475
Journal of Physiology (1990), 423, pp. 475-484 With 2 figures Printed in Great Britain
VASOACTIVE INTESTINAL PEPTIDE STIMULATION OF HUMAN RENAL ADENYLATE CYCLASE IN VITRO BY B. G. CHARLTON, D. E. NEAL* AND N. L. SIMMONS From the Department of Physiological Sciences, the Medical School, University of Newcastle upon Tyne, NE2 4HH and *Department of Urology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN
(Received 9 June 1989) SUMMARY
1. A direct action of vasoactive intestinal peptide (VIP) upon human kidney was sought by measurement of renal adenylate cyclase in tissue homogenates and plasma membranes isolated from tissue samples excised for therapeutic reasons. 2. VIP (1 ,M) produced a mean stimulation of adenylate cyclase activity of 3 5-fold compared to basal values in cortical plasma membranes; comparative stimulations of 2-8-fold and 27 3-fold were obtained with 1 puM-glucagon and 1 /tM-h(1-34) parathyroid hormone respectively. 3. Half-maximal stimulation of human renal cortical plasma membrane adenylate cyclase was observed with a mean value of 35 nM-VIP. 4. The stimulation of renal adenylate cyclase by VIP appeared to be specific because stimulation by glucagon was additive to that obtained with VIP, and the VIP receptor antagonist (4 Cl-D-Phe6, Leul7)-VIP inhibited the VIP-dependent stimulation of adenylate cyclase activity. INTRODUCTION
Vasoactive intestinal peptide (VIP) is a twenty-eight amino acid, straight chain peptide (Bodanszky, Klausner & Said, 1973) initially isolated from pig small intestine (Said & Mutt, 1970) but later found to be widespread within the gut and brain (Bryant, Polak, Modlin, Bloom, Albuquerque & Pearse, 1976). There is evidence from several sources that VIP has a role in regulating renal function. Intravenous VIP infusion in the rabbit alters renal haemodynamics, increases fractional excretions of sodium, potassium and chloride and decreases urinary pH (Dimaline, Peart & Unwin, 1983). The natriuretic action of VIP in conscious rabbits has been confirmed by Duggan & Macdonald (1987) using both renal arterial and intravenous VIP infusions. VIP has been detected in a variety of mammalian kidneys using immunohistochemical techniques (Barajas, Sokolski & Lechago, 1983; Knight, Beal, Yuan & Fournet, 1987; Reinecke & Forssmann, 1988) where it is located in nerves principally associated with blood vessels. VIP is known to act through stimulation of adenylate cyclase activity and a stimulatory effect of VIP on the renal enzyme has been previously demonstrated in the rabbit. (Griffiths & Simmons, 1987), cat (Griffiths, Rivier & Simmons, 1988 a) and MS 7744
476
B. G. CHARLTON, D. E. NEAL AND N. L. SIMMONS
dog (Griffiths, Rugg & Simmons, 1989a). Furthermore, VIP-sensitive adenylate cyclase is found to be located within rabbit kidney in glomeruli (Griffiths & Simmons, 1987) and in specific and distinctive regions of the nephron (in particular the bright portion of the distal convoluted tubule and the inner stripe of the outer medullary collecting tubule; Griffiths, Chabardes, Imbert-Teboul, Siaume-Perez, Morel & Simmons, 1988 b). Specific VIP receptors have been demonstrated using both 1251-labelled VIP binding to isolated feline plasma membranes (Griffiths & Simmons, 1988) and by 1251-labelled VIP autoradiography in rat kidney sections (Magistretti, Hof, Martin, Dietl & Palacios, 1988). There have been few studies on VIP modulation of renal function in the human. In human volunteers intravenous VIP infusions result in modification of urinary output of sodium, potassium chloride and phosphate, as well as causing increased urinary acidification (Calam, Dimaline, Peart, Singh & Unwin, 1983). As pointed out by these authors, however, it is difficult to assess whether the renal effects arise wholly or in part from a primary alteration of cardiovascular parameters. There is no information concerning VIP distribution or action within human kidney. The present study describes the measurement of VIP-stimulated adenylate cyclase activity in human renal tissue. A preliminary report of the present findings has been presented (Charlton, Neal & Simmons, 1989). METHODS
Renal material Renal tissue was obtained from patients undergoing nephrectomy for hypernephroma (n = 4), renal cyst (n = 1) and transitional cell carcinoma of the renal pelvis (n = 1) or ureter (n = 2). Apparently normal tissue was taken from a part of the kidney remote from the primary lesion. Patients with gross hydronephrosis or with advanced neoplasms (from whom normal kidney tissue could not be obtained) were excluded from the study. Tissue sample size ranged from 0-6 to 18 g. Seven samples of renal cortical material were obtained and one (18 g) sample of a complete normal pole which was subdivided into cortex and medulla. The cortical samples were immediately chilled and snap frozen in liquid nitrogen. Tissue was thawed on ice prior to membrane preparation. For the 18 g sample it was possible to chill the tissue and immediately prepare plasma membranes.
Preparation of plasma membranes from cortical and medullary tissue Methods used were similar to those previously described for the preparation of plasma membranes from rabbit kidneys (Griffiths & Simmons, 1987). Tissue samples were placed in ice-cold assay buffer containing sucrose (0 25 M), Tris-HEPES (2 mM), phenylmethylsulphonyl fluoride (0 1 mM), EDTA (1 mM), pH 7-4; and processed on an ice-cool plate. Adherent fat and capsule were removed and cortical tissue carefully dissected from medullary tissue (for the 18 g sample). Kidney tissue was chopped with scissors and coarsely homogenized using a Polytron homogenizer (setting 3 for 15 s) in the proportion of 1 g of tissue to 10 cm3 of assay buffer. Fine homogenization was achieved using a motor-driven Teflon-glass homogenizer (Braun, Potter S, five strokes at 800 r.p.m.). Samples of homogenate were retained for comparison with plasma membranes. The remaining homogenate was centrifuged for 10 min at 1000 g (centrifuge brake off) and the pellet discarded. The supernatant was then centrifuged for 15 min at 22000 g to form a triple-layered pellet. The upper white fluffy layer was then carefully harvested using gentle aspiration with Pasteur pipettes with a buffer containing 0-25 M-sucrose and 2 mM-Tris-HEPES (pH 7 4). After gentle homogenization (five strokes of a Dounce Teflon-glass homogenizer), all samples were snap frozen in liquid nitrogen and then transferred to a -90 °C freezer until use. The extent of purification of plasma membranes from homogenate may be assessed by the enrichment of the forskolin-stimulated values of adenylate cyclase (minus basal values). This
VIP AND HUMAN KIDNEY
477
increased from 353 + 23 (S.D., n = 3) in homogenates to 576 + 144 pmol cyclic AMP (mg protein)-' 15 min- for three subjects.
