludopd = y-L-glutamyl-L-dopa HS = high-salt diet NE = norepmephrme NS = normal-salt diet

RIF = renal mterstmal flmd UDAV = urmary DA excretron UN,V = urmary sochum excretion

UV = urmary flow rate

In Vivo Renal Microdialysis Studies were performed on female Sprague-Dawley rats (body

weight 200 to 250 g, Harlan Sprague Dawley Inc, Harlan Teklad, Madison, WI) All procedures on animals were approved by the Umverslty of Virginia Animal Care Committee With the animal under general anesthesia (pentobarbital sodium, 50 mg/kg mtra- pentoneally), the rat was tracheotormzed and placed on a heated pad to keep body temperature at 37°C to 38°C The right Jugular vein was cannulated with polyethylene tubing (PE-20), and m- travenous mfuslon of lactated Ringer’s solution was admmlstered at 0 5 mL/h per 100 g body weight throughout the experiment The left kidney was exposed via a midline abdominal mCislon The renal capsule was penetrated with 31-gauge needle that was tunneled mto the outer renal cortex approximately 2 to 3 mm from the outer renal surface for 0 5 cm before it exited by pen- etrating the capsule again The tip of the needle was inserted into one end of the dialysis probe, and the needle was pulled together with the dialysis tube until the dialysis fiber was situated m the renal cortex The inflow tube of the chalysls probe was connected to a gas-tight syringe filled with lactated Rmger’s solution and perfused at 1 IL/mm The effluent was collected from the outflow tube A 60mmute equilibration period elapsed before the exper- imental protocol was initiated In vlvo recovery of mtravenously infused 3H muhn m the anesthetized rat has demonstrated that ‘H muhn appearing in the RIF sample IS 2% of urinary 3H muhn, mdlcatmg that the rhalysate 1s not significantly contaminated by renal tubule fluid 9 Moreover, microdialysis probe implantation m the cortex of the rat kidney chd not significantly alter renal cortical blood flow in the area directly adjacent to the dialysis probe, or glomerular filtration rate or fractional U,,V lY

In Vivo Equilibrium Microdialysis To estimate the RIF DA level m the anesthetized rat on

standard diet (0.28% NaCl, Bioserve) and tap water ad hbltum, a gradient dialysis technique was employed, m which exo- genous DA was added to the perfusate, as described previ- ously 9 13 Rats (n=6) were prepared surgically as described above After 60 minutes of eqmhbnum, the probe was perfused at 1 pL/rnm with different concentrations of DA ranging from 0 to 25 pg/pL The dialysate fluid was collected during perfusion at each concentration (90 minutes each) and Its DA level was determmed A linear regression analysts was performed to deter- mme the relationship between the net loss or gam of DA m the collected chalysate and mttial DA concentration m the perfusate The concentration at which there 1s no net flux of DA across the dialysis membrane IS considered a valid estimate of the RIF DA concentration

Effect of Chronic Salt Loading on Renal DA Production and Distribution

In this study, rats (n=18, 9 m each group) were given a diet (Bioserve) containing either 0 28% (normal salt, NS) or 4 0% (high salt, HS) NaCl and tap water ad hbltum for 5 consecutive days Both right and left ureters of the rats, otherwlse prepared m an identical manner as described above, were cannulated with polyethylene tubing (PE-IO) After 60 minutes of eqmhbrmm, RIF and urine samples were collected for 90 minutes and stored at -80°C until assayed

Effect of Acute Gludopa Administration on Renal DA Production/Distribution, UN,V and Intrarenal Blood Flow Effect of Gludopa on Renal DA Production/ Distribution and U,,V

In this study, rats (n=28) consuming standard &et and tap water ad hbitum were divided Into four groups (n=7 m each group) Both right and left ureters of the rats, otherwise prepared m an identical manner as described above, also were cannu- lated A period of 60 minutes was allowed for eqmhbrmm RIF samples were collected for 90 minutes and urine samples were collected every 45 minutes for 90 minutes before and after IV bolus mJection of gludopa (UCB Bioproducts) at 3, 5, or 7 5 nmol/kg m 5% dextrose or vehicle only m the four groups of rats At the end of the experiments the right kidneys of the rats receiving gludopa at 7 5 nmol/kg or vehicle alone were col- lected, weighed, and stored at -80°C until assayed for renal tissue NE content

