Maturation LAURA Department
of cGMP response to ANP by isolated glomeruli L. NORLING,
CATHY
A. VAUGHAN,
Norling, Laura L., Cathy A. Vaughan, and Robert L. Chevalier. Maturation of cGMP response to ANP by isolated glomeruli. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): Fl38-F143, 1992.-Experiments were done to determine whether there is a maturational increase in production of guanosine 3’,5’-cyclic monophosphate (cGMP) by glomeruli or in egression of cGMP out of glomerular cells. Both preweaned and adult isolated rat glomeruli responded with an acute rise in intracellular cGMP after 0.5min exposure to 0.1 PM ANP. However, at 4 h extracellular cGMP was significantly greater in ANP-treated adult compared with preweaned glomeruli (P < 0.005). In the absence of 3-isobutyl-1-methylxanthine (IBMX) intracellular cGMP was significantly higher in preweaned glomeruli (P < 0.05). Moreover, the specific activity of phosphodiesterases for cGMP hydrolysis was twofold less in preweaned glomerular membranes (P < 0.004). Finally, probenecid decreased export of adult glomerular cGMP by 60 t 4%, whereas preweaned glomerular cGMP export decreased by only 27 -+ 4% (P < 0.05). In conclusion, compared with adult, ANPtreated preweaned glomeruli export less cGMP out of glomerular cells, have a higher concentration of intracellular cGMP, and have lower cGMP-specific phosphodiesterase activity, and the organic ion transporter in preweaned glomerular cells exports cGMP less effectively. The limited transport of cGMP out of preweaned glomeruli may account for the blunted natriuretic and diuretic response following ANP stimulation of young rats. egression of guanosine 3’,5’-cyclic monophosphate; diesterase activity; renal development
phospho-
NATRIURETIC PEPTIDE (ANP) is a 2%amino acid peptide, which is important in the regulation of extracellular fluid volume (28). This regulatory effect is primarily mediated by its action on the kidney, which responds to ANP by increasing excretion of water and sodium (28). In addition, the vasculature also responds to ANP by vasodilation (11, 14). The cellular mechanisms underlying the renal response to ANP are not clearly defined. ANP infusion in adult anesthetized rats produces a large rise in glomerular filtration rate and an increase in filtration fraction, with renal plasma flow remaining constant (2, 18). Guanosine 3’,5’-cyclic monophosphate (cGMP) may mediate the intracellular response to ANP (17). The recently cloned biological receptor for ANP includes the catalytic subunit of the membrane-bound guanylyl cyclase (9, 27). Therefore, levels of intracellular cGMP are influenced by binding of ANP to its biological receptor. In the kidney, ANP increases intracellular cGMP levels in the glomeruli (17) and in collecting ducts (29), where cGMP inhibits electrogenic sodium absorption (21). ANP infusion is also associated with increases in plasma and urinary cGMP concentrations (8,16). The natriuretic and diuretic response of the immature rat kidney to volume expansion and to ANP is decreased compared with that of the mature rat kidney (5, 8). In addition, the urinary cGMP levels of immature rats are lower than those of adults in response to increased circulating levels of plasma ANP (7). Therefore,
ATRIAL
F138
AND
ROBERT
L. CHEVALIER
of Pediatrics, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908
0363-6127/92 $2.00 Copyright
0
this study was designed to characterize the intracellular and extracellular glomerular cGMP response to ANP in preweaned and adult rats and to investigate specific cellular mechanisms that may be responsible for the maturational differences of the glomerular response to ANP. MATERIALS
AND METHODS
Animals. Two groups of animals were used for these experiments. Adult female Sprague-Dawley rats weighing 150-250 g (Hilltop Lab Animals, Scottdale, PA) were allowed free access to regular rat chow (Purina laboratory rat chow 5012; St. Louis, MO) and water. Preweaned rat litters were studied at 15-18 days of age, and rats weighed 25-75 g. They were housed with the mother rat who was fed identically to the other adult rats used in these experiments. Neither adult nor preweaned rats were fasted before experiments. They were anesthetized with 50 mg/kg ip phenobarbital sodium prior to nephrectomy. Tissue preparation. Renal cortex was removed from the kidneys of one adult rat or from 6-12 preweaned rats. It was then minced and mechanically dispersed in modified Dulbecco’s phosphate-buffered saline, 4°C containing (in mM) 130 NaCl, 2.5 KCl, 7.7 NaZHP04, 1.4 KHZP04, 5.2 glucose, 0.85 CaCla, 0.5 MgC12, 4.75 NaHCO,, 9.5 N-Z-hydroxyethylpiperazine-W-Zethanesulfonic acid (HEPES), pH 7.4 with NaOH, and 0.2 g/ dl bovine serum albumin). Glomeruli were isolated by selective sieving through stacked screens with sequential pore sizes of 180, 106, and 75 pm by the method of Misra (22). Glomeruli retained on the 75-pm sieve were collected, rinsed with modified Dulbecco’s buffer, pelleted by centrifugation (500 g for 15 min, 4”C), and resuspended in lo-15 ml of fresh modified Dulbecco’s buffer. Glomerular assays. Assay tubes were prepared by counting 100 glomeruli per siliconized glass tube using a dissecting microscope. Modified Dulbecco’s buffer was aspirated from the glomeruli and replaced with a known volume (160-180 ~1) of fresh buffer. Glomeruli were kept in buffer at 4°C until the experiments were started. Glomeruli were preequilibrated at 37°C for 2 min (acute study) or 15 min (chronic study). In some experiments, 3-isobutyl-l-methylxanthine (IBMX) (0.25 or 1.0 mM) and probenecid (1.0 mM) were present during this equilibration period, then rat ANP (Peninsula Laboratories, Belmont, CA) was added to each tube at a final concentration of 0.1 or 0.02 PM. Incubations were terminated at designated times over a O-4 h interval by separation of supernatant from glomeruli. Supernatant (200 ~1) was combined with 50 ~1 stop solution (100 mM EDTA, 5 mM IBMX) kept at 4°C and then analyzed for cGMP content. Glomeruli were combined with 200 ~1 trichloroacetic acid (TCA), 6% (wt/vol) at 4°C and then were sonicated (10 pulses) with a Branson Sonifier (Branson Ultrasonics, Danbury, CT) using a microtip, duty cycle 30%, and output control setting 3. This material was then extracted with ether and was analyzed for cGMP content. Viability of glomeruli was documented by linear incorporation of L-[““Slmethionine (New England Nuclear, Boston, MA) into proteins over a 4-h period (r = 0.997, adult; and r = 0.985, preweaned). Cyclic nucleotide measurements. cGMP was measured using a commercial radioimmunoassay kit (New England Nuclear, Boston, MA). Results were within the limits of detection of the
