Interaction of atria1 natriuretic and angiotensin II in proximal G. NASCIMENTO
GOMES
AND
factor HCO; reabsorption
M. MELLO
AIRES
Department of Physiology of Escola Paulista de Medicina, and Department of Physiology and Biophysics, Instituto de Cikncias Biomgdicas, Universidade de S&o Paulo, CEP 05508 S&o Paula, Brazil Gomes, G. Nascimento, and M. Mello Aires. Interaction of atria1 natriuretic factor and angiotensin in II in proximal HCO; reabsorption. An. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F303-F308, 1992.-Bicarbonate reabsorption was evaluated by the acidification kinetics technique in middle proximal tubule in Munich-Wistar rats. Atria1 natriuretic factor (ANF) and angiotensin II (ANG II) were infused into the jugular vein (ANF, 0.5 pg. min. kg-l after a prime of 10 pg/ kg; ANG II, 20 ng min-l *kg-l) or added to luminal or peritubular perfusion fluid ( lOa M ANF; lo-l2 M ANG II). In the presence of ANF, in each condition, no significant differences in net HCO; reabsorption or in acidification half time were observed compared with the control group. In the presence of ANG II, a significant increase in HCO; reabsorption was observed, expressed by a fall in acidification half time from a mean of 4.75 t 0.20 (n = 86) to 2.47 * 0.18 s (n = 32) in systemically infused rats or to 2.30 k 0.15 s (n = 35) in luminally perfused tubules and from 4.57 t 0.32 (n = 44) to 2.04 & 0.10 s (n = 50) during capillary perfusion. However, when ANG II was systemically infused or perfused in lumen or in peritubular capillaries, addition of ANF to lumen or capillaries by perfusion or systemic infusion abolished the effects observed with ANG II alone. These studies confirm that ANG II stimulates proximal HCO: reabsorption and show that ANF alone does not affect this process, but impairs the stimulation caused by ANG II. l
kinetics;
tubular
acidification
ATRIAL NATRIURETIC FACTORS (ANFs) are a family of peptides originating from mammalian heart atria, which present strong natriuretic and diuretic properties (9). The mechanism of this effect remains controversial. Several studies indicate that ANF acts directly on the kidney, probably by enhancing glomerular filtration rate (GFR) (8, 24, 30). However, clearance studies in rats, showed an increased phosphaturia even without an increase in GFR, suggesting a direct inhibition of Na+PO:- cotransport in proximal convoluted tubule (PCT) (19). Hammond et al. (16) found that Na+-PO:- cotransport and Na+-H+ exchange in brush-border vesicles from ANF-treated rats were inhibited compared with controls. More recently, Yusufi et al. (36) have demonstrated that the inhibition of phosphate transport occurs in brushborder vesicles from both superficial and juxtamedullary nephrons. It remains unclear whether ANF has a direct effect on PCT or whether the observed diuresis and natriuresis are the result of an increased fluid flow along this nephron segment caused by the increased GFR. Because several of these studies were performed in vivo and functional cholinergic receptors on the basolateral side of the PCT can regulate HCO; reabsorption (Jn& and fluid flux (JJ (34), systemic effects of ANF and the influence of the renal nerves could mask a direct action of ANF on the proximal nephron. THE
0363-6127/92
$2.00 Copyright
Angiotensin II (ANG II) is an octapeptide naturally found in blood, with a physiological concentration of the order of 1-5 X 10-l’ M (1, 11, 15). Evidence obtained from in vivo micropuncture studies in isolated perfused tubules and receptor binding studies indicate that ANG II exerts an important influence on both renal hemodynamics and tubular reabsorptive function (14, 17, 30). Liu and Cogan (21) have shown that ANG II caused a substantial increase in HCO; reabsorption in the proximal nephron, and thus it may act as a regulator of renal acidification. A recent report (13) suggests that ANG II stimulates both Na+-H+ exchange and Na+-HCOT cotransport in perfused Sl segments of proximal tubules isolated from superficial nephrons of the rabbit kidney. An interaction between ANF and ANG II has been observed in a variety of tissues. ANF inhibits the vasoconstrictor effect of ANG II on blood vessels in vitro (20), as well as the systemic pressor action of ANG II (10) and ANG II-stimulated aldosterone synthesis (2). Using the split-droplet technique, Harris et al. (18) found that ANF had an inhibitory effect on proximal fluid absorption stimulated by preliminary ANG II perfusion. Similar results were obtained by Garvin (12) in isolated perfused proximal straight tubules. However, the same effect was not observed by Liu and Cogan (22) during in vivo free-flow micropuncture experiments. This study was performed to examine the effects and interaction of ANG II and ANF on the kinetics of HCO; reabsorption in middle proximal tubule during in vivo stopped-flow microperfusion experiments. In this preparation the role of the systemic effects of both hormones were eliminated during luminal or peritubular capillary perfusion. METHODS
Female Munich-Wistar rats weighing 180-220 g, anesthetized with Inactin (100 mg/kg ip), were prepared for in vivo micropuncture (25). The kinetics of acidification were studied by means of a stationary microperfusion technique with continuous measurement of intratubular pH, as previously described (25). Briefly, the PCT was perfused by means of a double-barrelled micropipette, one barrel filled with Sudan black-colored castor oil and the other with the perfusion solution containing (in mM) 100 NaCl, 25 NaHC03, 5 KCl, 1 CaC12, and 1.2 MgS04. The osmolality was adjusted to 300 mosmol/kgH20 with raffinose. The intratubular pH was measured by means of an Sb microelectrode (33), continuously recorded on a polygraph (Beckman RS Dynograph), and digitized by a PC-type microcomputer system (Microtec XT 2002) with an analog-to-digital conversion board (Data Translation DT 2801) by which data were acquired and processed. The kinetics of HCO; reabsorption were measured by inject-
0 1992 the American
Physiological
Society
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F303
F304
ANF AND ANG II IN PROXIMAL