Measurement of adenylate cyclase and protein determinations These were performed as described previously (Griffiths & Simmons, 1987). Materials
[2,8-3H]cyclic AMP was obtained from Amersham International, Amersham. Forskolin, porcine VIP, peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), natural porcine glucagon, secretin and h(1-34) parathyroid hormone (PTH) were all obtained from Sigma Chemical Company (Poole, Dorset) and CRB (Harlton, Cambridgeshire). (4C1-D-Phe6, Leul7)-VIP was a generous gift of Dr J. Rivier (Salk Institute, San Diego, CA, USA). Stock solutions of peptide hormones were made up in buffer (80 mM-Tris-maleate, 4 mM-MgSO4, 0-2 mM-EGTA, pH 7-5) in Eppendorf polypropylene tubes, except for glucagon which was dissolved in 10 mM-sodium chloride solution. Stocks were maintained at 4 TC or frozen at -20 TC until required. Salts and other biochemicals were obtained from BDH Chemicals (Poole, Dorset) or the Sigma Chemical Company and were of Analar grade or equivalent. Statistics Variations in mean data are expressed as the standard deviation (S.D.) or if stated, standard error of the mean (S.E.M.) of results. Within experiment error was determined by replication (n = 3); mean values for individual subjects were pooled to give data for n subjects. Tests of significance of differences between mean values were made using a two-tailed Student's t test (paired means solution) where appropriate. Dose-response curves were analysed using the Allfit program (Delean, Munson & Rodbard, 1978). Estimates of the error of standard parameters are given for single dose-response curves as standard errors of the mean (S.E.M.) (Delean et al. 1978). RESULTS
Table 1 summarizes the response of human renal cortical plasma membrane adenylate cyclase activity to VIP, glucagon, h(1-34) PTH (all at 1 /tM) and 100 /iM)forskolin. The stimulation observed with PTH is of a similar order of magnitude to that observed with forskolin (except for subject 2). In all samples, 1 JiM-VIP gave a marked stimulation of adenylate cyclase activity. The mean fold stimulation observed over basal levels for the seven subjects was 3-5-fold (range 2-4 9). Structurally related peptides to VIP were also tested for their ability to stimulate human renal adenylate cyclase at 1 JUM. Glucagon (Table 1) stimulates human cortical renal adenylate cyclase; in each case the stimulation observed was similar to or lower than that with VIP (Table 1). In contrast to glucagon, secretin was largely ineffective in stimulation of human renal cortical adenylate cyclase (1-3+0-2-fold over basal values, in four subjects); the peptides PHI and PHM gave 2-1 + 0-8- (four subjects) and 2-4 + 05- (three subjects) fold stimulations of adenylate cyclase respectively. Samples of plasma membranes were snap frozen and stored at -90 'C. We tested whether a single cycle of freeze-thaw combined with 48 h storage influenced hormonal stimulation of adenylate cyclase. In fresh plasma membranes prepared from the 18 g sample, stimulations observed by 1 JuM-VIP, glucagon, PTH and 100 JaM-forskolin were (freeze-thaw membrane values in parentheses) 3-8 (4 4), 2 0 (0 6), 18-9 (20 5) and 30 7 (39 6) times the baseline value respectively. For one tissue sample (0-6 g) it was impossible to prepare plasma membranes.
¢
-£o,;
.I-_ . . .
B. G. CHARLTON, D. E. NEAL ANI) Xr L. SIMMOINIS
478
-
o o -
_
J-
[: lo s s X s X
S ._
.
:-~
£L
U-
t~Q~
21..
I. *p -4 t- P 4 -.I -. ce; 'I
; _ e -
-4
E
tc < O X X < < e
: e X e e - X X X K e s N e _*
-
+l +l
._
Ct
C.)
$
t
Ct cs3
_
_I
a:1
CQ
ce
C;
._
£
W
Ce
-
>
O: U: * m O:
2
: + c: es * o e .
.
.
.
.
.
.
t- t- 00 1m t- Cm 1-
--fc:
+l
cq
-4-_e
x
._
*)
-
c;
._
8
C) W
s
OeD
btO
z
X
Ct o = -o _ _ ptc
~~
~~~ C)
p+
¢e C; V
H
Z
~cqt
- CQ
-,
D t-
LC
VIP AND HUMAN KIDNEY
479
Instead, the action of VIP, glucagon, PTH and forskolin were tested on adenylate cyclase activities in a cortical homogenate. VIP-, glucagon- and PTH-stimulated values of adenylate cyclase were similar to those observed in plasma membranes; however, due to markedly lower basal values, fold stimulations (23-0, 9-1 and 174-5, respectively) were higher than in plasma membranes. 70.
60
E Il, In
C
50
0.
0
L-
Ea
40
E L-l ._
30
0
._
ca 0 U)
20
-
C.)
(a
10 -
0
-10
-9
-8
-7
-6
Iogo[VIP] (M) Fig. 1. Illustrative example of the effects of stimulation of human renal plasma membrane adenylate cyclase by VIP of cortical (@) or medullary (A) origin. Data are the mean+S.D. of three replicate determinations per point. Half-maximal stimulation was observed at 1X73 + 2*1 x 10-8 M for cortical plasma membranes and 6-4+ 1-3 nm for medullary membranes.
In three subjects, the action of VIP was tested on both cortical homogenate and cortical plasma membranes prepared from the same sample. Basal activities of adenylate cyclase were (mean + S.D. of replicate readings) 18-2 + 5-2, 9-3 + 1F1 and 4-8 + 1-8 pmol (mg protein)-' 15 min' for homogenates and 77-1 + 7 3, 23-9 + 5-5 and 14-7 + 2-8 pmol (mg protein)-1 15 min- for plasma membranes respectively. Stimulation by VIP in homogenates (80 + 5-8-fold over basal values) was correspondingly lower in plasma membranes (3 3 + 1*5-fold) due to the difference in basal values. The reason for the elevated levels of basal activity in the plasma membrane sample is unknown, but may involve co-purification of an endogenous agonist for adenylate cyclase.
480
B. G. CHARLTON, D. K NEAL AND N. L. SIMMONS
In only one subject was it possible to prepare medullary plasma membranes; a 2-4fold stimulation of adenylate cyclase by VIP in human renal medullary plasma membranes was observed in comparison to a 3-5-fold stimulation in cortical plasma membranes. In addition, the structurally related peptides glucagon (19-fold), PHI (1P8-fold) and secretin (1P2-fold) gave stimulations comparable to cortical membranes. 6
U)
01)
5
0
-
U1) (a
0)
4
0
4) U)
co
3 a)
(U)
4-a
(_
0 c
2
co
0 (U
A
E U)
1 U.
0
-9
-8
-7
-6
loglo[VIP] (M) Fig. 2. Antagonism of VIP-stimulated adenylate cyclase (0) by 5 /LM-(4C1-D-Phe6, Leul7)-VIP (-). Data are expressed as the mean of triplicate determinations from a single individual. Half-maximal stimulation by VIP was observed at 5-6 + 4-1 nM-VIP; this was increased to 41-6 + 3-0 nM by 5 ,uM-(4Cl-D-Phe6, Leu'7)-VIP.