1992 the American
Physiological
Society
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MATURATION
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F139
kit (2.5 fmol 7GMP). The average extraction efficiency of cGMP measured in glomerular tissue was 76% (range 71-80%). This was determined by addition of a known quantity of [‘HIcGMP to samples during the extraction process. Protein measurements. Protein determinations of glomeruli and glomerular membraneswere measuredusing the method of Bradford (4). Although preweanedglomeruli are known to be smallerin volume and in diameterthan adult glomeruli (24), the protein concentration of equal numbers of manually counted adult and preweanedglomeruli were not significantly different (0.99 & 0.06 and 1.02 & 0.09pg/lOO glomeruli, respectively, n = 3 adult rats, 14 measurements;n = 3 litters, 10 measurements). Phosphodiesteruse actiuity. Membranes of isolated glomeruli were preparedby sonication (40 pulses)with a Branson microtip (duty cycle 30%, output control setting 3), in isotonic TIME (mid TIME (mid membranebuffer containing (in mM) 250 mM sucrose,50 mM 2-(N-morpholino)ethanesulfonic acid, and 1 mM EDTA, pH D ** C 7.2). Membranes were processedat 4°C and stored at -80°C 40 until phosphodiesteraseactivity was measuredby the method of Kincaid and Manganiello (19). Briefly, a reaction mixture 33 containing 1.5-2 x lo6 counts/min (cpm) [&“H]cGMP, am- fiI 30 monium salt (New England Nuclear), 0.75 mM cGMP, 3.0 mM 0 MgCl,, 0.3 mg/ml bovine serumalbumin (BSA), and 0.15 mM d N,N-bis[2-hydroxyethyll-2-aminoethanesulfonic acid (BES), pH 7.2, was prepared and warmed to 30°C. Glomerular membranes (40 ~1)were combinedwith 60 ~1 enzyme dilutent (250 mM BES, 2.5 mg/ml ovalbumin, and 0.5 mg/ml soy trypsin inhibitor, pH 7.2). Assay tubes were preincubated at 30°C for 2 min. The experiment was initiated by the addition of 100 ~1 warmed reaction mixture. The assaywasterminated at 15 min by adding 100 ~1 0.25 M HCl and cooling to 4°C. Guanine 2 2 5 nucleotide wasconverted to free nucleosidein a secondreaction TIME (mid TIME (min) step after neutralization of the assaywith 100~10.25 M NaOH and addition of 0.2 mg/tube 5’nucleotidase. This reaction was Fig. 1. Characterization of acute glomerular reponse to atria1 natriterminated after a 30-min incubation at 30°C. The assaytube uretic peptide (ANP). Adult and preweaned rat glomeruli were incucontents were applied to a diethylaminoethyl (DEAE) -A25 bated with 1.0 mM 3-isobutyl-l-methylxanthine (IBMX) and with or Sephadexcolumn and eluted. Free nucleosidein the eluate was without 0.1 PM ANP as described in MATERIALS AND METHODS. Shown quantitated by scintillation counting. The specific activity of is concentration of extracellular (E.C., solid lines) and intracellular cGMP phosphodiesterase is expressedaspicomolescGMP per (I.C., dashed lines) guanosine 3’,5’-cyclic monophosphate (cGMP) for control preweaned glomeruli (A), ANP-treated preweaned glomeruli milligram protein per minute. adult glomeruli (D) Statistical analysis. Data are expressed as means * SE. (B), control adult glomeruli (C), and ANP-treated cGMP/lOO Statistical comparisonswere made using Student’s t test for over a 5-min period. Data are means ~fr SE in femtomoles for n = 4 adults, 4 litters, and 8 determinations for each time unpaired data and paired data. Multiple analysis of variance glomeruli point at each age. *P c 0.05 E.C. vs. I.C. [cGMP]; **P < 0.001 vs. time was used for comparison of variables of age, ANP, IBMX, 0 [cGMP]. probenecid, and time on glomerular cGMP production. The Scheffe post hoc test wasusedto identify individual differences cantly change during the 5-min exposure to ANP. In the between groups. Statistical significance was defined as P < absence of IBMX, there was no change in preweaned or 0.05.
RESULTS
Acute glomerular cGMP response to ANP. Preweaned and adult rat glomeruli were incubated with ANP (0.1 PM) in the presence of phosphodiesterase inhibitor, IBMX (1.0 mM), to characterize the acute intracellular and extracellular changes in glomerular cGMP that occurred in response to ANP over a 5min period. In both age groups, intracellular cGMP increased significantly after O.5-min exposure to ANP (4.1 fold, adult, and 2.2 fold, preweaned) and remained significantly higher than extracellular levels of cGMP during the 5-min incubation period (Fig. 1, B and 0). In control experiments, basal intracellular levels of cGMP in preweaned glomeruli were significantly higher than in adult glomeruli at all times (P < 0.05) (Fig. 1A and C). Extracellular levels of cGMP for both adult and preweaned glomeruli did not signifi-
adult glomerular cGMP in response to ANP during the acute 5-min incubations (data not shown). Chronic glomerular cGMP response to ANP. The chronic glomerular response to ANP differed from that of the acute response. In chronic experiments, preweaned and adult glomeruli were incubated with ANP (0.1 PM) but without IMBX. When intracellular and extracellular glomerular cGMP was measured over a 4-h period, extracellular cGMP was found to increase significantly over time for both preweaned and adult glomeruli even in the absence of ANP (Fig. 2, A and C). Intracellular cGMP did not change over time but did remain significantly higher in preweaned glomeruli than in adult glomeruli for control and ANP-treated glomeruli (P < 0.05). The most dramatic difference in chronic glomerular response to ANP was the increase of extracellular cGMP from adult glomeruli over a 4-h period compared with zero time (8.9-fold, adult and U-fold, preweaned Fig. 2, B
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F140
MATURATION
60
A
60
1
B 1
TIME 60
OF GLOMERULAR
(HR)
TIME
c
(HR) ** T
60
1
i
3 B z 4 (3
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40-
50 40
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1
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4 TIME
(HR)
Fig. 2. Characterization of chronic glomerular response to ANP. Adult and preweaned rat glomeruli were incubated with 0.1 PM ANP but without phosphodiesterase inhibition. Results show E.C. (solid line) and I.C. (dashed line) cGMP for control preweaned glomeruli (A), ANP-treated preweaned glomeruli (B), control adult glomeruli (C), and ANP-treated adult glomeruli (D) over 4-h period. Data are expressed as means t SE for n = 4 adults or 4 litters and 8 determinations for each time point at each age. *P < 0.05E.C. vs. I.C. [cGMP]; **P < 0.05vs. time 0 [cGMP].