Effect of Renal Denervation on Gludopa-Induced Renal DA Production/Distribution and UNOV

In this study, female Sprague-Dawley rats (body weight 200 to 250 g, n=lO) with prior bilateral renal denervatlon were pm- chased from Z~VK Miller During bilateral renal denervation, the renal artery on both sides was stripped of the adventitta and coated with a solution of 10% phenol m absolute alcohol The rats were placed on standard diet and tap water ad hbltum and allowed to recover for 3 days after the surgery. On the experiment day, both right and left ureters of the rats, otherwise prepared m an ldentlcal manner as described above, also were cannulated A period of 60 minutes was allowed for eqmhbrmm The above protocol was repeated with IV bolus inJection of gludopa at 7 5 nmol/kg m 5% dextrose or vehicle only m two groups of rats with prior bilateral renal denervation (n=5 m each group) At the end of the experiments the right kidneys were collected, weighed, and stored at -8O“C for measurement of kidney NE content to verify the effectiveness of the chronic renal denervation

Effect of Gludopa and Angiotensin II on I&arena1 Blood Flow Distribution

In this study, rats (n=12) consurnmg standard diet and tap water ad hbltum were divided mto two groups (n=6 m each group) Rats were surgically prepared as described above The left kidney was exposed and laser-Doppler probes (Advance Co Ltd) were applied on the ventral surface of the kidney (super- ficial probe, type C) or inserted mto the renal parenchyma at a depth of 4 mm (needle probe, type N) The laser-Doppler probes were connected to a laser-Doppler flowmeter (ALF 21D dual channel flowmeter, Advance Co Ltd) allowing simultaneous measurements of cortical and medullary blood flow as previ- ously described 1415 An eqmhbrmm period of 30 minutes was allowed before the experimental protocol was initiated Blood flow signals from the renal cortex and medulla were recorded every 10 minutes for a period of 90 minutes before and after the IV bolus mJection of gludopa (7.5 nmol/kg m 5% dextrose) (n=6) In another group of six rats, blood flow signals from the renal cortex and medulla were recorded every 10 minutes for a period of 30 minutes before and during the IV mfuslon of an- giotensm II at 100 ng kg-’ mn-‘, followed by a 30-minute recovery period

Analytical Methods RIF sample was collected mto a rmcrocentrlfuge tube contam-

mg 10 /JL of 4% acetic acid Urine was collected into a micro- centrifuge tube contammg 20 PL of 6N HCl Urine volume was calculated gravtmetrlcally Urine sodium was measured by flame photometry (IL943, Instrumentation Laboratory) Ahquots of urine were extracted by ion exchange on Eho-Rex 70 resin (50 to 100 mesh, sochum form) and absorption on alumina, followed by

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230 Hypertension Vol29, No 1, Part 2 January 1997

elutlon with 4% acetic acid.‘” Dlhydroxybenzylamme was used as the internal standard The kidneys were minced and homoge- mzed m 0 1 mol/L perchlorlc acid The supernatants obtained by centrifuge (20 OOOg, 20 minutes) wele then extracted ‘6 DA m both dlalysate sample and extracted urme and NE m the extracted kidney tlsque were separated with reverse-phase hlgh-pertor- mance hqmd chromatography and detected by the current pro- duced on exposure of the column effluent to oxldlzmg and then reducmg potentials m series (Coulochem 5 1OOA detector coupled with guard cell 5020 and analytlcal cell 5010, ESA) ~17 The overall recovery of the urine extractIon averaged 7528% The detectlon hmlt was 20 pg per volume assayed The mtra-assay and mterassay coefficients of varlatlon were 5 6% and 8.7%, respectively

All data are expressed as mean?SEM StatIstIcal analysis was performed with a Macmtosh StatVIew program (Abacus Concept?) Comparisons were made with either t test or analysis of variance, followed by ScheftC’s test for multiple comparisons when appropn- ate P< 05 was consldered statlstlcally slgmficant

Results In Vitro and In Vivo Validation of Renal Interstitial Microdialysis

Studies of m vitro recovery of DA demonstrated that DA concentrations In the dialysate were Inversely propor- tional to the flow rate through the dlalysls tubing The best relative recovery (percentage of DA concentration in dl- alysate/DA concentration m beaker) was observed with a perfusion rate of 1 PLlmm and was 92 722 0%, compared with 73 9+-4 9% and 44.3t3.3% at 3 and 5 pL/mm, re- spectively (Fig 1A) Using an equlhbrmm dlalysls method, we demonstrated that the theoretically derived point at which there 1s no net transmembrane flux (the steady state mterstltlal DA concentration) was similar to the interstitial DA concentration measured directly durmg normal salt m- take (Fig 1, Panel B)