Figure 1 is an illustrative example of the dose dependence of VIP stimulation of human renal adenylate cyclase in cortical and medullary plasma membranes from a single individual. In five such subjects the mean value of half-maximal stimulation in renal cortex by VIP was 35 + 1-9 (S.D.) X 10-8M. The slope factor (Delean et al. 1987) for these sigmoid curves was 0-79+0-29. The sensitivity of human renal adenylate cyclase to VIP is thus similar to that observed previously in rabbit (Griffiths & Simmons, 1987), dog (Griffiths et al. 1989a) and cat (Griffiths, Simmons & Rivier, 1989b). In order to test whether VIP and glucagon stimulate human renal adenylate cyclase via the same receptor, we tested the effects of VIP and glucagon stimulation alone and in conjunction (Table 2). For six subjects, the effects of VIP and glucagon
VIP AND HUMAN KIDNEY
481
were additive, with the combined effect of both hormones together being not significantly different to the sum of each of their individual effects (P < 0f25, paired data, n = 6). This implies that the two hormones act via separate receptors. The specificity of the observed stimulation of human renal adenylate cyclase by VIP was tested further by the use of the VIP receptor antagonist (4C1-D-Phe6, Leu17)-VIP (Pandol, Dharmsathaphorn, Schoeffield, Vale & Rivier, 1986). Figure 2 demonstrates that (4CI-D-Phe6, Leu17)-VIP is an effective competitive antagonist of VIP-stimulated activity and produces a significant right shift (approximately one order of magnitude) in the VIP dose-response curve (similar to that seen in the cat; Griffiths et al. 1989b). Furthermore 5 ,uM-(4Cl-D-Phe6. Leu17)-VIP has no significant intrinsic agonist activity (Fig. 2). In six separate subjects, 5 gM-(4Cl-D-Phe6, Leu17)-VIP reduced VIP-stimulated activity (10 nM-VIP) from 2'2-, 3-8-, 1P6-, 1P8-, 1-5- and 1-9-fold over basal values to 1-8-, 1'5-, 1-9-, 1-3-, 1P1- and 1P2-fold over basal values respectively. In one experiment 5 fLM-(4Cl-D-Phe6, Leu17)-VIP was without effect upon glucagon-stimulated adenylate cyclase activity (24-4 + 3-2 compared with 24-6 + 2'8 pmol (mg protein)-' 15 min-' plus antagonist). DISCUSSION
Human renal tissue, obtained from kidneys excised because of suspected tumours, has been used in the present study to assess whether vasoactive intestinal peptide (VIP) has a direct action on the human kidney. Previous studies on the action of renally acting hormones have also used tissue excised for similar therapeutic indications (Mulvehill, Hui, Barnes, Palumbo & Dousa, 1976; Kim, Frohnert, Hui, Barnes, Farrow & Dousa, 1977). These authors reported stimulation of tissue adenylate cyclase by hormones which are known to act directly on the kidney (e.g. PTH, glucagon, arginine vasopressin and calcitonin). Such early results have been substantiated by studies using isolated, microdissected nephron segments taken from normal human kidneys (Chabardes, Gagnan-Brunette, Imbert-Teboul, Gontcharevskaia, Montegut, Clique & Morel, 1980). The methods of the present study are consistent with those of Kim et al. (1977), who reported parathyroid hormone (8-fold) and glucagon (1'5-fold) stimulation of adenylate cyclase activity in renal cortex. In the present experiments forskolin has also been used as an internal experimental control. Forskolin activates the catalytic subunit of adenylate cyclase directly and so provides an estimate of the maximal range of adenylate cyclase present within the plasma membranes (Seamon & Daly, 1983). The magnitude of the stimulation of cortical plasma membrane adenylate cyclase by PTH is similar to that observed with forskolin and to the stimulation by PTH observed by Kim et al. (1977). In one case (subject 2), the reduction of PTH versus forskolin stimulation may indicate tissue damage due to obstruction of the ureter and reduction of glomerular filtration. We are therefore confident that the observed stimulation of adenylate cyclase by VIP is a feature of the normal human kidney. The magnitude of the stimulation of adenylate cyclase observed with VIP is a fraction of that observed with a forskolin or PTH. The mean value of stimulation by VIP in human renal material is similar to that found in other species (1-5-fold in rabbit, Griffiths & Simmons, 1987; 1-7-fold in dog, Griffiths et al. 1989a; 1'8-fold in guinea-pig and 4'1-fold in cat, Griffiths et al. 16
PH Y 423
482 482
B. G. CHARLTON, D.E. NEAL AND N. L. SIMMONS
1989b). It is likely that VIP-stimulated adenylate cyclase is restricted to discrete and specific loci not involving the bulk of the renal mass in the human kidney. This is directly analogous to the situation found in the rabbit (Griffiths & Simmons, 1987; Griffiths et al. 1988b). It is noteworthy that the magnitude of the VIP-stimulated adenylate cyclase response, although only a fraction of that observed with parathyroid hormone, is similar to that observed with glucagon. It is important to attempt to assess whether the observed effects of VIP upon human renal adenylate cyclase are indeed specific for VIP. Half-maximal stimulation of human renal adenylate cyclase by VIP was observed at 35nm, comparable to that previously observed in plasma membranes prepared from the kidneys of other mammals (Griffiths & Simmons, 1987; Griffiths et al. 1988a; Griffiths, Simmons & Rivier, 1989b) and from small bowel (Dharmsathaphorn, Harms, Yamashiro, Hughes, Binder & Wright, 1983). The action of VIP is subject to competitive inhibition by the VIP receptor antagonist (4C1-D-Phe6, Leu17)-VIP (Pandol et al. 1986) and though the related peptide glucagon may also stimulate human renal adenylate cyclase (see Results, also Kim et al. 1977) it is apparent that this stimulation is additive to that stimulation observed with VIP (suggesting that VIP and glucagon act via different receptors). Thus it seems likely that VIP has a specific stimulative action on human renal adenylate cyclase activity. In only one case was it possible to demonstrate that the VIP-responsive adenylate cyclase was not confined to renal cortex, but was also present in renal medulla. More work thus needs to be performed to investigate the intra-renal locus of the VIPsensitive adenylate cyclase in human kidney. It is possible that VIP-stimulated adenylate cyclase in the human is restricted to the vasculature; however, in other species this is not the case. In the rabbit the VIP-sensitive adenylate cyclase was confined to glomeruli, the 'bright' and portions of the distal convoluted tubule and the outer medullary collecting tubule (Griffiths et al. 1988b). More recent work in the cat has shown that the pars recta of the proximal tubule in the outer medulla was also responsive to VIP (N. M. Griffiths and N. L. Simmons, unpublished data). The reason for this difference in intra-renal locus for VIP action is unclear, but interspecies differences in the hormonal control of nephron function are well documented (Morel, 1981) and caution must be exercised in extrapolating data from one species to another in this regard. There is a single previous study in the human where the effects of intravenous VIP infusions on renal function have been measured (Calam et al. 1983). In this study, endogenous basal levels of VIP of 0-05 were roughly doubled by VIP infusion to 01 nm. Such infusions produced an increase in heart rate and a decrease in blood pressure consistent with generalized vasodilatation, but renal blood flow and glomerular filtration rate were unaffected. The observed fall in urinary water, Na+, K+, Cl- and Ca2+ outputs observed in this study may have been secondary to the cardiovascular alterations. In a similar study using the rabbit (Dimaline et al. 1983) much higher VIP plasma levels were achieved (0-6-2'0 and there was a reduction in renal blood flow and filtration rate: in however, direct tubular actions of VIP were addition, glomerular evident and the fractional excretion of Na+, K+ and Cl- increased significantly. The finding of natriuresis has been substantiated by Duggan & Macdonald (1987) using
'granular'
nm
nM)
VIP AND HUMAN KIDNEY
483
both intravenous and intrarenal infusions of VIP in conscious rabbits. Dimaline et al. (1983) reported that even at the lowest infusion dose (004-O-07 nm, plasma VIP concentration), a significant urinary acidification was evident. Urinary acidification was also observed in the human study (Calam et al. 1983). Since urine flow altered in a disparate way in these two species after VIP infusions, it seems that a direct tubular action of VIP giving rise to increased urinary acidification, even at this low plasma level, is evident. For the rabbit we have argued that VIP stimulation of adenylate cyclase in the collecting tubule of the outer stipe activates a Cl conductance in the intercalated cells, so augmenting H+ secretion (Griffiths et al. 1988b). It is possible that a similar mechanism underlies the VIP-dependent urinary acidification in man. In both man and rabbit plasma renin activity was increased by VIP (Calam et al. 1983; Dimaline et al. 1983). Though indirect effects cannot be excluded in both cases, and work in dogs would argue against a direct role for VIP in controlling renin secretion (Porter, Thrasher, Said & Ganong, 1985), we would draw attention to our own data which shows VIP stimulation of glomerular adenylate cyclase (Griffiths & Simmons, 1987). It is readily apparent from the preceding discussion that future progress in elucidating the direct role of VIP in the control of renal function requires the availability of stable and effective VIP agonists and antagonists. The antagonist (4C1-D-Phe6, Leu17)-VIP will be of limited use in man due to its low potency. The dose-dependent activation of VIP-sensitive renal adenylate cyclase reported here suggests that normal circulating levels of VIP are not physiologically important in activating renal adenylate cyclase and that VIP must be released intra-renally, most probably from nerves. Though VIP immunoreactivity has been reported in other mammalian kidneys (Barajas et al. 1983; Knight et al. 1987; Reinecke & Forssmann, 1988), direct confirmation of the presence of VIP-containing nerves within human kidney should now be sought. This work was supported by the Wellcome Trust (Grant 16764/15) and by Newcastle upon Tyne Small Grant award to B. G. C.