and 0). Extracellular cGMP was significantly greater than intracellular cGMP at 1 h and remained so throughout the rest of the incubation time for adult glomeruli. For preweaned glomeruli an increase of extracellular cGMP was seen at 4 h of glomerular exposure to ANP but this was not significantly different compared with extracellular cGMP in control incubations. The combined effect of age and ANP on intracellular and extracellular glomerular cGMP is further confirmed when the ratio of extracellular to intracellular (EC:IC) is analyzed over time by two-way analysis of variance comparing ANP treatment and age groups (P < 0.01, F = 9.1 at 1 h; P < 0.05, F = 4.8 at 4 h). Effect of phosphodiesterase inhibition on glomerular cGMP response.To investigate whether differences in preweaned and adult glomerular intracellular and extracellular cGMP could be due to phosphodiesterase hydrolysis of cGMP, the phosphodiesterase inhibitor, IBMX (1.0 mM) was added to glomerular incubations for up to 1 h. For preweaned control incubations in the presence of IBMX, extracellular glomerular cGMP increased significantly (P < 0.05) compared with incubations in the absence of IBMX (Fig. 3A). Control adult glomerular intracellular and extracellular cGMP levels
RESPONSE
TO ANP
increased significantly (P < O.OOOl), compared with incubations in the absence of IBMX (Fig. 3C). For adult glomeruli, incubated with IBMX and ANP (0.1 PM), extracellular cGMP at time 0 and intracellular cGMP at 0 and 1 h were significantly greater (P < 0.01) than incubations done in the presence of ANP alone (Fig. 30). For preweaned glomeruli, the only significant change in the presence of IBMX and ANP was an increase in extracellular cGMP at time 0 (Fig. 3B). Phosphodiesterase activity in glomeruli. Because of the greater increase in adult glomerular intracellular and extracellular cGMP than in preweaned glomeruli in the presence of IBMX, phosphodiesterase activity specific for hydrolysis of cGMP was measured directly in adult and preweaned glomerular membranes. Adult glomerular membranes were found to have twofold greater phosphodiesterase activity for cGMP compared with preweaned glomerular membranes (Fig. 4). Effect of probenecid on glomerular cGMP export. To determine if there is a difference in the capability of the organic ion transporter in adult and preweaned glomeruli to export cGMP, glomeruli were incubated with 1.0 mM probenecid, a competitive antagonist of organic acid transport, and then exposed to 0.02 PM ANP. Probenecid inhibited export of 60 t 4% of ANP-stimulated cGMP from adult glomeruli, whereas the export of cGMP was inhibited by only 27 t 4% in preweaned glomeruli (Fig. 5, B and 0). Multiple analysis of variance shows that the combined effects of ANP and probenecid on cGMP export are significantly greater in adult glomeruli than in preweaned glomeruli (F = 5.6, P < 0.02). DISCUSSION
This study represents the first investigation of the intracellular and extracellular effects of ANP on generation of cGMP by isolated glomeruli. In addition, the results demonstrate two dramatic maturational differences in the glomerular response to ANP: 1) preweaned rat glomeruli contain a significantly greater amount of intracellular cGMP than adult rat glomeruli, and 2) preweaned rat glomeruli export significantly less extracellular cGMP than adult glomeruli in response to stimulation by ANP. The results suggest that increased intracellular cGMP in preweaned glomeruli is due at least in part to decreased cGMP-specific phosphodiesterase available to hydrolyze the cyclic nucleotide. The experiments also indicate that increased intracellular cGMP in preweaned rat glomeruli may result from a diminished export of cGMP to the extracellular space by a glomerular organic ion transporter. A maturational difference in the activity of the glomerular biological ANP receptors is also possible. Maturation differences in renal response to ANP have been reported (5, 8, 25). Physiological studies suggest that natriuretic and diuretic responses to exogenous ANP increase with age (5, 8). It has been reported that in the presence of IBMX, isolated glomeruli from 2- to &day-old rats produce as much as fourfold more cGMP in response to ANP than adult rat glomeruli (25). This response reportedly decreased during the suckling period and persisted at the lower level in adult animals, although
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MATURATION
3
40
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10
dz
0
3
40
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ANP
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4 8
GLOMERULAR
20
f 8
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OF
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Fig. 3. Effect of phosphodiesterase inhibition on Adult and preweaned glomeruli were preincubated ANP or buffer were added to the glomeruli for intracellular cGMP at time 0 (solid bar) and at 1 preweaned glomeruli (B), control adult glomeruli for n = 3 adults or 3 litters and 6-8 determinations
PREWEANED
rat glomerular extracellular (E.C.) and intracellular (I.C.) cGMP. with or without 1.0 mM IBMX for 15 min. After that time, 0.1 PM a period of 1 h. Shown are concentrations of extracellular and h (hatched bar) for control preweaned glomeruli (A), ANP-treated (C), and ANP-treated adult glomeruli (D). Data are means k SE for each time point. *P c 0.05, vs. no IBMX.
ADULT
Fig. 4. Glomerular cGMP-specific phosphdiesterase activity. Adult and preweaned rat glomerular membranes were assayed for cGMPspecific phosphodiesterase activity. Results show specific activity for cGMP-specific phosphodiesterase in preweaned and adult glomerular membranes (solid bar) and amount of nonspecific hydrolysis of cyclic nucleotide in heat-inactivated membranes (hatched bar). Data are means t, SE for glomerular membranes from n = 4 adults or 4 litters. *P < 0.005, adult vs. preweaned.
intracellular and extracellular distribution of cGMP was not evaluated (25). In the present study in which intracellular and extracellular distribution of glomerular cGMP is examined, adult and infant glomerular response to acute stimulation with ANP is similar. It is the chronic ANP-stimulated glomerular extrusion of cGMP that is significantly different between preweaned and adult glomeruli. This glomerular response to ANP supports the observed age-related physiological differences in renal response to ANP. The chronic glomerular response to ANP is character-
ized by accumulation of extracellular cGMP. Our results show that the decreased export of cGMP by preweaned glomeruli is not due to increased hydrolysis of the cyclic nucleotide by phosphodiesterases. Therefore, a developmental alteration in glomerular cell transport of cGMP could explain the difference in extracellular cGMP accumulation in preweaned glomeruli compared with adult. It may be that more than one cGMP transport mechanism exists. This is suggested by the observation that control extracellular levels of cGMP in both adult and preweaned glomeruli do not decrease in the presence of probenecid. However, the limited effect of the organic acid transport antagonist, probenecid, in blocking export of cGMP in ANP-stimulated preweaned glomeruli suggests that the decreased egression of cGMP from preweaned rat glomeruli may be due to a difference in the ability of young glomerular cells to transport cGMP actively out of cells. A specific probenecid-sensitive active transport system for cGMP has been described in ANP-treated vascular smooth muscle (15), in endothelial cells (X5), and in a kidney epithelial cell line (26). In contrast to cellular transport of CAMP, which is short in duration and parallels the change in intracellular CAMP, cGMP transport is characterized by a slow continuous increase in extracellular cGMP from cells lasting hours after exposure to ANP. This raises the speculation that transport of cGMP from ANP-treated cells and glomeruli may have a function other than to regulate the intracellular concentration of cGMP. It may have a role as an extracellular second messenger (15). At present, the identity of the individual glomerular
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F142
MATURATION
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60
E.C.