ing a droplet of the perfusion solution betweenoil columnsand following the intratubular pH changestoward the steady-state level (pHs). From thesepH valuesand from the systemicpartial pressureof carbon dioxide (Pco~) the concentration of HCO; was calculated in intervals of 0.2 s by use of the HendersonHasselbalchequation. We have previously demonstrated that renal cortical PCO~ is similar to that of arterial blood (26). The concentration of HCO: fell exponentially toward its stationary level. The velocity of tubular acidification was evaluated by meansof the half time of reabsorption of the injected HCO,. Net HCO: reabsorption wascalculated from the equation J HCO, = k([HCO& - [HCO;]s)rl2 where lz is the rate constant of the reduction of luminal bicarbonate [12= ln2/( t/2), where t/2 is the half time of reabsorption of the injected HCO;], r is the tubule radius, and [HCO& and [HCO& are the concentrations of the injected HCO, and HCO; at the stationary level, respectively. Sb microelectrodes were calibrated in HP04 Ringer buffer similar in composition to that usedto fill the glassdouble-barrelled micropipette, but HCOi was substituted for phosphate and adjusted to pH 6.5, 7.0, and 7.4. Appropriate anion correction wasperformed when measuringHCOT-containing solutions (23). Peritubular capillary perfusion was performed with a solution containing (in mM) 140 NaCl, 20 NaHCOa, 5.0 KCl, 1.0 CaC&, 1.2 MgSO,, and 5.0 NaCH&02, at pH 7.4. This solution waspreequilibrated with 5% COZin air. Twenty-eight-amino acid ANF (Bachem Fine Chemicals, New Haven, CT) was infused at 0.5 pgomin-lgkg-l, after a prime of 10 pg/kg, or was added to luminal or peritubular perfusion fluid (lo-" M). ANG II (1,046 mol wt; Biophysical Laboratory, Escola Paulista de Medicina, Brazil) was either infused at a rate of 20 ng min. kg-l or addedto luminal or to the peritubular perfusion solution (lo-l2 M). To measurethe GFR, inulin was infused at 5 mg*min. kg-l after a prime of 300 mg/kg. The groupsof rats studiedwere selectedto combine severalpossibilitiesof drug administration, i.e., Control; control with peritubular capillary perfusion; ANF systemically infused; ANF luminal perfusion; ANF peritubular capillary perfusion; ANG II systemically infused; ANG II luminal perfusion; ANG II peritubular capillary perfusion; ANG II systemically infused plus ANF peritubular capillary perfusion; ANF systemically infused plus ANG II peritubular capillary perfusion; ANF plus ANG II luminal perfusion; and ANF plus ANG II peritubular capillary’perfusion. The pH and PCO~ in samplesof blood collected from the carotid artery were measuredwith a Radiometer PHM 72 MK2 digital acid-baseanalyzer. Na+ in urine collected from urinary bladder was measuredby flame photometry. Inulin in plasma and urine wasmeasuredby calorimetry. Data are means t SE; n is the number of measurements. Differences between experimental groups were evaluated by Student’s t test and by analysis of variance with the Scheffe contrast test when more than two groupswere compared (29).
HCO;
REABSORPTION
Table
1. Effects of systemic administration of ANF of ANG II on urine flow, glomerular filtration rate, and excreted amount of sodium
GFR, uIG+t X n ml min-l .kg-’ ml. min-l kg-l peg min-l kg-l Control 0.078t0.009 7.53kO.61 8.62k1.33 11 ANF 0.168*0.067* lO.lt0.73* 19.5+3.23t 9 ANG II 0.05lt0.007" 5.49kO.62" 4.22t0.79" 12 Values are means t SE; n, number of measurements. V, urine flow; GFR, glomerular filtration rate; UNa+V, sodium excretion; ANF, atria1 natriuretic factor. Both ANF and ANG II were systemically infused. * Significantly different from control value (P < 0.05). t Significantly different from control value (P < 0.01). l
l
l
l
-4--- AP
l
RESULTS
We confirmed that systemic administration of ANF increased urinary flow, GFR, and Na+ excretion in a highly significant manner, whereas ANG II decreased urinary flow and GFR, as well as Na+ excretion (Table 1). During luminal or capillary perfusion with ANF, ANG II, or ANF + ANG II, the urinary flow and the Na+ excretion were similar to the control values (Fig. 1). In all experimental groups no significant differences between pHs or [HCO& were observed, suggesting that the main driving force for H+ secretion (i.e., the Na+
I ANF Fig. 1. Sodium mental/control) sions with atria1 during 5 min; n
AN8
It
ANF+ANB
IT:
excretion (&+V) and urine flow (V) ratios (experiin rats receiving luminal (0) or peritubular (A) perfunatriuretic factor (ANF) and/or ANG II for 2-3 periods = 5 measurements in each group.
gradient across the apical membrane) is not altered. Table 2 gives acidification data in PCT of rats infused systemically with ANF or with luminal or peritubular capillary perfusion of ANF-containing solution. The controls of the latter group were PCT undergoing peritubular capillary perfusion with the same solution but without addition of ANF. In these experiments no significant differences between J Hco; or half time of acidification (t/2) was noted. Table 2 also gives acidification data in PCT of rats systemically infused with ANG II and in tubules undergoing luminal or capillary perfusion with ANG IIcontaining solution. A significant increase in &co3_ was found both in systemically ANG II-infused rats or during luminal or capillary perfusion with ANG II. This increase was due to a fall in acidification half time with the use of ANG II. Figure 2 compares the results of experiments in which proximal tubules were first perfused in control condi-
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ANF AND ANG II IN PROXIMAL
Table 2. Acidification data obtained in proximal convoluted tubule during systemic, luminal, or peritubular administration of ANF or ANG II t/2,
HCO,
0
CONT
q ANF-SYST
m
ANG II:-SYST
a
CONT-PERI
ANG II-PER)
u
ANF-SYST+ANG
J l-E059
n crnB2.s-’ Control 4.75kO.20 2.30t0.10 86 Control, peritubular 4.57kO.32 1.83t0.14 44 ANF, systemic 3.89kO.30 2.20t0.24 27 ANF, lumen 4.05kO.32 2.18t0.25 33 ANF, peritubular 3.85k0.24 1.88t0.16 50 ANG II, systemic 2.47*0.18* 3.37kO.22" 32 ANG II, lumen 2.30+0.15* 3.94t0.35" 35 ANG II, peritubular 2.04+0.10t 4.10+0.22t 50 Values are means t SE; n, number of measurements. Jncos_, net bicarbonate reabsorption; t/2, half time of acidification. * Significantly different from control value (P < 0.01). t Significantly different from peritubular control value (P < 0.01). S
nmol
l
4
CI 1
(86)
cn2 N‘ :
0 v I 3
0 I
(8)
(8)
I C
ANGII- SYST
I
(8) I 1 ANCII-SST’
ANFhRI Fig. 2. Stimulatory effect of systemic administration of ANG II on kinetics of proximal tubule acidification is immediately reversed by addition of ANF in peritubular (Peri) capillary perfusion. C, control; ANG II-Syst, effect of ANG II infused at a rate of 20 ngomin-lg kg? ANG II-Syst + ANF-Peri, effect of 10m6M ANF added to peritubular Capillary perfusion maintaining systemic infusion of ANG II. Values are means * SE; number of measurements is shown in parentheses. &co;, net HCO; reabsorption; t/2, half time of acidification. Eightpointed star, significantly different from control value (P < 0.05). * Significantly different from ANG II-Syst value (P < 0.05).