a
University of
REFERENCES
BARAJAS, L., SOKOLSKI, K. N. & LECHAGO, J. (1983). Vasoactive intestinal polypeptideimmunoreactive nerves in the kidney. Neuroscience Letters 43, 263-269. BODANSZKY, M., KLAUSNER, M. & SAID, S. I. (1973). Biological activities of synthetic peptides corresponding to fragments of and to the entire sequence of vasoactive intestinal peptide. Proceedings of the National Academy of Sciences of the USA 70, 382-384. BRYANT, M. G., POLAK, J. M., MODLIN, I., BLOOM, S. R., ALBUQUERQUE, R. H. & PEARSE, A. G. E. (1976). Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet i, 991-993. CALAM, J., DIMALINE, R., PEART, W. S., SINGH, J. & UNWIN, R. J. (1983). Effects of vasoactive intestinal polypeptide on renal function in man. Journal of Physiology 345. 496-475. CHABARDES, D., GAGNAN-BRUNETTE, M., IMBERT-TEBOUL, M., GONTCHAREVSKAIA, 0., MONTEGUT, M., CLIQUE, A. & MOREL, F. (1980). Adenylate cyclase responsiveness to hormones in various portions of the human nephron. Journal of Clinical Investigation 65, 439-448. CHARLTON, B. G., NEAL, D. E. & SIMMONS, N. L. (1989). Vasoactive intestinal peptide stimulation of human renal adenylate cyclase in vitro. Journal of Physiology 416, 32P. 16-2
B. G. CHARLTON, D. E. NEAL AND N. L. SIMMONS 484 DELEAN, A., MUNSON, P. J. & RODBARD, D. (1978). Simultaneous analysis of families of sigmoid curves: application to bioassay, radioligand assay and physiological dose-response curves. American Journal of Physiology 235, E97-102. DHARMSATHAPHORN, K., HARMS, V., YAMASHIRO, D. J., HUGHES, R. J., BINDER, H. J. & WRIGHT, E. M. (1983). Preferential binding of vasoactive intestinal polypeptide to basolateral membrane or rat enterocytes. Journal of Clinical Investigations 71, 27-35. DIMALINE, R., PEART, W. S. & UNWIN, R. J. (1983). Effects of vasoactive intestinal polypeptide (VIP) on renal function and plasma renin activity in the conscious rabbit. Journal of Physiology 344, 379-388. DUGGAN, K. A. AND MACDONALD, G. J. (1987). VIP: a direct renal natriuretic substance. Clinical Science 72, 195-200. GRIFFITHS, N. M., CHABARDES, D., IMBERT-TEBOUL, M., SIAUME-PEREZ, S., MOREL, F. & SIMMONS, N. L. (1988b). Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron. Pfiigers Archiv 412, 363-368. GRIFFITHS, N. M., RIVIER, J. & SIMMONS, N. L. (1988a). Vasoactive intestinal peptide stimulation of feline renal adenylate cyclase; inhibitory effects of (4CI-D-Phe6, Leul7)-VIP. Annals of the New York Academy of Sciences 527, 640-643. GRIFFITHS, N. M., RUGG, E. L. & SIMMONS, N. L. (1989a). Vasoactive intestinal peptide control of renal adenylate cyclase: in vitro studies of canine renal membranes and cultured renal epithelial (MDCK) cells. Quarterly Journal of Experimental Physiology 74, 339-353. GRIFFITHS, N. M. & SIMMONS, N. L. (1987). Vasoactive intestinal peptide regulation of rabbit renal adenylate cyclase activity in vitro. Journal of Physiology 387, 1-17. GRIFFITHS, N. M. & SIMMONS, N. L. (1988). Characterization of [3-iodotyrosyl '25I]vasoactive intestinal peptide binding to feline renal plasma membranes in vitro. Journal of Physiology 406,
118P.
GRIFFITHS, N. M., SIMMONS, N. L. & RIVIER, J. (1989b). Vasoactive intestinal peptide stimulation of renal adenylate cyclase and antagonism by (4CI-D-Phe6, Leul7)-VIP. Pfiugers Archiv 414, 222-227.
KIM, J. K., FROHNERT, P. P., HUI, Y. S. F., BARNES, L. D., FARROW, G. M. & DOUSA, T. P. (1977). Enzymes of cyclic 3',5'-nucleotide metabolism in human renal cortex and renal adenocarcinoma. Kidney International 12, 172- 183. KNIGHT, D. S., BEAL, J. A., YUAN, Z. P. & FOURNET, T. S. (1987). Vasoactive intestinal peptideimmunoreactive nerves in the rat kidney. Anatomical Record 219, 193-203. MAGISTRETTI, P. J., HOF, P. R., MARTIN, J. L., DIETL, M. & PALACIOS, J. M. (1988). High- and low-affinity binding sites for vasoactive intestinal peptide (VIP) in the rat kidney revealed by light microscopic autoradiography. Regulatory Peptides 23, 145-152. MOREL, F. (1981). Sites of hormone action in the mammalian nephron. American Journal of Physiology 240, 159-164. MULVEHILL, J. B., HUI, Y. S. F., BARNES, L. D., PALUMBO, P. J. & DOUSA, T. P. (1976). Glucagonsensitive adenylate cyclase in human renal medulla. Journal of Clinical Endocrinology and Metabolism 42, 380-384. PANDOL, S. J., DHARMSATHAPHORN, K., SCHOEFFIELD, M. S., VALE, W. & RIVIER, J. (1986). Vasoactive intestinal peptide receptor antagonist (4-Cl-D-Phe6, Leu'7) VIP. American Journal of Physiology 250, G553-557. PORTER, J. P., THRASHER, T. N., SAID, S. I. & GANONG, W. F. (1985). Vasoactive intestinal peptide in the control of renin secretion. American Journal of Physiology 249, F84-89.
REINECKE, M. & FORSSMANN, W. G. (1988). Neuropeptide (neuropeptide Y, neurotensin, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, somatostatin) immunohistochemistry and ultrastructure of renal nerves. Histochemistry 89, 1-9. SAID, S. I. & MUTT, V. (1970). Polypeptide with broad biological activity: isolation from small intestine. Science 169, 1217-1218. SEAMON, K. B. & DALY, J. W. (1983). Forskolin, cyclic AMP and cellular physiology. Trends in Pharmacological Sciences 2, 120-123.