OF
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cB
I.C.
E.C.
I.C.
E.C.
I.C.
60
0 E.C.
I.C.
Fig. 5. Effect of probenecid on transport of glomerular cGMP. Adult and preweaned rat glomeruli were incubated with 0.25 mM IBMX in presence (hatched bars) or absence (solid bars) of 1.0 mM probenecid and 0.02 PM ANP for 1 h. Results show concentration of extracellular cGMP and intracellular cGMP for control preweaned (A) and adult rat (C) glomeruli and for ANP-treated preweaned (B) and adult (D) rat glomeruli. Data are means t SE from n = 4 adult and n = 5 litters and with 8-14 measurements for each condition. *P < 0.05 vs. no probenecid.
cells responding to ANP by cGMP production remains uncertain. Cultured glomerular cells expressing ANP receptors and responding with increase in cGMP have been described for mesangial (1, 6) and epithelial cells (6). We have recently identified immunoreactive cGMP in glomerular epithelial podocytes (but not mesangial cells) in kidneys from rats perfused in vivo with ANP (12). In contrast, stimulation of soluble guanylate cyclase by infusion of sodium nitroprusside resulted in localization of cGMP to mesangial cells rather than podocytes (12). These results suggest that in vivo podocytes are primarily responsible for the glomerular response to ANP. Micropuncture studies have shown that ANP enhances glomerular production of cGMP in glomerular ultrafiltrate without a significant increase of systemic arterial or renal venous cGMP (17). Therefore, egression of cGMP from glomerular cells presumably accounts for cGMP found in glomerular ultrafiltrate. In addition, the increase in urinary cGMP seen after administration of ANP is directly attributable to glomerular production of cGMP (17). Systemic infusion of cGMP in dogs has been shown to result in diuresis, natriuresis, and an increase in glomerular filtration rate (23). The infused cGMP is eliminated only in glomerular filtrate (23). It has also been reported that addition of exogenous cGMP to the bath of rat cortical collecting duct inhibits NaCl resorp-
RESPONSE
TO
ANP
tion and vasopressin-stimulated fluid absorption, mimicking the action of ANP on these segments of the nephron (24). The identical rena, response to direct infusion of cGMP and to ANP strongly suggests a role for cGMP as a second messenger in the biological action of ANP on the kidney. The known biological actions of cGMP include stimulation of cGMP-dependent protein kinase (lo), activation of phosphodiesterase II (3), and gating of sodium channels in retinal rods (13) and renal intramedullary collecting ducts (20). These responses are generally felt to be mediated by increased intracellular cGMP. However, it is possible that extracellular cGMP in the glomerular filtrate may act at additional sites within the nephron to facilitate sodium and water excretion. This could be accomplished by direct action of cGMP on proximal and distal tubule epithelia or after uptake of cGMP into tubular cells. Therefore, the decrease in export of cGMP from preweaned glomerular cells may be physiologically relevant to the observed limitation of natriuresis and diuresis in this age. In summary, our data indicate that intracellular cGMP content is increased and phosphodiesterase activity is decreased in preweaned glomeruli compared with adult glomeruli. Moreover, in response to ANP, the preweaned glomeruli export less cGMP to the extracellular space. If the biological action of ANP is dependent on transport of cGMP out of the glomerular cells, then these differences may be responsible in part for blunted natriuresis and diuresis in preweaned rats. In turn, this adaptation may facilitate conservation of sodium in the rapidly growing preweaned rat with limited access to exogenous sodium and increased sodium requirements for growth. This study was supported by National Heart, Lung, and Blood Institute Grant HL-40209 (to R. L. Chevalier), American Heart Association Grant-in-Aid 91006380 (to R. L. Chevalier), Division of Research Resources Biomedical Research Support Grant RR-05431-28 (to L. L. Norling), and Research Center of Excellence in Pediatric Nephrology and Urology Grant P50-DK-44765-01 (to R. L. Chevalier and L. L. Norling). Portions of this work were presented at the Annual Meeting of the Southern Society for Pediatric Research in New Orleans, LA, in January 1991. Address for reprint requests: L. L. Norling, Dept. of Pediatrics, Univ. of Virginia, MR4/2034, Charlottesville, VA 22908. Received
3 July
1991; accepted
in final
form
24 September
1991.
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TO ANP
that cGMP is the second messenger for atria1 natriuretic
factor.
Proc. NatZ. Acad. Sci. USA 83: 8015-8018, 1986. 18. Huang, C.-L., J. Lewicki, L. K. Johnson, and M. G. Cogan. Renal mechanism of action of rat atria1 natriuretic factor. J. CZin. Invest. 75: 769- 773, 1985. 19. Kincaid, R. L., and V. C. Manganiello. Assay of cyclic nucleo-
tide phosphodiesterase using radiolabeled and fluorescent substrates. Methods Enzymol. 159: 457-470, 1988. 20. Light, D. B., J. D. Corbin, and B. A. Stanton. Dual ionchannel regulation by cyclic GMP and cyclic GMP-dependent protein kinase. Nature Lond. 344: 336- 339, 1990. 21. Light, D. B., E. M. Schwiebert, K. H. Karlson, and B. A. Stanton. Atria1 natriuretic peptide inhibits a cation channel in renal inner medullary collecting duct cells. Science Wash. DC 243: 383-385,1989. 22. Misra, R. P. Isolation of glomeruli from mammalian kidneys by graded sieving. Am. J. CZin. Pathol. 58: 135-139, 1972. 23. Osswald, H. Cyclic guanosine-3’: 5’-monophosphate induced diuresis in rats. Arch. Pharmacol. 284: 207-214, 1974. 24. Nonoguchi, H., J. M. Sands, and M. A. Knepper. ANF inhibits
NaCl and fluid absorption in cortical collecting duct of rat kidney.
Am. J. Physiol. 256 (Renal Fluid F186,1989. 25. Semmekrot, B., D. Chabardes, and D. Butlen. Developmental
Electrolyte
Physiol.
25): F179-
S. Roseau,
S. Siaume-Perez,
pattern of cyclic guanosine monophosphate production stimulated by atria1 natriuretic peptide in glomeruli microdissected from kidneys of young rats. Pfluegers
Arch. 416: 519-525,199O. 26. Woods, M., and M. D. Houslay.