tions, then during systemic infusion of ANG II, and finally during capillary perfusion with ANF when the systemic infusion of ANG II is maintained. These experiments show that ANF in the capillary perfusion inhibits the stimulation of proximal tubular HCO: transport induced by systemic administration of ANG II in the same PCT. We also studied acidification data in PCT of rats systemically infused with ANF and with capillary perfusion containing ANG II. The results obtained are shown in Fig. 3. Also in these conditions, ANF caused an inhibition of the stimulatory effect of ANG II on the half time of acidification, which increased from 2.04 t 0.10 (n = 50) to 4.52 t 0.30 s (n = 42) and consequently on &co,, which fell from 4.10 t 0.22 (n = 50) to 1.55 t 0.16 nmol . cmD2 s-l (n = 42). Figure 4 gives data obtained in PCT undergoing peril
F305
REABSORPTION
* I* P (32)
(44)
It-PER1
(SO)
Fig. 3. Systemic infusion of ANF impairs stimulatory effect of peritubular capillary perfusion of ANG II on kinetics of proximal tubule acidification. Top: effect on acidification half time. Bottom: effect on net HCOi reabsorption. Cont, control. ANF-Syst, effect of ANF infused at a rate of 0.5 pgomin-’ *kg-’ after a prime of 10 pg/kg. ANG II-Syst, effect of ANG II infused at a rate of 20 ng= min-‘. kg-‘. Cont-Peri, effect of control peritubular capillary perfusion. ANG II-Peri, effect of lo-l2 M ANG II added to peritubular capillary perfusion. ANF-Syst + ANG II-Peri, effect of ANG II added to peritubular capillary perfusion maintaining systemic infusion of ANF. Values are means t SE; number of measurements is shown in parentheses. * Significantly different from Cont value (P < 0.01). Eight-pointed star, significantly different from Cont-Peri value (P < 0.01). * Significantly different from ANG II-Peri value (P < 0.01).
tubular capillary perfusion with ANF and ANG II. The perfusion of ANF in the peritubular capillary also caused an inhibition of the stimulatory effect of ANG II in peritubular capillary perfusion; the half time of acidification increased to 3.96 t 0.43 s (n = 34), and the J HCo; decreased to 1.56 t 0.11 nmol . crne2 s-l (n = 34). Figure 5 shows experiments that demonstrate that ANF perfused in the lumen also inhibits the stimulation of proximal HCO: transport induced by luminal perfusion of ANG II. The half time of acidification increased from 2.30 t 0.15 s (n = 35) during luminal perfusion with ANG II to 4.07 t 0.31 s (n = 46) when ANF was added, and the J HCo; decreased from 3.94 t 0.35 (n = 35) with ANG II to 2.21 t 0.15 nmo10cm-2s-1 (n = 46) with ANG II and ANF. l
DISCUSSION
Effect of ANF on tubular HCO; reabsorption. Since the discovery of ANF in 1981 (9), there has been substantial controversy regarding its diuretic and natriuretic action mechanisms. Several studies performed in different species have demonstrated a large increase in GFR after ANF infusion
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F306
ANF AND ANG II IN PROXIMAL
HCO,
REABSORPTION
0
CONT.PERI
q ANF-PERI
0
w
ANO
m
l ANGII-LUMEN
II-PER)
ANF-TERI ANO
‘IT-PER1
0:?:: ANF-LUMEN
CONT
ANF-LUMEN + ANG IT-LUMEN
W a
LI
l .* ...
1:: .‘.:.:.:.: ::: ....... ....... : : : .‘.‘.‘. .*.::::*:* :::
.“‘::: .
.*.:.: :::
’ . ....
l
.‘.‘.‘.
.‘.I.: . . . . . .*.. ::I.‘.‘.‘. :y::. *::: .*.::::::: : : : .‘.‘.‘.. . . . . . . . . . .. .. . ::I.‘.’ .*:* ::::::A’ .*.* :::::::y :y::.*.* . . .“‘::: :::.*.* . . ......
Fig. 4. Stimulatory effect of peritubular capillary perfusion of ANG II on kinetics of proximal tubule acidification is abolished by addition of ANF to peritubular capillary perfusion. Top: effect on acidification half time. Bottom: effect on net HCO; reabsorption. ANF-Peri, effect of low6 M ANF added to peritubular capillary perfusion. ANF-Peri + ANG II-Peri, effect of ANF plus ANG II added to peritubular capillary perfusion. Values are means t SE; number of measurements is shown in parentheses. Eight-pointed star, significantly different from ContPeri value (P < 0.01). * Significantly different from ANG II-Peri value (P c 0.01). Cont-Peri and ANG II-Peri groups are same as included in Fig. 3.
(4-6, 32). Attempts to identify a direct tubular effect have led to conflicting results. Free-flow micropuncture studies are difficult to interpret because of the marked effect of ANF on GFR and the tight link between GFR and proximal tubular Na+ reabsorption. Studies in vitro that rule out the influence of GFR yielded conflicting results. One study showed decreased activity of Na+PO:- and Na+-H+ transport in vesicles obtained from animals treated with ANF (16); on the other hand, studies in isolated perfused tubules failed to demonstrate any direct tubular effect of ANF (3). To test the possibility that ANF is active when applied in vivo but ineffective if administered to isolated systems, Capasso et al. (7) performed studies using the shrinking-droplet technique in PCT in vivo and measured the oxygen consumption of in vitro proximal tubules under the influence of ANF; but their results indicated that the enhancement of renal Na+ excretion induced by ANF is not related to a direct inhibition of Na+ transport in proximal tubule in both preparations. We examined the effect of systemic infusion of ANF in vivo, by a stopped-flow microperfusion technique, which is not affected by GFR. We also examined the action of ANF infused into the lumen or into the peritubular capillary. This procedure avoids systemic alterations caused by ANF infusion confirmed by absence of changes in urine flow and Na+ excretion unde r these conditions. Another ad vantage of the luminal or peritubular perfusion is to ascertain that the measurements of
[I (86)
. . .. . . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . . ........ . . ... ... ... ... ... ... .. .. ........ . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . . ........ . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . .
. . .. .
rl
(33)
(46)
Fig. 5. Stimulatory effect of luminal perfusion of ANG II on kinetics of proximal tubule acidification is abolished by addition of ANF to luminal perfusion. Top: effect on acidification half time. Bottom: effect on net HCO; reabsorption. ANF-Lumen, effect of 10D6 M ANF added to luminal perfusion. ANG II-Lumen, effect of lo-l2 M ANG II added to luminal perfusion. ANF-Lumen + ANG II-Lumen, effect of ANF plus ANG II added to luminal perfusion. Values are means t SE; number of measurements is shown in parentheses. Eight-pointed star, significantly different from Cont value (P < 0.01). * Significantly different from ANG II-Lumen value (P < 0.01). Cont group is same as included in Fig. 3.
acidification kinetics are performed during the period of highest activity of ANF, as it is known that this peptide has a transient action when injected parenterally. In all experimental groups, we failed to demonstrate a direct effect of ANF on proximal HCO: reabsorption. Role of ANG II in HCO: reabsorption. The effects of ANG II on the kinetics of bicarbonate reabsorption infused systemically, in the lumen or into the peritubular capillary, were also studied. A significant increase in HCO: reabsorption was observed, expressed by a fall in acidification half time from a mean of 4.75 to 2.47 s in systemically infused rats, or to 2.30 s during luminal perfusion, and from 4.57 to 2.04 s in capillary-perfused tubules. In normal conditions ANG II may be stimulating HCO: reabsorption in PCT. However, it is possible that this stimulation might be absent in our experimental conditions because of the moderate volume expansion to which the rats were subjected. Our results agree with the studies of Liu and Cogan (21, 22), who have shown by microperfusion and determination of total COn by microcalorimetry an important stimulation of HCO: reabsorption in the proximal tubule induced by systemic infusion of ANG II. This effect was
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ANF
AND
ANG
II IN
PROXIMAL
amiloride sensitive, which implies that the Na+-H+ antiporter is an important participant in the enhanced acidification stimulated by ANG II (22). The results by Valega et al. (31), showing activation of Na+-H+ exchange by ANG II in culture of vascular smooth muscle, and by Wang and Chan (35), indicating that ANG II increases HCO: and fluid reabsorption in PCT, support this hypothesis. The effect of ANG II and ANF during luminal perfusion raises the question of the distribution of receptors for these peptides on the cell surface. There appear to be such receptors also on the luminal cell membrane; it is, however, possible that these substances may reach the basolateral membrane via the paracellular shunt path during luminal perfusion. Interaction of ANF with ANG II. Harris et al. (18) studied the effect of ANF on proximal fluid absorption in rats pretreated with ANG II using the shrinkingdroplet technique. When they first treated proximal tubules with lo-l2 M ANG II, 2 x 10-l’ M ANF inhibited fluid absorption by 27%. This result agrees with the 35% inhibition reported by Garvin (12) for the proximal straight tubule. In contrast, Liu and Cogan (22) were not able to detect inhibition of fluid absorption, with or without angiotensin, during in vivo free-flow micropuncture experiments; however, they infused both hormones intravenously, raising the possibility that systemic effects of the hormones might have affected their data. Our data indicate that the stimulatory effect of ANG II on the kinetics of proximal tubule acidification is abolished by systemic infusion of ANF or by addition of ANF in luminal or peritubular capillary perfusion. According to Harris et al. (18), the range of angiotensin concentrations that permit ANF to inhibit fluid absorption lies between lo-l2 and 10mfOM; the concentration used in our study in lumen or in peritubular capillaries is within this range. During systemic infusion, the exact concentration of ANF in the kidney is unknown, but the results show that ANF does inhibit ANG II-stimulated HCOT reabsorption in the proximal nephron by more than 50%. Our results agree with the idea that the proximal nephron effects of ANF could account for a significant portion of the natriuretic and diuretic action as proposed by Harris et al. (18), Garvin (12), and Salazar et al. (28). The interaction of ANG II and ANF is not yet understood, but guanosine 3’,5’-cyclic monophosphate appears to be part of the second messenger cascade for ANF in the proximal nephron, as it is in other tissues (12). Because ANG II causes a direct and substantial stimulation of both luminal Na+-H+ exchange and basolateral Na+-HCO; cotransport (13), it is possible that ANF may inhibit the luminal and basolateral steps of transepithelial HCO; reabsorption induced by ANG II. We thank Dr. Gerhard Malnic, Universidade de S&o Paulo, Brazil, for critical reading of the manuscript. Address for reprint requests: M. Mello Aires, Dept. of Physiology and Biophysics, Instituto de Ciencias Biomedicas, Universidade de S&o Paulo, CP 4365 Sgo Paulo, Brazil. Received 1 October 1990; accepted in final form 10 September 1991. REFERENCES 1. Aguilera, G. A., A. Schirar, A. Baukal, and K. J. Catt. Angiotensin II receptors: properties and regulation in adrenal
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J. Physiol. 1988. 23.
1987.
Liu, F. Y., and M. G. Cogan. Atria1 natriuretic factor does not inhibit basal or angiotensin II-stimulated proximal transport. Am. 255 (Renal
Fluid
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Physiol.
24): F434-F437,
Lopes, A. G., M. Mello Aires, and G. Malnic. Behavior of the antimony microelectrode in different buffer solutions. Bruz. J. Med.
Biol.
Res. 14: 29-36,
1981.
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ANF
AND
ANG
II IN
PROXIMAL
24. Maack, T., M. J. F. Camargo, H. D. Kleinert, J. H. Laragh, and S. H. Atlas. Atria1 natriuretic factor: structure and functional properties. Kidney Int. 27: 607-615,1985. 25. Malnic, G., and M. Mello Aires. Kinetics study of bicarbonate reabsorption in proximal tubule of the rat. Am. J. Physiol. 220: 1759-1767,197l. 26. Mello Aires, M., M. J. Lopes, and G. Malnic. PCO~ in renal cortex. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28):
F357-F365,1990. 27. Navar,
L.
angiotensin
and L. Rosivall. Contribution of the reninsystem to the control of intrarenal hemodynamics.
G.,
Kidney Int. 25: 857-868,1984. 28. Salazar, F. J., J. P. Granger, Bentley, and J. C. Romero.
M.
J. Fisken-Olsen,
M.
D.
Possible modulatory role of angiotensin II on atria1 peptide-induced natriuresis. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F880-F883, 1987.
29. Snedecor,
G.
W.,
and
W.
G.
Cochran.
Statistical
Methods.