Journal of Physiology (1990), 423, pp. 475-484 With 2 figures Printed in Great Britain
VASOACTIVE INTESTINAL PEPTIDE STIMULATION OF HUMAN RENAL ADENYLATE CYCLASE IN VITRO BY B. G. CHARLTON, D. E. NEAL* AND N. L. SIMMONS From the Department of Physiological Sciences, the Medical School, University of Newcastle upon Tyne, NE2 4HH and *Department of Urology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN
(Received 9 June 1989) SUMMARY
1. A direct action of vasoactive intestinal peptide (VIP) upon human kidney was sought by measurement of renal adenylate cyclase in tissue homogenates and plasma membranes isolated from tissue samples excised for therapeutic reasons. 2. VIP (1 ,M) produced a mean stimulation of adenylate cyclase activity of 3 5-fold compared to basal values in cortical plasma membranes; comparative stimulations of 2-8-fold and 27 3-fold were obtained with 1 puM-glucagon and 1 /tM-h(1-34) parathyroid hormone respectively. 3. Half-maximal stimulation of human renal cortical plasma membrane adenylate cyclase was observed with a mean value of 35 nM-VIP. 4. The stimulation of renal adenylate cyclase by VIP appeared to be specific because stimulation by glucagon was additive to that obtained with VIP, and the VIP receptor antagonist (4 Cl-D-Phe6, Leul7)-VIP inhibited the VIP-dependent stimulation of adenylate cyclase activity. INTRODUCTION
Vasoactive intestinal peptide (VIP) is a twenty-eight amino acid, straight chain peptide (Bodanszky, Klausner & Said, 1973) initially isolated from pig small intestine (Said & Mutt, 1970) but later found to be widespread within the gut and brain (Bryant, Polak, Modlin, Bloom, Albuquerque & Pearse, 1976). There is evidence from several sources that VIP has a role in regulating renal function. Intravenous VIP infusion in the rabbit alters renal haemodynamics, increases fractional excretions of sodium, potassium and chloride and decreases urinary pH (Dimaline, Peart & Unwin, 1983). The natriuretic action of VIP in conscious rabbits has been confirmed by Duggan & Macdonald (1987) using both renal arterial and intravenous VIP infusions. VIP has been detected in a variety of mammalian kidneys using immunohistochemical techniques (Barajas, Sokolski & Lechago, 1983; Knight, Beal, Yuan & Fournet, 1987; Reinecke & Forssmann, 1988) where it is located in nerves principally associated with blood vessels. VIP is known to act through stimulation of adenylate cyclase activity and a stimulatory effect of VIP on the renal enzyme has been previously demonstrated in the rabbit. (Griffiths & Simmons, 1987), cat (Griffiths, Rivier & Simmons, 1988 a) and MS 7744
476
B. G. CHARLTON, D. E. NEAL AND N. L. SIMMONS
dog (Griffiths, Rugg & Simmons, 1989a). Furthermore, VIP-sensitive adenylate cyclase is found to be located within rabbit kidney in glomeruli (Griffiths & Simmons, 1987) and in specific and distinctive regions of the nephron (in particular the bright portion of the distal convoluted tubule and the inner stripe of the outer medullary collecting tubule; Griffiths, Chabardes, Imbert-Teboul, Siaume-Perez, Morel & Simmons, 1988 b). Specific VIP receptors have been demonstrated using both 1251-labelled VIP binding to isolated feline plasma membranes (Griffiths & Simmons, 1988) and by 1251-labelled VIP autoradiography in rat kidney sections (Magistretti, Hof, Martin, Dietl & Palacios, 1988). There have been few studies on VIP modulation of renal function in the human. In human volunteers intravenous VIP infusions result in modification of urinary output of sodium, potassium chloride and phosphate, as well as causing increased urinary acidification (Calam, Dimaline, Peart, Singh & Unwin, 1983). As pointed out by these authors, however, it is difficult to assess whether the renal effects arise wholly or in part from a primary alteration of cardiovascular parameters. There is no information concerning VIP distribution or action within human kidney. The present study describes the measurement of VIP-stimulated adenylate cyclase activity in human renal tissue. A preliminary report of the present findings has been presented (Charlton, Neal & Simmons, 1989). METHODS
Renal material Renal tissue was obtained from patients undergoing nephrectomy for hypernephroma (n = 4), renal cyst (n = 1) and transitional cell carcinoma of the renal pelvis (n = 1) or ureter (n = 2). Apparently normal tissue was taken from a part of the kidney remote from the primary lesion. Patients with gross hydronephrosis or with advanced neoplasms (from whom normal kidney tissue could not be obtained) were excluded from the study. Tissue sample size ranged from 0-6 to 18 g. Seven samples of renal cortical material were obtained and one (18 g) sample of a complete normal pole which was subdivided into cortex and medulla. The cortical samples were immediately chilled and snap frozen in liquid nitrogen. Tissue was thawed on ice prior to membrane preparation. For the 18 g sample it was possible to chill the tissue and immediately prepare plasma membranes.
Preparation of plasma membranes from cortical and medullary tissue Methods used were similar to those previously described for the preparation of plasma membranes from rabbit kidneys (Griffiths & Simmons, 1987). Tissue samples were placed in ice-cold assay buffer containing sucrose (0 25 M), Tris-HEPES (2 mM), phenylmethylsulphonyl fluoride (0 1 mM), EDTA (1 mM), pH 7-4; and processed on an ice-cool plate. Adherent fat and capsule were removed and cortical tissue carefully dissected from medullary tissue (for the 18 g sample). Kidney tissue was chopped with scissors and coarsely homogenized using a Polytron homogenizer (setting 3 for 15 s) in the proportion of 1 g of tissue to 10 cm3 of assay buffer. Fine homogenization was achieved using a motor-driven Teflon-glass homogenizer (Braun, Potter S, five strokes at 800 r.p.m.). Samples of homogenate were retained for comparison with plasma membranes. The remaining homogenate was centrifuged for 10 min at 1000 g (centrifuge brake off) and the pellet discarded. The supernatant was then centrifuged for 15 min at 22000 g to form a triple-layered pellet. The upper white fluffy layer was then carefully harvested using gentle aspiration with Pasteur pipettes with a buffer containing 0-25 M-sucrose and 2 mM-Tris-HEPES (pH 7 4). After gentle homogenization (five strokes of a Dounce Teflon-glass homogenizer), all samples were snap frozen in liquid nitrogen and then transferred to a -90 °C freezer until use. The extent of purification of plasma membranes from homogenate may be assessed by the enrichment of the forskolin-stimulated values of adenylate cyclase (minus basal values). This
VIP AND HUMAN KIDNEY
477
increased from 353 + 23 (S.D., n = 3) in homogenates to 576 + 144 pmol cyclic AMP (mg protein)-' 15 min- for three subjects.