Desensitization of atriopeptin stimulated accumulation and extrusion of cyclic GMP from a kidney epithelial cell line (MDCK). Biochem. Pharmacol. 41: 385-
394,1991. 27. Yamaguchi,
M., L. J. Rutledge, and D. L. Garbers. The primary structure of the rat guanylyl cyclase A/atria1 natriuretic peptide receptor gene. J. BioZ. Chem. 265: 20414-20420,199O. 28. Zeidel, M. L. Renal actions of atria1 natriuretic peptide. In: Atrial Natriuretic Peptides, Contemporary Issues in NephroZogy, edited by B. M. Brenner, and J. H. Stein. New York: Churchill Livingstone, 1990, p. 191-207. 29. Zeidel, M. L., P. Silva, B. M. Brenner, and J. L. Seifter. cGMP mediates effects of atria1 peptides on medullary collecting duct cells. Am. J. Physiol. 252 (Renal FZuid Electrolyte Physiol. 21): F551-F559,1987.
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of cGMP response to ANP by isolated glomeruli L. NORLING,
CATHY
A. VAUGHAN,
Norling, Laura L., Cathy A. Vaughan, and Robert L. Chevalier. Maturation of cGMP response to ANP by isolated glomeruli. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): Fl38-F143, 1992.-Experiments were done to determine whether there is a maturational increase in production of guanosine 3’,5’-cyclic monophosphate (cGMP) by glomeruli or in egression of cGMP out of glomerular cells. Both preweaned and adult isolated rat glomeruli responded with an acute rise in intracellular cGMP after 0.5min exposure to 0.1 PM ANP. However, at 4 h extracellular cGMP was significantly greater in ANP-treated adult compared with preweaned glomeruli (P < 0.005). In the absence of 3-isobutyl-1-methylxanthine (IBMX) intracellular cGMP was significantly higher in preweaned glomeruli (P < 0.05). Moreover, the specific activity of phosphodiesterases for cGMP hydrolysis was twofold less in preweaned glomerular membranes (P < 0.004). Finally, probenecid decreased export of adult glomerular cGMP by 60 t 4%, whereas preweaned glomerular cGMP export decreased by only 27 -+ 4% (P < 0.05). In conclusion, compared with adult, ANPtreated preweaned glomeruli export less cGMP out of glomerular cells, have a higher concentration of intracellular cGMP, and have lower cGMP-specific phosphodiesterase activity, and the organic ion transporter in preweaned glomerular cells exports cGMP less effectively. The limited transport of cGMP out of preweaned glomeruli may account for the blunted natriuretic and diuretic response following ANP stimulation of young rats. egression of guanosine 3’,5’-cyclic monophosphate; diesterase activity; renal development
phospho-
NATRIURETIC PEPTIDE (ANP) is a 2%amino acid peptide, which is important in the regulation of extracellular fluid volume (28). This regulatory effect is primarily mediated by its action on the kidney, which responds to ANP by increasing excretion of water and sodium (28). In addition, the vasculature also responds to ANP by vasodilation (11, 14). The cellular mechanisms underlying the renal response to ANP are not clearly defined. ANP infusion in adult anesthetized rats produces a large rise in glomerular filtration rate and an increase in filtration fraction, with renal plasma flow remaining constant (2, 18). Guanosine 3’,5’-cyclic monophosphate (cGMP) may mediate the intracellular response to ANP (17). The recently cloned biological receptor for ANP includes the catalytic subunit of the membrane-bound guanylyl cyclase (9, 27). Therefore, levels of intracellular cGMP are influenced by binding of ANP to its biological receptor. In the kidney, ANP increases intracellular cGMP levels in the glomeruli (17) and in collecting ducts (29), where cGMP inhibits electrogenic sodium absorption (21). ANP infusion is also associated with increases in plasma and urinary cGMP concentrations (8,16). The natriuretic and diuretic response of the immature rat kidney to volume expansion and to ANP is decreased compared with that of the mature rat kidney (5, 8). In addition, the urinary cGMP levels of immature rats are lower than those of adults in response to increased circulating levels of plasma ANP (7). Therefore,
ATRIAL
F138
AND
ROBERT
L. CHEVALIER
of Pediatrics, University of Virginia, Health Sciences Center, Charlottesville, Virginia 22908
0363-6127/92 $2.00 Copyright
0
this study was designed to characterize the intracellular and extracellular glomerular cGMP response to ANP in preweaned and adult rats and to investigate specific cellular mechanisms that may be responsible for the maturational differences of the glomerular response to ANP. MATERIALS
AND METHODS
Animals. Two groups of animals were used for these experiments. Adult female Sprague-Dawley rats weighing 150-250 g (Hilltop Lab Animals, Scottdale, PA) were allowed free access to regular rat chow (Purina laboratory rat chow 5012; St. Louis, MO) and water. Preweaned rat litters were studied at 15-18 days of age, and rats weighed 25-75 g. They were housed with the mother rat who was fed identically to the other adult rats used in these experiments. Neither adult nor preweaned rats were fasted before experiments. They were anesthetized with 50 mg/kg ip phenobarbital sodium prior to nephrectomy. Tissue preparation. Renal cortex was removed from the kidneys of one adult rat or from 6-12 preweaned rats. It was then minced and mechanically dispersed in modified Dulbecco’s phosphate-buffered saline, 4°C containing (in mM) 130 NaCl, 2.5 KCl, 7.7 NaZHP04, 1.4 KHZP04, 5.2 glucose, 0.85 CaCla, 0.5 MgC12, 4.75 NaHCO,, 9.5 N-Z-hydroxyethylpiperazine-W-Zethanesulfonic acid (HEPES), pH 7.4 with NaOH, and 0.2 g/ dl bovine serum albumin). Glomeruli were isolated by selective sieving through stacked screens with sequential pore sizes of 180, 106, and 75 pm by the method of Misra (22). Glomeruli retained on the 75-pm sieve were collected, rinsed with modified Dulbecco’s buffer, pelleted by centrifugation (500 g for 15 min, 4”C), and resuspended in lo-15 ml of fresh modified Dulbecco’s buffer. Glomerular assays. Assay tubes were prepared by counting 100 glomeruli per siliconized glass tube using a dissecting microscope. Modified Dulbecco’s buffer was aspirated from the glomeruli and replaced with a known volume (160-180 ~1) of fresh buffer. Glomeruli were kept in buffer at 4°C until the experiments were started. Glomeruli were preequilibrated at 37°C for 2 min (acute study) or 15 min (chronic study). In some experiments, 3-isobutyl-l-methylxanthine (IBMX) (0.25 or 1.0 mM) and probenecid (1.0 mM) were present during this equilibration period, then rat ANP (Peninsula Laboratories, Belmont, CA) was added to each tube at a final concentration of 0.1 or 0.02 PM. Incubations were terminated at designated times over a O-4 h interval by separation of supernatant from glomeruli. Supernatant (200 ~1) was combined with 50 ~1 stop solution (100 mM EDTA, 5 mM IBMX) kept at 4°C and then analyzed for cGMP content. Glomeruli were combined with 200 ~1 trichloroacetic acid (TCA), 6% (wt/vol) at 4°C and then were sonicated (10 pulses) with a Branson Sonifier (Branson Ultrasonics, Danbury, CT) using a microtip, duty cycle 30%, and output control setting 3. This material was then extracted with ether and was analyzed for cGMP content. Viability of glomeruli was documented by linear incorporation of L-[““Slmethionine (New England Nuclear, Boston, MA) into proteins over a 4-h period (r = 0.997, adult; and r = 0.985, preweaned). Cyclic nucleotide measurements. cGMP was measured using a commercial radioimmunoassay kit (New England Nuclear, Boston, MA). Results were within the limits of detection of the