Ames: Iowa State Univ. Press, 1967, p. 271-275. 30. Sosa, Atlas,
R. E., M. Volpe, J. Marion, J. H. Laragh, and T. Maack.
hemodynamics and natriuretic
E. D. Vaughan,
HCO;
Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F520F524,1986. 31. Vallega, G. A., M. L. Canessa, B. C. Berk, T. A. Brock, and R. W. Alexander. Vascular smooth muscle Na’/H+ exchanger kinetics and its activation by angiotensin II. Am. J. Physiol. 254 (Cell Physiol. 23): C571-C578, 1988. A., K. Blouch, and R. J. Janison. Effect of 32* Van De Stolpe,
atria1 natriuretic peptid (ANP) on the superficial proximal tubule and loop of Henle (Abstract). Kidney Int. 33: 289, 1988. 33. Vieira, F. L., and G. Malnic. Hydrogen ion secretion by rat renal cortical tubules as studied by an antimony microelectrode. Am. J. Physiol. 214: 710-718, 1968. 34. Wang, T., and Y. L. Chan. Cholinergic effect on rat proximal convoluted tubule. Pfluegers Arch. 415: 533-539, 1990. 35 Wang, T., and Y. L. Chan. Mechanism of angiotensin II action on proximal tubular transport. J. Pharmacol. Exp. Ther. 252: 689l
36
695,199O.
. Yusufi, P. Dousa,
Jr.,
S. A.
Relationship between renal effects of atria1 natriuretic factor.
REABSORPTION
A. N. K., T. J. Berndt, H. Moltaji, V. Donovan, and F. G. Knox. Rat atria1 natriuretic factor
T.
(ANPmembrane from
III) inhibits phosphate transport in brush-border superficial and justamedullary cortex. Proc. Sot. Exp.
Biol.
190: 87-90,1989.
Downloaded from www.physiology.org/journal/ajprenal at Karolinska Institutet University Library (130.237.122.245) on February 12, 2019.
Med.
GOMES
AND
factor HCO; reabsorption
M. MELLO
AIRES
Department of Physiology of Escola Paulista de Medicina, and Department of Physiology and Biophysics, Instituto de Cikncias Biomgdicas, Universidade de S&o Paulo, CEP 05508 S&o Paula, Brazil Gomes, G. Nascimento, and M. Mello Aires. Interaction of atria1 natriuretic factor and angiotensin in II in proximal HCO; reabsorption. An. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F303-F308, 1992.-Bicarbonate reabsorption was evaluated by the acidification kinetics technique in middle proximal tubule in Munich-Wistar rats. Atria1 natriuretic factor (ANF) and angiotensin II (ANG II) were infused into the jugular vein (ANF, 0.5 pg. min. kg-l after a prime of 10 pg/ kg; ANG II, 20 ng min-l *kg-l) or added to luminal or peritubular perfusion fluid ( lOa M ANF; lo-l2 M ANG II). In the presence of ANF, in each condition, no significant differences in net HCO; reabsorption or in acidification half time were observed compared with the control group. In the presence of ANG II, a significant increase in HCO; reabsorption was observed, expressed by a fall in acidification half time from a mean of 4.75 t 0.20 (n = 86) to 2.47 * 0.18 s (n = 32) in systemically infused rats or to 2.30 k 0.15 s (n = 35) in luminally perfused tubules and from 4.57 t 0.32 (n = 44) to 2.04 & 0.10 s (n = 50) during capillary perfusion. However, when ANG II was systemically infused or perfused in lumen or in peritubular capillaries, addition of ANF to lumen or capillaries by perfusion or systemic infusion abolished the effects observed with ANG II alone. These studies confirm that ANG II stimulates proximal HCO: reabsorption and show that ANF alone does not affect this process, but impairs the stimulation caused by ANG II. l
kinetics;
tubular
acidification
ATRIAL NATRIURETIC FACTORS (ANFs) are a family of peptides originating from mammalian heart atria, which present strong natriuretic and diuretic properties (9). The mechanism of this effect remains controversial. Several studies indicate that ANF acts directly on the kidney, probably by enhancing glomerular filtration rate (GFR) (8, 24, 30). However, clearance studies in rats, showed an increased phosphaturia even without an increase in GFR, suggesting a direct inhibition of Na+PO:- cotransport in proximal convoluted tubule (PCT) (19). Hammond et al. (16) found that Na+-PO:- cotransport and Na+-H+ exchange in brush-border vesicles from ANF-treated rats were inhibited compared with controls. More recently, Yusufi et al. (36) have demonstrated that the inhibition of phosphate transport occurs in brushborder vesicles from both superficial and juxtamedullary nephrons. It remains unclear whether ANF has a direct effect on PCT or whether the observed diuresis and natriuresis are the result of an increased fluid flow along this nephron segment caused by the increased GFR. Because several of these studies were performed in vivo and functional cholinergic receptors on the basolateral side of the PCT can regulate HCO; reabsorption (Jn& and fluid flux (JJ (34), systemic effects of ANF and the influence of the renal nerves could mask a direct action of ANF on the proximal nephron. THE
0363-6127/92
$2.00 Copyright
Angiotensin II (ANG II) is an octapeptide naturally found in blood, with a physiological concentration of the order of 1-5 X 10-l’ M (1, 11, 15). Evidence obtained from in vivo micropuncture studies in isolated perfused tubules and receptor binding studies indicate that ANG II exerts an important influence on both renal hemodynamics and tubular reabsorptive function (14, 17, 30). Liu and Cogan (21) have shown that ANG II caused a substantial increase in HCO; reabsorption in the proximal nephron, and thus it may act as a regulator of renal acidification. A recent report (13) suggests that ANG II stimulates both Na+-H+ exchange and Na+-HCOT cotransport in perfused Sl segments of proximal tubules isolated from superficial nephrons of the rabbit kidney. An interaction between ANF and ANG II has been observed in a variety of tissues. ANF inhibits the vasoconstrictor effect of ANG II on blood vessels in vitro (20), as well as the systemic pressor action of ANG II (10) and ANG II-stimulated aldosterone synthesis (2). Using the split-droplet technique, Harris et al. (18) found that ANF had an inhibitory effect on proximal fluid absorption stimulated by preliminary ANG II perfusion. Similar results were obtained by Garvin (12) in isolated perfused proximal straight tubules. However, the same effect was not observed by Liu and Cogan (22) during in vivo free-flow micropuncture experiments. This study was performed to examine the effects and interaction of ANG II and ANF on the kinetics of HCO; reabsorption in middle proximal tubule during in vivo stopped-flow microperfusion experiments. In this preparation the role of the systemic effects of both hormones were eliminated during luminal or peritubular capillary perfusion. METHODS