Effect of Chronic Salt Loading on Renal DA Production/Distribution

UV and UN,V in HS (n=9) were greater than in NS (n=9) rats (UV 7 220 6 versus 3 8?0 3 pL/mm, P< 01, UN,V 497&66 versus 265?27 nmol/mm, P< 01) UDAV increased m HS compared with NS rats (6Ol-t68 versus 420237 pg/mm, P< 05) In contrast, RIF DA was slgmf- scantly lower m HS than NS rats (1 25+0 36 versus 3 68 20.49 pg/mm, P< 01) (Fig 2).

Effect of Acute Gludopa Administration on Renal DA Production/Distribution and UN,V

Basal UoAV and RIF DA from rats with prior bilateral renal denervatlon (n= 10) were smular to rats with intact renal innervation (n=l4) (UDAV 476230 versus 394228 pg/mm, P> 05, RIF DA 2 920 2 versus 3 4kO.3 pg/mm, P> 05) UV tended to be lugher m rats with prior bilateral renal de- nervatlon (4 1 +O 2 versus 3.420 2 pL/mm, P= 05), while UN,V was not slgmficantly different between the two groups (286% 13 versus 2652 16 nmol/mm, P> 05) (Fig 3)

In rats with Intact renal innervation (n=7 m each group), IV inJectIon of gludopa at 3,5, and 7 5 nmol/kg produced a larger increase m UDAV than RIF DA. Only the highest dose of gludopa (7 5 nmol/kg), which resulted m a 7.3- fold increase in UDAV and 1 7-fold increase m RIF DA, was associated with slgmficant dmresls and natrluresls (UV 3 4tO 4 versus 7 4+-O 5 pL/mm, P< 01; UN,V 265+-27 versus 71 l? 120 nmol/mm, P< 01) (Fig 4)

In rats with prior bilateral renal denervation (n=5 m each group), gludopa at 7 5 nmol/kg produced slgmficant

Increase m UDAV (8 3-fold) and RIF DA (1 8-fold), accom- panied by significant dmresls and natrmresls (UV 3 9?0 2 versus 8.250.5 pL/mm, P< 01, UN&V 2892~s 778t56 nmol/mm, P< 01) (Rg 5)

Tissue NE content of chronically denervated ktdneys (n=5) was slgmficantly lower than mtact kidneys (n=6) (13 823 8 versus 103 5+ 10 rig/g tissue m vehicle-control rats, and 10 8t2 1 versus 135 52 15 8 rig/g tissue in glu- dopa-treated rats, both P< 01)

Effect of Acute Gludopa or Angiotensin II Administration on Intrarenal Blood Flow Distribution

Renal blood flow of the cortex and medulla did not m- crease m rats recelvmg gludopa (7 5 nmol/kg) (n=6, Fig 6A), while anglotensm II (100 ng kg-’ mn-‘) induced significant reduction m cortical (42 6%) and medullary (28 1%) blood flow, which gradually returned toward preanglotensm levels when the anglotensm infusion was stopped (n=6) (Fig 6B).

Discussion Using a novel mterstltlal mlcrodlalysls techmque, we

momtored changes m RIF and urme DA excretion m re- sponse to chronic sodium loading and acute gludopa ad-

mmlstratlon m anesthetized rats It 1\ well documented that the kidney Itself 1s the main source of urinary DA The

Perfusion rate (ul/mtn)

-5 00 -

-7 50 -

-10 00 ’ 0 5 10 15 20 25 30

Perfusate DA concent (pg/ul)

Valldatton of the In VIVO renal dlalysls technique A, In vitro recovery of DA by individual mtcrodtalysls probes (n=8) with different perfusion flow rates Perfusion of a probe at high flow rate results In a decrease in the amount of DA passing through the dialysis membrane (percent relative recovery) B, Linear re- gression analysis of in VIVO gradient DA dialysis Estimated renal interstitial DA concentration from the cortex when there IS no net flux across the dtalysls membrane IS 4 1 pg/bL