1992 the American
Physiological
Society
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MATURATION
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RESPONSE
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F139
kit (2.5 fmol 7GMP). The average extraction efficiency of cGMP measured in glomerular tissue was 76% (range 71-80%). This was determined by addition of a known quantity of [‘HIcGMP to samples during the extraction process. Protein measurements. Protein determinations of glomeruli and glomerular membraneswere measuredusing the method of Bradford (4). Although preweanedglomeruli are known to be smallerin volume and in diameterthan adult glomeruli (24), the protein concentration of equal numbers of manually counted adult and preweanedglomeruli were not significantly different (0.99 & 0.06 and 1.02 & 0.09pg/lOO glomeruli, respectively, n = 3 adult rats, 14 measurements;n = 3 litters, 10 measurements). Phosphodiesteruse actiuity. Membranes of isolated glomeruli were preparedby sonication (40 pulses)with a Branson microtip (duty cycle 30%, output control setting 3), in isotonic TIME (mid TIME (mid membranebuffer containing (in mM) 250 mM sucrose,50 mM 2-(N-morpholino)ethanesulfonic acid, and 1 mM EDTA, pH D ** C 7.2). Membranes were processedat 4°C and stored at -80°C 40 until phosphodiesteraseactivity was measuredby the method of Kincaid and Manganiello (19). Briefly, a reaction mixture 33 containing 1.5-2 x lo6 counts/min (cpm) [&“H]cGMP, am- fiI 30 monium salt (New England Nuclear), 0.75 mM cGMP, 3.0 mM 0 MgCl,, 0.3 mg/ml bovine serumalbumin (BSA), and 0.15 mM d N,N-bis[2-hydroxyethyll-2-aminoethanesulfonic acid (BES), pH 7.2, was prepared and warmed to 30°C. Glomerular membranes (40 ~1)were combinedwith 60 ~1 enzyme dilutent (250 mM BES, 2.5 mg/ml ovalbumin, and 0.5 mg/ml soy trypsin inhibitor, pH 7.2). Assay tubes were preincubated at 30°C for 2 min. The experiment was initiated by the addition of 100 ~1 warmed reaction mixture. The assaywasterminated at 15 min by adding 100 ~1 0.25 M HCl and cooling to 4°C. Guanine 2 2 5 nucleotide wasconverted to free nucleosidein a secondreaction TIME (mid TIME (min) step after neutralization of the assaywith 100~10.25 M NaOH and addition of 0.2 mg/tube 5’nucleotidase. This reaction was Fig. 1. Characterization of acute glomerular reponse to atria1 natriterminated after a 30-min incubation at 30°C. The assaytube uretic peptide (ANP). Adult and preweaned rat glomeruli were incucontents were applied to a diethylaminoethyl (DEAE) -A25 bated with 1.0 mM 3-isobutyl-l-methylxanthine (IBMX) and with or Sephadexcolumn and eluted. Free nucleosidein the eluate was without 0.1 PM ANP as described in MATERIALS AND METHODS. Shown quantitated by scintillation counting. The specific activity of is concentration of extracellular (E.C., solid lines) and intracellular cGMP phosphodiesterase is expressedaspicomolescGMP per (I.C., dashed lines) guanosine 3’,5’-cyclic monophosphate (cGMP) for control preweaned glomeruli (A), ANP-treated preweaned glomeruli milligram protein per minute. adult glomeruli (D) Statistical analysis. Data are expressed as means * SE. (B), control adult glomeruli (C), and ANP-treated cGMP/lOO Statistical comparisonswere made using Student’s t test for over a 5-min period. Data are means ~fr SE in femtomoles for n = 4 adults, 4 litters, and 8 determinations for each time unpaired data and paired data. Multiple analysis of variance glomeruli point at each age. *P c 0.05 E.C. vs. I.C. [cGMP]; **P < 0.001 vs. time was used for comparison of variables of age, ANP, IBMX, 0 [cGMP]. probenecid, and time on glomerular cGMP production. The Scheffe post hoc test wasusedto identify individual differences cantly change during the 5-min exposure to ANP. In the between groups. Statistical significance was defined as P < absence of IBMX, there was no change in preweaned or 0.05.
RESULTS
Acute glomerular cGMP response to ANP. Preweaned and adult rat glomeruli were incubated with ANP (0.1 PM) in the presence of phosphodiesterase inhibitor, IBMX (1.0 mM), to characterize the acute intracellular and extracellular changes in glomerular cGMP that occurred in response to ANP over a 5min period. In both age groups, intracellular cGMP increased significantly after O.5-min exposure to ANP (4.1 fold, adult, and 2.2 fold, preweaned) and remained significantly higher than extracellular levels of cGMP during the 5-min incubation period (Fig. 1, B and 0). In control experiments, basal intracellular levels of cGMP in preweaned glomeruli were significantly higher than in adult glomeruli at all times (P < 0.05) (Fig. 1A and C). Extracellular levels of cGMP for both adult and preweaned glomeruli did not signifi-
adult glomerular cGMP in response to ANP during the acute 5-min incubations (data not shown). Chronic glomerular cGMP response to ANP. The chronic glomerular response to ANP differed from that of the acute response. In chronic experiments, preweaned and adult glomeruli were incubated with ANP (0.1 PM) but without IMBX. When intracellular and extracellular glomerular cGMP was measured over a 4-h period, extracellular cGMP was found to increase significantly over time for both preweaned and adult glomeruli even in the absence of ANP (Fig. 2, A and C). Intracellular cGMP did not change over time but did remain significantly higher in preweaned glomeruli than in adult glomeruli for control and ANP-treated glomeruli (P < 0.05). The most dramatic difference in chronic glomerular response to ANP was the increase of extracellular cGMP from adult glomeruli over a 4-h period compared with zero time (8.9-fold, adult and U-fold, preweaned Fig. 2, B
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F140
MATURATION
60
A
60
1
B 1
TIME 60
OF GLOMERULAR
(HR)
TIME
c
(HR) ** T
60
1
i
3 B z 4 (3
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i
3 B I 2 Q
40-
50 40
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4 TIME
(HR)
Fig. 2. Characterization of chronic glomerular response to ANP. Adult and preweaned rat glomeruli were incubated with 0.1 PM ANP but without phosphodiesterase inhibition. Results show E.C. (solid line) and I.C. (dashed line) cGMP for control preweaned glomeruli (A), ANP-treated preweaned glomeruli (B), control adult glomeruli (C), and ANP-treated adult glomeruli (D) over 4-h period. Data are expressed as means t SE for n = 4 adults or 4 litters and 8 determinations for each time point at each age. *P < 0.05E.C. vs. I.C. [cGMP]; **P < 0.05vs. time 0 [cGMP].