Female Munich-Wistar rats weighing 180-220 g, anesthetized with Inactin (100 mg/kg ip), were prepared for in vivo micropuncture (25). The kinetics of acidification were studied by means of a stationary microperfusion technique with continuous measurement of intratubular pH, as previously described (25). Briefly, the PCT was perfused by means of a double-barrelled micropipette, one barrel filled with Sudan black-colored castor oil and the other with the perfusion solution containing (in mM) 100 NaCl, 25 NaHC03, 5 KCl, 1 CaC12, and 1.2 MgS04. The osmolality was adjusted to 300 mosmol/kgH20 with raffinose. The intratubular pH was measured by means of an Sb microelectrode (33), continuously recorded on a polygraph (Beckman RS Dynograph), and digitized by a PC-type microcomputer system (Microtec XT 2002) with an analog-to-digital conversion board (Data Translation DT 2801) by which data were acquired and processed. The kinetics of HCO; reabsorption were measured by inject-
0 1992 the American
Physiological
Society
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F303
F304
ANF AND ANG II IN PROXIMAL
ing a droplet of the perfusion solution betweenoil columnsand following the intratubular pH changestoward the steady-state level (pHs). From thesepH valuesand from the systemicpartial pressureof carbon dioxide (Pco~) the concentration of HCO; was calculated in intervals of 0.2 s by use of the HendersonHasselbalchequation. We have previously demonstrated that renal cortical PCO~ is similar to that of arterial blood (26). The concentration of HCO: fell exponentially toward its stationary level. The velocity of tubular acidification was evaluated by meansof the half time of reabsorption of the injected HCO,. Net HCO: reabsorption wascalculated from the equation J HCO, = k([HCO& - [HCO;]s)rl2 where lz is the rate constant of the reduction of luminal bicarbonate [12= ln2/( t/2), where t/2 is the half time of reabsorption of the injected HCO;], r is the tubule radius, and [HCO& and [HCO& are the concentrations of the injected HCO, and HCO; at the stationary level, respectively. Sb microelectrodes were calibrated in HP04 Ringer buffer similar in composition to that usedto fill the glassdouble-barrelled micropipette, but HCOi was substituted for phosphate and adjusted to pH 6.5, 7.0, and 7.4. Appropriate anion correction wasperformed when measuringHCOT-containing solutions (23). Peritubular capillary perfusion was performed with a solution containing (in mM) 140 NaCl, 20 NaHCOa, 5.0 KCl, 1.0 CaC&, 1.2 MgSO,, and 5.0 NaCH&02, at pH 7.4. This solution waspreequilibrated with 5% COZin air. Twenty-eight-amino acid ANF (Bachem Fine Chemicals, New Haven, CT) was infused at 0.5 pgomin-lgkg-l, after a prime of 10 pg/kg, or was added to luminal or peritubular perfusion fluid (lo-" M). ANG II (1,046 mol wt; Biophysical Laboratory, Escola Paulista de Medicina, Brazil) was either infused at a rate of 20 ng min. kg-l or addedto luminal or to the peritubular perfusion solution (lo-l2 M). To measurethe GFR, inulin was infused at 5 mg*min. kg-l after a prime of 300 mg/kg. The groupsof rats studiedwere selectedto combine severalpossibilitiesof drug administration, i.e., Control; control with peritubular capillary perfusion; ANF systemically infused; ANF luminal perfusion; ANF peritubular capillary perfusion; ANG II systemically infused; ANG II luminal perfusion; ANG II peritubular capillary perfusion; ANG II systemically infused plus ANF peritubular capillary perfusion; ANF systemically infused plus ANG II peritubular capillary perfusion; ANF plus ANG II luminal perfusion; and ANF plus ANG II peritubular capillary’perfusion. The pH and PCO~ in samplesof blood collected from the carotid artery were measuredwith a Radiometer PHM 72 MK2 digital acid-baseanalyzer. Na+ in urine collected from urinary bladder was measuredby flame photometry. Inulin in plasma and urine wasmeasuredby calorimetry. Data are means t SE; n is the number of measurements. Differences between experimental groups were evaluated by Student’s t test and by analysis of variance with the Scheffe contrast test when more than two groupswere compared (29).
HCO;
REABSORPTION
Table
1. Effects of systemic administration of ANF of ANG II on urine flow, glomerular filtration rate, and excreted amount of sodium
GFR, uIG+t X n ml min-l .kg-’ ml. min-l kg-l peg min-l kg-l Control 0.078t0.009 7.53kO.61 8.62k1.33 11 ANF 0.168*0.067* lO.lt0.73* 19.5+3.23t 9 ANG II 0.05lt0.007" 5.49kO.62" 4.22t0.79" 12 Values are means t SE; n, number of measurements. V, urine flow; GFR, glomerular filtration rate; UNa+V, sodium excretion; ANF, atria1 natriuretic factor. Both ANF and ANG II were systemically infused. * Significantly different from control value (P < 0.05). t Significantly different from control value (P < 0.01). l
l
l
l
-4--- AP
l
RESULTS
We confirmed that systemic administration of ANF increased urinary flow, GFR, and Na+ excretion in a highly significant manner, whereas ANG II decreased urinary flow and GFR, as well as Na+ excretion (Table 1). During luminal or capillary perfusion with ANF, ANG II, or ANF + ANG II, the urinary flow and the Na+ excretion were similar to the control values (Fig. 1). In all experimental groups no significant differences between pHs or [HCO& were observed, suggesting that the main driving force for H+ secretion (i.e., the Na+
I ANF Fig. 1. Sodium mental/control) sions with atria1 during 5 min; n
AN8
It
ANF+ANB
IT:
excretion (&+V) and urine flow (V) ratios (experiin rats receiving luminal (0) or peritubular (A) perfunatriuretic factor (ANF) and/or ANG II for 2-3 periods = 5 measurements in each group.