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NS HS

NS HS

Wang et nl Renal Dopamine Production and Sodium Excretion 231

10

i

renal venous concentration of DA has been shown to be Our study confirmed that UDAV mcreases m response to stgmficantly htgher than m the artery ‘8 The concentratron chrome salt loading, one of the most powerful sttmuh of DA m renal lymph, however, 1s not higher than m the known to increase renal DA production Surprtsmgly, artery 19 In rats on a normal salt duet in the present study, however, RIF DA levels in rats on high sodium diet were RIF DA levels were much lower than UDAV, suggesting stgnificantly lower than durmg normal salt balance The that mtrarenally produced DA is released preferentially mechanism underlying this reduction with chrome salt mto the tubule lumen rather than peritubular space Cel- loading 1s not apparent. Acute isotomc salme loadmg m lular mechanisms of renal DA secretton are not under- conscious rabbits has been shown to mcrease urme DA stood DA tmmunoreacttve granules and L-dopa-induced excretton (threefold) without concurrent mcrease m renal fluorescence are mamly concentrated in the region of the DA spillover mto plasma 23 The DA prodrug gludopa pro- apical membrane of proximal tubule cells 20,*1 There may duced an 800-fold increase m UDAV without srgmficant be lumenal orgamc cation transporters with high affimty change m renal venous DA concentration in consctous rab- for DA m porcine proximal tubule (LLC-PK,) cells.22 btts 24 RIF DA may derive from tubular outward transport

INK DNK

NS HS FIG 2 UV, U,,V, U,,V, and RIF DA in anesthetized rats consum- Ing a normal salt (NS) or high salt (HS) d/et (n=9 In each group) *PC 05, **P< 01 vs N.S.

NS HS

INK DNK FIG 3 UV, &,V, UonV, and RIF DA in anesthetized rats with (INK, n=14) and without (DNK, n=iO) intact renal lnnervatlon *P= 05 vs INK

INK DNK INK DNK

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232 Hypertension Vo129, No 1, Part 2 January 1997

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z E = 5 2.

5

0 -90 -45 45 90

Time (min)

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P E

s E 6 500

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0

0 -90 -45 45 90

Time (min)

-90 -45 45 90

Time (min)

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of DA through the basolateral membrane. Histofluorescent entially into the luminal space where it may act on apical and nemochemical findings suggest the presence of do- DA receptors as an autocoid or paracrine factor. Therefore, paminergic neurons in the kidney and adrenergic nerves renal DA may act as an intrarenal regulator of kidney func- may also become dopaminergic under certain circum- stances.1 .Vagal afferents have been shown to stimulate

tion in highly compartmentalized fashion. DA receptors located in the brush border membrane may be exposed to

renal release of DA and produce a neurogenically medi- DA originating in the tubule with the physiological con- ated natriuresis.25 Our data confirm that the main source sequence of natriuresis and diuresis.6 On the other hand, of DA in the urine is nonneuronal, and further demonstrate DA receptors in the basolateral membrane, renal vascu- that renal nerve activity does not contribute significantly lature, and glomerulus may be exposed to DA released into to either urinary or interstitial fluid DA. During chronic the renal interstitium and possibly have a compartmental- salt loading, intrarenally produced DA is released prefer- ized role in the control of glomerular filtration rate and/or

** **

FIG 4. Effect of gludopa (GD; 3, 5, and 7.5 nmol/kg IV) on UV, U,,V, U,,V, and RIF DA in anes- thetized rats with intact renal in- nervation (n=7 in each group). Control values at -90 and -45 minutes before gludopa adminis- tration, experimental values at 45 and 90 minutes after gludopa ad- ministration. l P<.O5, **P<.Ol vs vehicle control.

FIG 5. Effect of gludopa (GD, 7.5 nmol/ka Iv) on UV. UN-V. U,,V,

-90 -45 45 90 -90 -45 45 90 and RIF DA in anesthetized rats

Time (min) Time (min) with prior bilateral renal denerva- tion (n=5 in each group). Control values at -90 and -45 minutes before gludopa administration, exoerimental values at 45 and 90

10 **

minutes after gludopa administra- tion. *P<.O5, **P<.Ol vs vehicle control.