and 0). Extracellular cGMP was significantly greater than intracellular cGMP at 1 h and remained so throughout the rest of the incubation time for adult glomeruli. For preweaned glomeruli an increase of extracellular cGMP was seen at 4 h of glomerular exposure to ANP but this was not significantly different compared with extracellular cGMP in control incubations. The combined effect of age and ANP on intracellular and extracellular glomerular cGMP is further confirmed when the ratio of extracellular to intracellular (EC:IC) is analyzed over time by two-way analysis of variance comparing ANP treatment and age groups (P < 0.01, F = 9.1 at 1 h; P < 0.05, F = 4.8 at 4 h). Effect of phosphodiesterase inhibition on glomerular cGMP response.To investigate whether differences in preweaned and adult glomerular intracellular and extracellular cGMP could be due to phosphodiesterase hydrolysis of cGMP, the phosphodiesterase inhibitor, IBMX (1.0 mM) was added to glomerular incubations for up to 1 h. For preweaned control incubations in the presence of IBMX, extracellular glomerular cGMP increased significantly (P < 0.05) compared with incubations in the absence of IBMX (Fig. 3A). Control adult glomerular intracellular and extracellular cGMP levels
RESPONSE
TO ANP
increased significantly (P < O.OOOl), compared with incubations in the absence of IBMX (Fig. 3C). For adult glomeruli, incubated with IBMX and ANP (0.1 PM), extracellular cGMP at time 0 and intracellular cGMP at 0 and 1 h were significantly greater (P < 0.01) than incubations done in the presence of ANP alone (Fig. 30). For preweaned glomeruli, the only significant change in the presence of IBMX and ANP was an increase in extracellular cGMP at time 0 (Fig. 3B). Phosphodiesterase activity in glomeruli. Because of the greater increase in adult glomerular intracellular and extracellular cGMP than in preweaned glomeruli in the presence of IBMX, phosphodiesterase activity specific for hydrolysis of cGMP was measured directly in adult and preweaned glomerular membranes. Adult glomerular membranes were found to have twofold greater phosphodiesterase activity for cGMP compared with preweaned glomerular membranes (Fig. 4). Effect of probenecid on glomerular cGMP export. To determine if there is a difference in the capability of the organic ion transporter in adult and preweaned glomeruli to export cGMP, glomeruli were incubated with 1.0 mM probenecid, a competitive antagonist of organic acid transport, and then exposed to 0.02 PM ANP. Probenecid inhibited export of 60 t 4% of ANP-stimulated cGMP from adult glomeruli, whereas the export of cGMP was inhibited by only 27 t 4% in preweaned glomeruli (Fig. 5, B and 0). Multiple analysis of variance shows that the combined effects of ANP and probenecid on cGMP export are significantly greater in adult glomeruli than in preweaned glomeruli (F = 5.6, P < 0.02). DISCUSSION
This study represents the first investigation of the intracellular and extracellular effects of ANP on generation of cGMP by isolated glomeruli. In addition, the results demonstrate two dramatic maturational differences in the glomerular response to ANP: 1) preweaned rat glomeruli contain a significantly greater amount of intracellular cGMP than adult rat glomeruli, and 2) preweaned rat glomeruli export significantly less extracellular cGMP than adult glomeruli in response to stimulation by ANP. The results suggest that increased intracellular cGMP in preweaned glomeruli is due at least in part to decreased cGMP-specific phosphodiesterase available to hydrolyze the cyclic nucleotide. The experiments also indicate that increased intracellular cGMP in preweaned rat glomeruli may result from a diminished export of cGMP to the extracellular space by a glomerular organic ion transporter. A maturational difference in the activity of the glomerular biological ANP receptors is also possible. Maturation differences in renal response to ANP have been reported (5, 8, 25). Physiological studies suggest that natriuretic and diuretic responses to exogenous ANP increase with age (5, 8). It has been reported that in the presence of IBMX, isolated glomeruli from 2- to &day-old rats produce as much as fourfold more cGMP in response to ANP than adult rat glomeruli (25). This response reportedly decreased during the suckling period and persisted at the lower level in adult animals, although
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MATURATION
3
40
ii iE 03
30
g c
10
dz
0
3
40
iii 2 0d
30
d scc
RESPONSE
TO
ANP
F141
A
L
EC.
4 8
GLOMERULAR
20
f 8
g =:
OF
EamMx
LC.
lsJlBwX
C
20
10
0 EC.
LC.
Fig. 3. Effect of phosphodiesterase inhibition on Adult and preweaned glomeruli were preincubated ANP or buffer were added to the glomeruli for intracellular cGMP at time 0 (solid bar) and at 1 preweaned glomeruli (B), control adult glomeruli for n = 3 adults or 3 litters and 6-8 determinations
PREWEANED
rat glomerular extracellular (E.C.) and intracellular (I.C.) cGMP. with or without 1.0 mM IBMX for 15 min. After that time, 0.1 PM a period of 1 h. Shown are concentrations of extracellular and h (hatched bar) for control preweaned glomeruli (A), ANP-treated (C), and ANP-treated adult glomeruli (D). Data are means k SE for each time point. *P c 0.05, vs. no IBMX.