gradient across the apical membrane) is not altered. Table 2 gives acidification data in PCT of rats infused systemically with ANF or with luminal or peritubular capillary perfusion of ANF-containing solution. The controls of the latter group were PCT undergoing peritubular capillary perfusion with the same solution but without addition of ANF. In these experiments no significant differences between J Hco; or half time of acidification (t/2) was noted. Table 2 also gives acidification data in PCT of rats systemically infused with ANG II and in tubules undergoing luminal or capillary perfusion with ANG IIcontaining solution. A significant increase in &co3_ was found both in systemically ANG II-infused rats or during luminal or capillary perfusion with ANG II. This increase was due to a fall in acidification half time with the use of ANG II. Figure 2 compares the results of experiments in which proximal tubules were first perfused in control condi-
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ANF AND ANG II IN PROXIMAL
Table 2. Acidification data obtained in proximal convoluted tubule during systemic, luminal, or peritubular administration of ANF or ANG II t/2,
HCO,
0
CONT
q ANF-SYST
m
ANG II:-SYST
a
CONT-PERI
ANG II-PER)
u
ANF-SYST+ANG
J l-E059
n crnB2.s-’ Control 4.75kO.20 2.30t0.10 86 Control, peritubular 4.57kO.32 1.83t0.14 44 ANF, systemic 3.89kO.30 2.20t0.24 27 ANF, lumen 4.05kO.32 2.18t0.25 33 ANF, peritubular 3.85k0.24 1.88t0.16 50 ANG II, systemic 2.47*0.18* 3.37kO.22" 32 ANG II, lumen 2.30+0.15* 3.94t0.35" 35 ANG II, peritubular 2.04+0.10t 4.10+0.22t 50 Values are means t SE; n, number of measurements. Jncos_, net bicarbonate reabsorption; t/2, half time of acidification. * Significantly different from control value (P < 0.01). t Significantly different from peritubular control value (P < 0.01). S
nmol
l
4
CI 1
(86)
cn2 N‘ :
0 v I 3
0 I
(8)
(8)
I C
ANGII- SYST
I
(8) I 1 ANCII-SST’
ANFhRI Fig. 2. Stimulatory effect of systemic administration of ANG II on kinetics of proximal tubule acidification is immediately reversed by addition of ANF in peritubular (Peri) capillary perfusion. C, control; ANG II-Syst, effect of ANG II infused at a rate of 20 ngomin-lg kg? ANG II-Syst + ANF-Peri, effect of 10m6M ANF added to peritubular Capillary perfusion maintaining systemic infusion of ANG II. Values are means * SE; number of measurements is shown in parentheses. &co;, net HCO; reabsorption; t/2, half time of acidification. Eightpointed star, significantly different from control value (P < 0.05). * Significantly different from ANG II-Syst value (P < 0.05).
tions, then during systemic infusion of ANG II, and finally during capillary perfusion with ANF when the systemic infusion of ANG II is maintained. These experiments show that ANF in the capillary perfusion inhibits the stimulation of proximal tubular HCO: transport induced by systemic administration of ANG II in the same PCT. We also studied acidification data in PCT of rats systemically infused with ANF and with capillary perfusion containing ANG II. The results obtained are shown in Fig. 3. Also in these conditions, ANF caused an inhibition of the stimulatory effect of ANG II on the half time of acidification, which increased from 2.04 t 0.10 (n = 50) to 4.52 t 0.30 s (n = 42) and consequently on &co,, which fell from 4.10 t 0.22 (n = 50) to 1.55 t 0.16 nmol . cmD2 s-l (n = 42). Figure 4 gives data obtained in PCT undergoing peril
F305
REABSORPTION
* I* P (32)
(44)
It-PER1
(SO)
Fig. 3. Systemic infusion of ANF impairs stimulatory effect of peritubular capillary perfusion of ANG II on kinetics of proximal tubule acidification. Top: effect on acidification half time. Bottom: effect on net HCOi reabsorption. Cont, control. ANF-Syst, effect of ANF infused at a rate of 0.5 pgomin-’ *kg-’ after a prime of 10 pg/kg. ANG II-Syst, effect of ANG II infused at a rate of 20 ng= min-‘. kg-‘. Cont-Peri, effect of control peritubular capillary perfusion. ANG II-Peri, effect of lo-l2 M ANG II added to peritubular capillary perfusion. ANF-Syst + ANG II-Peri, effect of ANG II added to peritubular capillary perfusion maintaining systemic infusion of ANF. Values are means t SE; number of measurements is shown in parentheses. * Significantly different from Cont value (P < 0.01). Eight-pointed star, significantly different from Cont-Peri value (P < 0.01). * Significantly different from ANG II-Peri value (P < 0.01).
tubular capillary perfusion with ANF and ANG II. The perfusion of ANF in the peritubular capillary also caused an inhibition of the stimulatory effect of ANG II in peritubular capillary perfusion; the half time of acidification increased to 3.96 t 0.43 s (n = 34), and the J HCo; decreased to 1.56 t 0.11 nmol . crne2 s-l (n = 34). Figure 5 shows experiments that demonstrate that ANF perfused in the lumen also inhibits the stimulation of proximal HCO: transport induced by luminal perfusion of ANG II. The half time of acidification increased from 2.30 t 0.15 s (n = 35) during luminal perfusion with ANG II to 4.07 t 0.31 s (n = 46) when ANF was added, and the J HCo; decreased from 3.94 t 0.35 (n = 35) with ANG II to 2.21 t 0.15 nmo10cm-2s-1 (n = 46) with ANG II and ANF. l
DISCUSSION
Effect of ANF on tubular HCO; reabsorption. Since the discovery of ANF in 1981 (9), there has been substantial controversy regarding its diuretic and natriuretic action mechanisms. Several studies performed in different species have demonstrated a large increase in GFR after ANF infusion
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F306
ANF AND ANG II IN PROXIMAL
HCO,
REABSORPTION
0
CONT.PERI
q ANF-PERI
0
w
ANO
m
l ANGII-LUMEN
II-PER)
ANF-TERI ANO
‘IT-PER1
0:?:: ANF-LUMEN
CONT
ANF-LUMEN + ANG IT-LUMEN
W a
LI
l .* ...
1:: .‘.:.:.:.: ::: ....... ....... : : : .‘.‘.‘. .*.::::*:* :::
.“‘::: .
.*.:.: :::
’ . ....
l
.‘.‘.‘.
.‘.I.: . . . . . .*.. ::I.‘.‘.‘. :y::. *::: .*.::::::: : : : .‘.‘.‘.. . . . . . . . . . .. .. . ::I.‘.’ .*:* ::::::A’ .*.* :::::::y :y::.*.* . . .“‘::: :::.*.* . . ......
Fig. 4. Stimulatory effect of peritubular capillary perfusion of ANG II on kinetics of proximal tubule acidification is abolished by addition of ANF to peritubular capillary perfusion. Top: effect on acidification half time. Bottom: effect on net HCO; reabsorption. ANF-Peri, effect of low6 M ANF added to peritubular capillary perfusion. ANF-Peri + ANG II-Peri, effect of ANF plus ANG II added to peritubular capillary perfusion. Values are means t SE; number of measurements is shown in parentheses. Eight-pointed star, significantly different from ContPeri value (P < 0.01). * Significantly different from ANG II-Peri value (P c 0.01). Cont-Peri and ANG II-Peri groups are same as included in Fig. 3.