Measurement of adenylate cyclase and protein determinations These were performed as described previously (Griffiths & Simmons, 1987). Materials
[2,8-3H]cyclic AMP was obtained from Amersham International, Amersham. Forskolin, porcine VIP, peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), natural porcine glucagon, secretin and h(1-34) parathyroid hormone (PTH) were all obtained from Sigma Chemical Company (Poole, Dorset) and CRB (Harlton, Cambridgeshire). (4C1-D-Phe6, Leul7)-VIP was a generous gift of Dr J. Rivier (Salk Institute, San Diego, CA, USA). Stock solutions of peptide hormones were made up in buffer (80 mM-Tris-maleate, 4 mM-MgSO4, 0-2 mM-EGTA, pH 7-5) in Eppendorf polypropylene tubes, except for glucagon which was dissolved in 10 mM-sodium chloride solution. Stocks were maintained at 4 TC or frozen at -20 TC until required. Salts and other biochemicals were obtained from BDH Chemicals (Poole, Dorset) or the Sigma Chemical Company and were of Analar grade or equivalent. Statistics Variations in mean data are expressed as the standard deviation (S.D.) or if stated, standard error of the mean (S.E.M.) of results. Within experiment error was determined by replication (n = 3); mean values for individual subjects were pooled to give data for n subjects. Tests of significance of differences between mean values were made using a two-tailed Student's t test (paired means solution) where appropriate. Dose-response curves were analysed using the Allfit program (Delean, Munson & Rodbard, 1978). Estimates of the error of standard parameters are given for single dose-response curves as standard errors of the mean (S.E.M.) (Delean et al. 1978). RESULTS
Table 1 summarizes the response of human renal cortical plasma membrane adenylate cyclase activity to VIP, glucagon, h(1-34) PTH (all at 1 /tM) and 100 /iM)forskolin. The stimulation observed with PTH is of a similar order of magnitude to that observed with forskolin (except for subject 2). In all samples, 1 JiM-VIP gave a marked stimulation of adenylate cyclase activity. The mean fold stimulation observed over basal levels for the seven subjects was 3-5-fold (range 2-4 9). Structurally related peptides to VIP were also tested for their ability to stimulate human renal adenylate cyclase at 1 JUM. Glucagon (Table 1) stimulates human cortical renal adenylate cyclase; in each case the stimulation observed was similar to or lower than that with VIP (Table 1). In contrast to glucagon, secretin was largely ineffective in stimulation of human renal cortical adenylate cyclase (1-3+0-2-fold over basal values, in four subjects); the peptides PHI and PHM gave 2-1 + 0-8- (four subjects) and 2-4 + 05- (three subjects) fold stimulations of adenylate cyclase respectively. Samples of plasma membranes were snap frozen and stored at -90 'C. We tested whether a single cycle of freeze-thaw combined with 48 h storage influenced hormonal stimulation of adenylate cyclase. In fresh plasma membranes prepared from the 18 g sample, stimulations observed by 1 JuM-VIP, glucagon, PTH and 100 JaM-forskolin were (freeze-thaw membrane values in parentheses) 3-8 (4 4), 2 0 (0 6), 18-9 (20 5) and 30 7 (39 6) times the baseline value respectively. For one tissue sample (0-6 g) it was impossible to prepare plasma membranes.
¢
-£o,;
.I-_ . . .
B. G. CHARLTON, D. E. NEAL ANI) Xr L. SIMMOINIS
478
-
o o -
_
J-
[: lo s s X s X
S ._
.
:-~
£L
U-
t~Q~
21..
I. *p -4 t- P 4 -.I -. ce; 'I
; _ e -
-4
E
tc < O X X < < e
: e X e e - X X X K e s N e _*
-
+l +l
._
Ct
C.)
$
t
Ct cs3
_
_I
a:1
CQ
ce
C;
._
£
W
Ce
-
>
O: U: * m O:
2
: + c: es * o e .
.
.
.
.
.
.
t- t- 00 1m t- Cm 1-
--fc:
+l
cq
-4-_e
x
._
*)
-
c;
._
8
C) W
s
OeD
btO
z
X
Ct o = -o _ _ ptc
~~
~~~ C)
p+
¢e C; V
H
Z
~cqt
- CQ
-,
D t-
LC
VIP AND HUMAN KIDNEY
479
Instead, the action of VIP, glucagon, PTH and forskolin were tested on adenylate cyclase activities in a cortical homogenate. VIP-, glucagon- and PTH-stimulated values of adenylate cyclase were similar to those observed in plasma membranes; however, due to markedly lower basal values, fold stimulations (23-0, 9-1 and 174-5, respectively) were higher than in plasma membranes. 70.
60
E Il, In
C
50
0.
0
L-
Ea
40
E L-l ._
30
0
._
ca 0 U)
20
-
C.)
(a
10 -
0
-10
-9
-8
-7
-6
Iogo[VIP] (M) Fig. 1. Illustrative example of the effects of stimulation of human renal plasma membrane adenylate cyclase by VIP of cortical (@) or medullary (A) origin. Data are the mean+S.D. of three replicate determinations per point. Half-maximal stimulation was observed at 1X73 + 2*1 x 10-8 M for cortical plasma membranes and 6-4+ 1-3 nm for medullary membranes.
In three subjects, the action of VIP was tested on both cortical homogenate and cortical plasma membranes prepared from the same sample. Basal activities of adenylate cyclase were (mean + S.D. of replicate readings) 18-2 + 5-2, 9-3 + 1F1 and 4-8 + 1-8 pmol (mg protein)-' 15 min' for homogenates and 77-1 + 7 3, 23-9 + 5-5 and 14-7 + 2-8 pmol (mg protein)-1 15 min- for plasma membranes respectively. Stimulation by VIP in homogenates (80 + 5-8-fold over basal values) was correspondingly lower in plasma membranes (3 3 + 1*5-fold) due to the difference in basal values. The reason for the elevated levels of basal activity in the plasma membrane sample is unknown, but may involve co-purification of an endogenous agonist for adenylate cyclase.
480
B. G. CHARLTON, D. K NEAL AND N. L. SIMMONS
In only one subject was it possible to prepare medullary plasma membranes; a 2-4fold stimulation of adenylate cyclase by VIP in human renal medullary plasma membranes was observed in comparison to a 3-5-fold stimulation in cortical plasma membranes. In addition, the structurally related peptides glucagon (19-fold), PHI (1P8-fold) and secretin (1P2-fold) gave stimulations comparable to cortical membranes. 6
U)
01)
5
0
-
U1) (a
0)
4
0
4) U)
co
3 a)
(U)
4-a
(_
0 c
2
co
0 (U
A
E U)
1 U.
0
-9
-8
-7
-6
loglo[VIP] (M) Fig. 2. Antagonism of VIP-stimulated adenylate cyclase (0) by 5 /LM-(4C1-D-Phe6, Leul7)-VIP (-). Data are expressed as the mean of triplicate determinations from a single individual. Half-maximal stimulation by VIP was observed at 5-6 + 4-1 nM-VIP; this was increased to 41-6 + 3-0 nM by 5 ,uM-(4Cl-D-Phe6, Leu'7)-VIP.