ADULT
Fig. 4. Glomerular cGMP-specific phosphdiesterase activity. Adult and preweaned rat glomerular membranes were assayed for cGMPspecific phosphodiesterase activity. Results show specific activity for cGMP-specific phosphodiesterase in preweaned and adult glomerular membranes (solid bar) and amount of nonspecific hydrolysis of cyclic nucleotide in heat-inactivated membranes (hatched bar). Data are means t, SE for glomerular membranes from n = 4 adults or 4 litters. *P < 0.005, adult vs. preweaned.
intracellular and extracellular distribution of cGMP was not evaluated (25). In the present study in which intracellular and extracellular distribution of glomerular cGMP is examined, adult and infant glomerular response to acute stimulation with ANP is similar. It is the chronic ANP-stimulated glomerular extrusion of cGMP that is significantly different between preweaned and adult glomeruli. This glomerular response to ANP supports the observed age-related physiological differences in renal response to ANP. The chronic glomerular response to ANP is character-
ized by accumulation of extracellular cGMP. Our results show that the decreased export of cGMP by preweaned glomeruli is not due to increased hydrolysis of the cyclic nucleotide by phosphodiesterases. Therefore, a developmental alteration in glomerular cell transport of cGMP could explain the difference in extracellular cGMP accumulation in preweaned glomeruli compared with adult. It may be that more than one cGMP transport mechanism exists. This is suggested by the observation that control extracellular levels of cGMP in both adult and preweaned glomeruli do not decrease in the presence of probenecid. However, the limited effect of the organic acid transport antagonist, probenecid, in blocking export of cGMP in ANP-stimulated preweaned glomeruli suggests that the decreased egression of cGMP from preweaned rat glomeruli may be due to a difference in the ability of young glomerular cells to transport cGMP actively out of cells. A specific probenecid-sensitive active transport system for cGMP has been described in ANP-treated vascular smooth muscle (15), in endothelial cells (X5), and in a kidney epithelial cell line (26). In contrast to cellular transport of CAMP, which is short in duration and parallels the change in intracellular CAMP, cGMP transport is characterized by a slow continuous increase in extracellular cGMP from cells lasting hours after exposure to ANP. This raises the speculation that transport of cGMP from ANP-treated cells and glomeruli may have a function other than to regulate the intracellular concentration of cGMP. It may have a role as an extracellular second messenger (15). At present, the identity of the individual glomerular
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
F142
MATURATION
60
60
E.C.
OF
GLOMERULAR
cB
I.C.
E.C.
I.C.
E.C.
I.C.
60
0 E.C.
I.C.
Fig. 5. Effect of probenecid on transport of glomerular cGMP. Adult and preweaned rat glomeruli were incubated with 0.25 mM IBMX in presence (hatched bars) or absence (solid bars) of 1.0 mM probenecid and 0.02 PM ANP for 1 h. Results show concentration of extracellular cGMP and intracellular cGMP for control preweaned (A) and adult rat (C) glomeruli and for ANP-treated preweaned (B) and adult (D) rat glomeruli. Data are means t SE from n = 4 adult and n = 5 litters and with 8-14 measurements for each condition. *P < 0.05 vs. no probenecid.
cells responding to ANP by cGMP production remains uncertain. Cultured glomerular cells expressing ANP receptors and responding with increase in cGMP have been described for mesangial (1, 6) and epithelial cells (6). We have recently identified immunoreactive cGMP in glomerular epithelial podocytes (but not mesangial cells) in kidneys from rats perfused in vivo with ANP (12). In contrast, stimulation of soluble guanylate cyclase by infusion of sodium nitroprusside resulted in localization of cGMP to mesangial cells rather than podocytes (12). These results suggest that in vivo podocytes are primarily responsible for the glomerular response to ANP. Micropuncture studies have shown that ANP enhances glomerular production of cGMP in glomerular ultrafiltrate without a significant increase of systemic arterial or renal venous cGMP (17). Therefore, egression of cGMP from glomerular cells presumably accounts for cGMP found in glomerular ultrafiltrate. In addition, the increase in urinary cGMP seen after administration of ANP is directly attributable to glomerular production of cGMP (17). Systemic infusion of cGMP in dogs has been shown to result in diuresis, natriuresis, and an increase in glomerular filtration rate (23). The infused cGMP is eliminated only in glomerular filtrate (23). It has also been reported that addition of exogenous cGMP to the bath of rat cortical collecting duct inhibits NaCl resorp-
RESPONSE
TO
ANP
tion and vasopressin-stimulated fluid absorption, mimicking the action of ANP on these segments of the nephron (24). The identical rena, response to direct infusion of cGMP and to ANP strongly suggests a role for cGMP as a second messenger in the biological action of ANP on the kidney. The known biological actions of cGMP include stimulation of cGMP-dependent protein kinase (lo), activation of phosphodiesterase II (3), and gating of sodium channels in retinal rods (13) and renal intramedullary collecting ducts (20). These responses are generally felt to be mediated by increased intracellular cGMP. However, it is possible that extracellular cGMP in the glomerular filtrate may act at additional sites within the nephron to facilitate sodium and water excretion. This could be accomplished by direct action of cGMP on proximal and distal tubule epithelia or after uptake of cGMP into tubular cells. Therefore, the decrease in export of cGMP from preweaned glomerular cells may be physiologically relevant to the observed limitation of natriuresis and diuresis in this age. In summary, our data indicate that intracellular cGMP content is increased and phosphodiesterase activity is decreased in preweaned glomeruli compared with adult glomeruli. Moreover, in response to ANP, the preweaned glomeruli export less cGMP to the extracellular space. If the biological action of ANP is dependent on transport of cGMP out of the glomerular cells, then these differences may be responsible in part for blunted natriuresis and diuresis in preweaned rats. In turn, this adaptation may facilitate conservation of sodium in the rapidly growing preweaned rat with limited access to exogenous sodium and increased sodium requirements for growth. This study was supported by National Heart, Lung, and Blood Institute Grant HL-40209 (to R. L. Chevalier), American Heart Association Grant-in-Aid 91006380 (to R. L. Chevalier), Division of Research Resources Biomedical Research Support Grant RR-05431-28 (to L. L. Norling), and Research Center of Excellence in Pediatric Nephrology and Urology Grant P50-DK-44765-01 (to R. L. Chevalier and L. L. Norling). Portions of this work were presented at the Annual Meeting of the Southern Society for Pediatric Research in New Orleans, LA, in January 1991. Address for reprint requests: L. L. Norling, Dept. of Pediatrics, Univ. of Virginia, MR4/2034, Charlottesville, VA 22908. Received
3 July
1991; accepted
in final
form
24 September
1991.
REFERENCES 1. Ballermann, B. J., R. L. Hoover, M. J. Karnovsky, and B. M. Brenner. Physiologic regulation of atria1 natriuretic peptide receptors in rat renal glomeruli. J. Clin. Invest. 76: 2049-2056, 1985. 2. Beasley, D., and R. L. Malvin. Atria1 extracts increase glomerular filtration rate in vivo. Am. J. Physiol. 248 (Renal Fluid Electrolyte Physiol. 17): F24-F30, 1985. 3. Beavo, J. A., J. G. Hardman, and E. W. Sutherland. Hydrolysis of cyclic guanosine and adenosine 3’,5’-monophosphates by rat and bovine tissues. J. Biol. Chem. 245: 5649-5655, 1970. 4. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. 5. Braunlich, H., and S. Solomon. Renal effects of atria1 natriuretic factor in rats of different ages. Physiol. BohemosZov. 36: 119124,1987. 6. Chansel, D., P. Pham, M.-P. Nivez, and R. Ardaillou. Char-
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