I “ehlde

-90 -45 45 90 -90 90

Time (mln) Time (mln)

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Wang et al Renal Dopamine Production and Sodium Excretion 233

-so -60 -30 0 30 80 so

Time (mm)

B

Time (min)

FIG 6 Effects of gludopa (7 5 nmol/kg Iv) and angtotensin II (A II, 100 ng kg-’ mln-’ Iv) on rntrarenal corhcal (CBF) and med- ullaty (MBF) blood flow In anesthetized rats (n=6 in each group) *PC 05, **PC 01 vs predrug (Ttme 0)

sodium reabsorptlon.26 The functional slgmficance of ap- ical and basolateral DA receptors needs much further mvestlgatlon

Gludopa IS devoid of pharmacological activity per se, but 1s converted to L-dopa and then to DA by sequential actions of the brush border enzyme y-glutamyl transpep- tldase and cytosolic amino acid decarboxylase predomi- nantly m the proximal tubule cells, where both enzymes exist m abundance.27 Significant natrmresls and renal va- sodllatlon occurs m the whole animal and human after pharmacological increase of UoAV (more than 300-fold) engendered by gludopa. *6,27,2* Renal vasodilatlon 1s also observed in the isolated perfused rat ludney m response to micromolar range gludopa 29 In the present study m anes- thetized rats, much smaller quantities of gludopa (7 5 nmol/kg) resulted in a physlologlcal increase m UDAV (7.3-fold), accompanied by a slight increase m RIF DA (1.7-fold), and produced significant diuresls and natn- uresis without detectable changes m mtrarenal blood tlow It is of interest to note that the increase in renal DA pro- duction after chronic salt loading was associated with an Increase in U,,V but not when the increase was caused by low doses of gludopa Whether this 1s as a result of dietary salt loading-induced upregulation of kidney DA receptors or increased efficiency of receptor couplmg to signal trans- duction 1s unclear These results support preferential se- cretion of DA generated by proximal tubule cells mto the tubule lumen In the isolated perfused rat kidney, DA-m- duced natrmresis and dmresls was observed even when renal blood flow, glomerular filtration rate, and perfusion pressure remained constant 30 Our previous study m the

unmephrectomlzed conscious dog demonstrated that block- ade of the lenal DA-l receptor with mtrarenal infusion of SCH-23390 produced a 50% decrease m UV and UN,V, but there were no renal hemodynarnic changes accom- panying the antinatriuresls 6 Jose et a131 recently reported that a 5 2-fold increase in ludney DA content with gludopa administration was associated with decreased renal cortl- cal brush border Na+/H + antlporter activity and significant natriuresis (4 9-fold) and dmresls (2 &fold) without any change m glomerular filtration rate m anesthetized rats. Barendregt et al32 reported that low dose L-dopa mfuwon, which resulted m a fivefold to eightfold increase m ucme DA excretion, had a natriuretlc effect without any change in blood pressure or glomerular filtration rate in healthy subJects on low sodium diet. Taken together with the pres- ent results, these studies suggest that the natriuretic effect of gludopa 1s secondary to direct tubule inhibition of so- dium reabsorption by locally formed DA. These results strongly support the thesis that mtrarenal DA plays a role m the control of natrmresls through a tubule mechanism

The renal sympathetic nerve endings are closely related to theJuxtaglomerular apparatus and proximal tubule cells Whether the sympathetic nervous system influences m- trarenal DA generation and regulates renal DA-mediated natrmresis and dmresls 1s unknown Basal DA production (as reflected by UDAV and RIF DA) and UN,V and then responses to gludopa were similar between rats with and without chronic renal denervatlon. The observation 1s con- sistent with other studies, which showed that chronic renal denervation did not alter UDAV both basally and m re- sponse to increased dietary phosphate mtake.33$14 Our re- sults further demonstrate that mtrarenal productlon of DA (both UoAV and RIF DA) and its renal effects are not slg- mficantly modulated by renal sympathetic nerve activity

In summary, our data demonstrate renal mterstltlal ml- crodialysis can be used to monitor DA levels in the RIF m anesthetized rats. According to our results, DA pro- duced in the kidneys is released preferentially mto the tu- bule lumen and exerts a direct tubule effect m the control of UN,V Renal DA production and its renal effects were not significantly influenced by renal sympathetic nerve ac- tivity. The unique compartmentallzatlon of renal DA re- lease warrants further mvestlgatlon, especially at the cel- lular level.