Figure 1 is an illustrative example of the dose dependence of VIP stimulation of human renal adenylate cyclase in cortical and medullary plasma membranes from a single individual. In five such subjects the mean value of half-maximal stimulation in renal cortex by VIP was 35 + 1-9 (S.D.) X 10-8M. The slope factor (Delean et al. 1987) for these sigmoid curves was 0-79+0-29. The sensitivity of human renal adenylate cyclase to VIP is thus similar to that observed previously in rabbit (Griffiths & Simmons, 1987), dog (Griffiths et al. 1989a) and cat (Griffiths, Simmons & Rivier, 1989b). In order to test whether VIP and glucagon stimulate human renal adenylate cyclase via the same receptor, we tested the effects of VIP and glucagon stimulation alone and in conjunction (Table 2). For six subjects, the effects of VIP and glucagon
VIP AND HUMAN KIDNEY
481
were additive, with the combined effect of both hormones together being not significantly different to the sum of each of their individual effects (P < 0f25, paired data, n = 6). This implies that the two hormones act via separate receptors. The specificity of the observed stimulation of human renal adenylate cyclase by VIP was tested further by the use of the VIP receptor antagonist (4C1-D-Phe6, Leu17)-VIP (Pandol, Dharmsathaphorn, Schoeffield, Vale & Rivier, 1986). Figure 2 demonstrates that (4CI-D-Phe6, Leu17)-VIP is an effective competitive antagonist of VIP-stimulated activity and produces a significant right shift (approximately one order of magnitude) in the VIP dose-response curve (similar to that seen in the cat; Griffiths et al. 1989b). Furthermore 5 ,uM-(4Cl-D-Phe6. Leu17)-VIP has no significant intrinsic agonist activity (Fig. 2). In six separate subjects, 5 gM-(4Cl-D-Phe6, Leu17)-VIP reduced VIP-stimulated activity (10 nM-VIP) from 2'2-, 3-8-, 1P6-, 1P8-, 1-5- and 1-9-fold over basal values to 1-8-, 1'5-, 1-9-, 1-3-, 1P1- and 1P2-fold over basal values respectively. In one experiment 5 fLM-(4Cl-D-Phe6, Leu17)-VIP was without effect upon glucagon-stimulated adenylate cyclase activity (24-4 + 3-2 compared with 24-6 + 2'8 pmol (mg protein)-' 15 min-' plus antagonist). DISCUSSION
Human renal tissue, obtained from kidneys excised because of suspected tumours, has been used in the present study to assess whether vasoactive intestinal peptide (VIP) has a direct action on the human kidney. Previous studies on the action of renally acting hormones have also used tissue excised for similar therapeutic indications (Mulvehill, Hui, Barnes, Palumbo & Dousa, 1976; Kim, Frohnert, Hui, Barnes, Farrow & Dousa, 1977). These authors reported stimulation of tissue adenylate cyclase by hormones which are known to act directly on the kidney (e.g. PTH, glucagon, arginine vasopressin and calcitonin). Such early results have been substantiated by studies using isolated, microdissected nephron segments taken from normal human kidneys (Chabardes, Gagnan-Brunette, Imbert-Teboul, Gontcharevskaia, Montegut, Clique & Morel, 1980). The methods of the present study are consistent with those of Kim et al. (1977), who reported parathyroid hormone (8-fold) and glucagon (1'5-fold) stimulation of adenylate cyclase activity in renal cortex. In the present experiments forskolin has also been used as an internal experimental control. Forskolin activates the catalytic subunit of adenylate cyclase directly and so provides an estimate of the maximal range of adenylate cyclase present within the plasma membranes (Seamon & Daly, 1983). The magnitude of the stimulation of cortical plasma membrane adenylate cyclase by PTH is similar to that observed with forskolin and to the stimulation by PTH observed by Kim et al. (1977). In one case (subject 2), the reduction of PTH versus forskolin stimulation may indicate tissue damage due to obstruction of the ureter and reduction of glomerular filtration. We are therefore confident that the observed stimulation of adenylate cyclase by VIP is a feature of the normal human kidney. The magnitude of the stimulation of adenylate cyclase observed with VIP is a fraction of that observed with a forskolin or PTH. The mean value of stimulation by VIP in human renal material is similar to that found in other species (1-5-fold in rabbit, Griffiths & Simmons, 1987; 1-7-fold in dog, Griffiths et al. 1989a; 1'8-fold in guinea-pig and 4'1-fold in cat, Griffiths et al. 16
PH Y 423
482 482
B. G. CHARLTON, D.E. NEAL AND N. L. SIMMONS
1989b). It is likely that VIP-stimulated adenylate cyclase is restricted to discrete and specific loci not involving the bulk of the renal mass in the human kidney. This is directly analogous to the situation found in the rabbit (Griffiths & Simmons, 1987; Griffiths et al. 1988b). It is noteworthy that the magnitude of the VIP-stimulated adenylate cyclase response, although only a fraction of that observed with parathyroid hormone, is similar to that observed with glucagon. It is important to attempt to assess whether the observed effects of VIP upon human renal adenylate cyclase are indeed specific for VIP. Half-maximal stimulation of human renal adenylate cyclase by VIP was observed at 35nm, comparable to that previously observed in plasma membranes prepared from the kidneys of other mammals (Griffiths & Simmons, 1987; Griffiths et al. 1988a; Griffiths, Simmons & Rivier, 1989b) and from small bowel (Dharmsathaphorn, Harms, Yamashiro, Hughes, Binder & Wright, 1983). The action of VIP is subject to competitive inhibition by the VIP receptor antagonist (4C1-D-Phe6, Leu17)-VIP (Pandol et al. 1986) and though the related peptide glucagon may also stimulate human renal adenylate cyclase (see Results, also Kim et al. 1977) it is apparent that this stimulation is additive to that stimulation observed with VIP (suggesting that VIP and glucagon act via different receptors). Thus it seems likely that VIP has a specific stimulative action on human renal adenylate cyclase activity. In only one case was it possible to demonstrate that the VIP-responsive adenylate cyclase was not confined to renal cortex, but was also present in renal medulla. More work thus needs to be performed to investigate the intra-renal locus of the VIPsensitive adenylate cyclase in human kidney. It is possible that VIP-stimulated adenylate cyclase in the human is restricted to the vasculature; however, in other species this is not the case. In the rabbit the VIP-sensitive adenylate cyclase was confined to glomeruli, the 'bright' and portions of the distal convoluted tubule and the outer medullary collecting tubule (Griffiths et al. 1988b). More recent work in the cat has shown that the pars recta of the proximal tubule in the outer medulla was also responsive to VIP (N. M. Griffiths and N. L. Simmons, unpublished data). The reason for this difference in intra-renal locus for VIP action is unclear, but interspecies differences in the hormonal control of nephron function are well documented (Morel, 1981) and caution must be exercised in extrapolating data from one species to another in this regard. There is a single previous study in the human where the effects of intravenous VIP infusions on renal function have been measured (Calam et al. 1983). In this study, endogenous basal levels of VIP of 0-05 were roughly doubled by VIP infusion to 01 nm. Such infusions produced an increase in heart rate and a decrease in blood pressure consistent with generalized vasodilatation, but renal blood flow and glomerular filtration rate were unaffected. The observed fall in urinary water, Na+, K+, Cl- and Ca2+ outputs observed in this study may have been secondary to the cardiovascular alterations. In a similar study using the rabbit (Dimaline et al. 1983) much higher VIP plasma levels were achieved (0-6-2'0 and there was a reduction in renal blood flow and filtration rate: in however, direct tubular actions of VIP were addition, glomerular evident and the fractional excretion of Na+, K+ and Cl- increased significantly. The finding of natriuresis has been substantiated by Duggan & Macdonald (1987) using
'granular'
nm
nM)
VIP AND HUMAN KIDNEY
483
both intravenous and intrarenal infusions of VIP in conscious rabbits. Dimaline et al. (1983) reported that even at the lowest infusion dose (004-O-07 nm, plasma VIP concentration), a significant urinary acidification was evident. Urinary acidification was also observed in the human study (Calam et al. 1983). Since urine flow altered in a disparate way in these two species after VIP infusions, it seems that a direct tubular action of VIP giving rise to increased urinary acidification, even at this low plasma level, is evident. For the rabbit we have argued that VIP stimulation of adenylate cyclase in the collecting tubule of the outer stipe activates a Cl conductance in the intercalated cells, so augmenting H+ secretion (Griffiths et al. 1988b). It is possible that a similar mechanism underlies the VIP-dependent urinary acidification in man. In both man and rabbit plasma renin activity was increased by VIP (Calam et al. 1983; Dimaline et al. 1983). Though indirect effects cannot be excluded in both cases, and work in dogs would argue against a direct role for VIP in controlling renin secretion (Porter, Thrasher, Said & Ganong, 1985), we would draw attention to our own data which shows VIP stimulation of glomerular adenylate cyclase (Griffiths & Simmons, 1987). It is readily apparent from the preceding discussion that future progress in elucidating the direct role of VIP in the control of renal function requires the availability of stable and effective VIP agonists and antagonists. The antagonist (4C1-D-Phe6, Leu17)-VIP will be of limited use in man due to its low potency. The dose-dependent activation of VIP-sensitive renal adenylate cyclase reported here suggests that normal circulating levels of VIP are not physiologically important in activating renal adenylate cyclase and that VIP must be released intra-renally, most probably from nerves. Though VIP immunoreactivity has been reported in other mammalian kidneys (Barajas et al. 1983; Knight et al. 1987; Reinecke & Forssmann, 1988), direct confirmation of the presence of VIP-containing nerves within human kidney should now be sought. This work was supported by the Wellcome Trust (Grant 16764/15) and by Newcastle upon Tyne Small Grant award to B. G. C.