(4-6, 32). Attempts to identify a direct tubular effect have led to conflicting results. Free-flow micropuncture studies are difficult to interpret because of the marked effect of ANF on GFR and the tight link between GFR and proximal tubular Na+ reabsorption. Studies in vitro that rule out the influence of GFR yielded conflicting results. One study showed decreased activity of Na+PO:- and Na+-H+ transport in vesicles obtained from animals treated with ANF (16); on the other hand, studies in isolated perfused tubules failed to demonstrate any direct tubular effect of ANF (3). To test the possibility that ANF is active when applied in vivo but ineffective if administered to isolated systems, Capasso et al. (7) performed studies using the shrinking-droplet technique in PCT in vivo and measured the oxygen consumption of in vitro proximal tubules under the influence of ANF; but their results indicated that the enhancement of renal Na+ excretion induced by ANF is not related to a direct inhibition of Na+ transport in proximal tubule in both preparations. We examined the effect of systemic infusion of ANF in vivo, by a stopped-flow microperfusion technique, which is not affected by GFR. We also examined the action of ANF infused into the lumen or into the peritubular capillary. This procedure avoids systemic alterations caused by ANF infusion confirmed by absence of changes in urine flow and Na+ excretion unde r these conditions. Another ad vantage of the luminal or peritubular perfusion is to ascertain that the measurements of
[I (86)
. . .. . . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . . ........ . . ... ... ... ... ... ... .. .. ........ . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . . ........ . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . . . . .. .. .. .. .. .. . .
. . .. .
rl
(33)
(46)
Fig. 5. Stimulatory effect of luminal perfusion of ANG II on kinetics of proximal tubule acidification is abolished by addition of ANF to luminal perfusion. Top: effect on acidification half time. Bottom: effect on net HCO; reabsorption. ANF-Lumen, effect of 10D6 M ANF added to luminal perfusion. ANG II-Lumen, effect of lo-l2 M ANG II added to luminal perfusion. ANF-Lumen + ANG II-Lumen, effect of ANF plus ANG II added to luminal perfusion. Values are means t SE; number of measurements is shown in parentheses. Eight-pointed star, significantly different from Cont value (P < 0.01). * Significantly different from ANG II-Lumen value (P < 0.01). Cont group is same as included in Fig. 3.
acidification kinetics are performed during the period of highest activity of ANF, as it is known that this peptide has a transient action when injected parenterally. In all experimental groups, we failed to demonstrate a direct effect of ANF on proximal HCO: reabsorption. Role of ANG II in HCO: reabsorption. The effects of ANG II on the kinetics of bicarbonate reabsorption infused systemically, in the lumen or into the peritubular capillary, were also studied. A significant increase in HCO: reabsorption was observed, expressed by a fall in acidification half time from a mean of 4.75 to 2.47 s in systemically infused rats, or to 2.30 s during luminal perfusion, and from 4.57 to 2.04 s in capillary-perfused tubules. In normal conditions ANG II may be stimulating HCO: reabsorption in PCT. However, it is possible that this stimulation might be absent in our experimental conditions because of the moderate volume expansion to which the rats were subjected. Our results agree with the studies of Liu and Cogan (21, 22), who have shown by microperfusion and determination of total COn by microcalorimetry an important stimulation of HCO: reabsorption in the proximal tubule induced by systemic infusion of ANG II. This effect was
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ANF
AND
ANG
II IN
PROXIMAL
amiloride sensitive, which implies that the Na+-H+ antiporter is an important participant in the enhanced acidification stimulated by ANG II (22). The results by Valega et al. (31), showing activation of Na+-H+ exchange by ANG II in culture of vascular smooth muscle, and by Wang and Chan (35), indicating that ANG II increases HCO: and fluid reabsorption in PCT, support this hypothesis. The effect of ANG II and ANF during luminal perfusion raises the question of the distribution of receptors for these peptides on the cell surface. There appear to be such receptors also on the luminal cell membrane; it is, however, possible that these substances may reach the basolateral membrane via the paracellular shunt path during luminal perfusion. Interaction of ANF with ANG II. Harris et al. (18) studied the effect of ANF on proximal fluid absorption in rats pretreated with ANG II using the shrinkingdroplet technique. When they first treated proximal tubules with lo-l2 M ANG II, 2 x 10-l’ M ANF inhibited fluid absorption by 27%. This result agrees with the 35% inhibition reported by Garvin (12) for the proximal straight tubule. In contrast, Liu and Cogan (22) were not able to detect inhibition of fluid absorption, with or without angiotensin, during in vivo free-flow micropuncture experiments; however, they infused both hormones intravenously, raising the possibility that systemic effects of the hormones might have affected their data. Our data indicate that the stimulatory effect of ANG II on the kinetics of proximal tubule acidification is abolished by systemic infusion of ANF or by addition of ANF in luminal or peritubular capillary perfusion. According to Harris et al. (18), the range of angiotensin concentrations that permit ANF to inhibit fluid absorption lies between lo-l2 and 10mfOM; the concentration used in our study in lumen or in peritubular capillaries is within this range. During systemic infusion, the exact concentration of ANF in the kidney is unknown, but the results show that ANF does inhibit ANG II-stimulated HCOT reabsorption in the proximal nephron by more than 50%. Our results agree with the idea that the proximal nephron effects of ANF could account for a significant portion of the natriuretic and diuretic action as proposed by Harris et al. (18), Garvin (12), and Salazar et al. (28). The interaction of ANG II and ANF is not yet understood, but guanosine 3’,5’-cyclic monophosphate appears to be part of the second messenger cascade for ANF in the proximal nephron, as it is in other tissues (12). Because ANG II causes a direct and substantial stimulation of both luminal Na+-H+ exchange and basolateral Na+-HCO; cotransport (13), it is possible that ANF may inhibit the luminal and basolateral steps of transepithelial HCO; reabsorption induced by ANG II. We thank Dr. Gerhard Malnic, Universidade de S&o Paulo, Brazil, for critical reading of the manuscript. Address for reprint requests: M. Mello Aires, Dept. of Physiology and Biophysics, Instituto de Ciencias Biomedicas, Universidade de S&o Paulo, CP 4365 Sgo Paulo, Brazil. Received 1 October 1990; accepted in final form 10 September 1991. REFERENCES 1. Aguilera, G. A., A. Schirar, A. Baukal, and K. J. Catt. Angiotensin II receptors: properties and regulation in adrenal
HCO;
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REABSORPTION
glomerulosa cells. Circ. Res. 46, Suppl. I: 1-118-I-127, 1980. Atarashi, K., P. J. Mulrow, R. Franco-Saenz, R. Snajdar, and J. Rapp. Inhibition of aldosterone production by an atria1 extract. Science Wash. DC 224: 992-994, 1984. 3. Baum, M., and R. D. Toto. Lack of a direct effect of atria1 natriuretic factor in the rabbit proximal tubule. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 19): F66-F69, 1986. 4. Beasley, D., and R. L. Malvin. Atria1 extracts increase glomerular filtration rate in vivo. Am. J. Physiol. 248 (Renal Fluid 2.
Electrolyte 5.
Physiol.
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