Acknowledgments This work was supported m part by grant ROl-HL-49575 from

the National Institutes of Health to Dr R M Carey. The authors wish to thank Dawn M Fultz and Nancy L Howell for their skillful technical assistance

References Carey RM Dopamme, hypertension, and the potential for agomst therapy In Laragh JH, Brenner BM, eds Hypercenswn Pathophys- cology, Dzagnosrs, and Management 2nd ed New York, NY Raven Press Ltd, 1995 2937-2952 Carey RM, Slragy HM, Ragsdale NV, Howell NL, Felder RA, Peach MJ, Chevalier RL Dopamme-I and dopamme-2 mechanisms m the control of renal function Am J Hypertens 1990,3 59S-63s Soares-da-Sdva P Source and handlmg of renal dopamme Its phys- lological Importance News Physml Set 1994,9*128-134 Sun D, Schafer JA Dopamme mlublts AVP-dependent Nd+ transport and water permeablhty m iat CCD via a D-4-like receptor AWI J Physrol 1996,40 F391-F400 Hughes J, Beck T, Rose C Jr, Carey RM The effect of selective dopatnme-1 stlmulatlon on renal and adrenal fun&on m mdn d Chn Endocrmol Metab 1988,66 518-525

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Slragy HM, Felder RA, Howell NL, Chevaher RL, Peach MJ, Carey RM Evidence that mtrarenal dopamme acts as a paracrme substance dt the renal tubule Am J Phywl 1989,257 F469-F477 Siragy HM, Carey RM The subtype-2 (AT,) anglotensm receptor regulates renal cychc guanosine 3’,5’-monophoqphate and AT, re- ceptor-medlated prostaglandm E2 productmn m conscious rats J Clan Invest 1996,97 1978-1982 Sn’agy HM, Howell NL, Ragsdale NV, Carey RM Renal mterstttlal fluld angiotensm modulation by anesthesia, epmephrme, sodium de- pletion, and renm mhlbttton Hypertenston 1995:25 1021-1024 Stragy HM, Linden J Sodium intake markedly alters renal mterstmal flmd adenosme Hypertenszon 1996,27 404-407 Siragy HM, Johns RA, Peach MJ, Carey RM Nitric oxide alters renal function and guanosme 3’,5’-monophosphate Hype, tenston 1992, 19 775-779 Siragy HM, Jaffa AA, Margohus HS Stimulation of renal mterstttlal bradykmm by sodium depletion Am J Hypertens 1993,6 863-866 Siragy HM, Ibrahlm MM, Jaffa AA, Mayfield R, Margohus HS Rdt renal interstitial bradykmm, prostaglandm E2, and cychc guanosme 3’,5’-monophosphate effects of altered sodmm mtdke Hype! tenJum 1994,23 1068-1070 Baranowskl RL, WestenfelderC Estimation of renal mterstltlal aden- osme and purme metabohtes by microdlalysls Am J Phywl 1994, 267 F174-Fl82 Agmon Y, Dmour D, Brezts M Disparate effects of mtrarenal aden- osme Al and A2 agomsts upon mtrarenal blood flow Am .I Physrol 1993,265 F802-F806 Agmon Y, Peleg H, Greenfeld Z, Rosen S, Brezls M Nitric oxide and prostanolds protect the renal outer meddulla from radiocontrast toxi&y in the ra; J Clrn Invest 1994,94 1069-1075 Wang Z-Q, Way D, Shlmlzu K, Fona F, Trlga L, McGrath BP Bene&alacute-effects of selective midulatlon>f renal dopamme system by y-L-glutamyl-L-dopa m rabbits with congestive heart failure J Cardrovasc Pharmacol 1993,21 1004-1011 Sarre S, Mlchotte Y High-performance liquid chromatography with electrochemical detectton for the determmatlon of levodopa, cate- cholammes and their metabohtes in rat bram dlalysates J Chroma- togr 1992,575 207-212 Ball SG, Gunn IG, Douglas IHS Renal handling of dopa, nor- epmephrme, and epmephrme m the dog Am J Physrol 1982, 242 F56-F62 Stephenson RK, Sole MJ, Barnes AD Neural and extraneural cat- echolamme production by rat kidneys Am J Phystol 1982,242 F261-F266

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Zhi-Qin Wang, Helmy M. Siragy, Robin A. Felder and Robert M. CareySodium Excretion

Intrarenal Dopamine Production and Distribution in the Rat: Physiological Control of

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