a
University of
REFERENCES
BARAJAS, L., SOKOLSKI, K. N. & LECHAGO, J. (1983). Vasoactive intestinal polypeptideimmunoreactive nerves in the kidney. Neuroscience Letters 43, 263-269. BODANSZKY, M., KLAUSNER, M. & SAID, S. I. (1973). Biological activities of synthetic peptides corresponding to fragments of and to the entire sequence of vasoactive intestinal peptide. Proceedings of the National Academy of Sciences of the USA 70, 382-384. BRYANT, M. G., POLAK, J. M., MODLIN, I., BLOOM, S. R., ALBUQUERQUE, R. H. & PEARSE, A. G. E. (1976). Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance. Lancet i, 991-993. CALAM, J., DIMALINE, R., PEART, W. S., SINGH, J. & UNWIN, R. J. (1983). Effects of vasoactive intestinal polypeptide on renal function in man. Journal of Physiology 345. 496-475. CHABARDES, D., GAGNAN-BRUNETTE, M., IMBERT-TEBOUL, M., GONTCHAREVSKAIA, 0., MONTEGUT, M., CLIQUE, A. & MOREL, F. (1980). Adenylate cyclase responsiveness to hormones in various portions of the human nephron. Journal of Clinical Investigation 65, 439-448. CHARLTON, B. G., NEAL, D. E. & SIMMONS, N. L. (1989). Vasoactive intestinal peptide stimulation of human renal adenylate cyclase in vitro. Journal of Physiology 416, 32P. 16-2
B. G. CHARLTON, D. E. NEAL AND N. L. SIMMONS 484 DELEAN, A., MUNSON, P. J. & RODBARD, D. (1978). Simultaneous analysis of families of sigmoid curves: application to bioassay, radioligand assay and physiological dose-response curves. American Journal of Physiology 235, E97-102. DHARMSATHAPHORN, K., HARMS, V., YAMASHIRO, D. J., HUGHES, R. J., BINDER, H. J. & WRIGHT, E. M. (1983). Preferential binding of vasoactive intestinal polypeptide to basolateral membrane or rat enterocytes. Journal of Clinical Investigations 71, 27-35. DIMALINE, R., PEART, W. S. & UNWIN, R. J. (1983). Effects of vasoactive intestinal polypeptide (VIP) on renal function and plasma renin activity in the conscious rabbit. Journal of Physiology 344, 379-388. DUGGAN, K. A. AND MACDONALD, G. J. (1987). VIP: a direct renal natriuretic substance. Clinical Science 72, 195-200. GRIFFITHS, N. M., CHABARDES, D., IMBERT-TEBOUL, M., SIAUME-PEREZ, S., MOREL, F. & SIMMONS, N. L. (1988b). Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron. Pfiigers Archiv 412, 363-368. GRIFFITHS, N. M., RIVIER, J. & SIMMONS, N. L. (1988a). Vasoactive intestinal peptide stimulation of feline renal adenylate cyclase; inhibitory effects of (4CI-D-Phe6, Leul7)-VIP. Annals of the New York Academy of Sciences 527, 640-643. GRIFFITHS, N. M., RUGG, E. L. & SIMMONS, N. L. (1989a). Vasoactive intestinal peptide control of renal adenylate cyclase: in vitro studies of canine renal membranes and cultured renal epithelial (MDCK) cells. Quarterly Journal of Experimental Physiology 74, 339-353. GRIFFITHS, N. M. & SIMMONS, N. L. (1987). Vasoactive intestinal peptide regulation of rabbit renal adenylate cyclase activity in vitro. Journal of Physiology 387, 1-17. GRIFFITHS, N. M. & SIMMONS, N. L. (1988). Characterization of [3-iodotyrosyl '25I]vasoactive intestinal peptide binding to feline renal plasma membranes in vitro. Journal of Physiology 406,
118P.
GRIFFITHS, N. M., SIMMONS, N. L. & RIVIER, J. (1989b). Vasoactive intestinal peptide stimulation of renal adenylate cyclase and antagonism by (4CI-D-Phe6, Leul7)-VIP. Pfiugers Archiv 414, 222-227.
KIM, J. K., FROHNERT, P. P., HUI, Y. S. F., BARNES, L. D., FARROW, G. M. & DOUSA, T. P. (1977). Enzymes of cyclic 3',5'-nucleotide metabolism in human renal cortex and renal adenocarcinoma. Kidney International 12, 172- 183. KNIGHT, D. S., BEAL, J. A., YUAN, Z. P. & FOURNET, T. S. (1987). Vasoactive intestinal peptideimmunoreactive nerves in the rat kidney. Anatomical Record 219, 193-203. MAGISTRETTI, P. J., HOF, P. R., MARTIN, J. L., DIETL, M. & PALACIOS, J. M. (1988). High- and low-affinity binding sites for vasoactive intestinal peptide (VIP) in the rat kidney revealed by light microscopic autoradiography. Regulatory Peptides 23, 145-152. MOREL, F. (1981). Sites of hormone action in the mammalian nephron. American Journal of Physiology 240, 159-164. MULVEHILL, J. B., HUI, Y. S. F., BARNES, L. D., PALUMBO, P. J. & DOUSA, T. P. (1976). Glucagonsensitive adenylate cyclase in human renal medulla. Journal of Clinical Endocrinology and Metabolism 42, 380-384. PANDOL, S. J., DHARMSATHAPHORN, K., SCHOEFFIELD, M. S., VALE, W. & RIVIER, J. (1986). Vasoactive intestinal peptide receptor antagonist (4-Cl-D-Phe6, Leu'7) VIP. American Journal of Physiology 250, G553-557. PORTER, J. P., THRASHER, T. N., SAID, S. I. & GANONG, W. F. (1985). Vasoactive intestinal peptide in the control of renin secretion. American Journal of Physiology 249, F84-89.
REINECKE, M. & FORSSMANN, W. G. (1988). Neuropeptide (neuropeptide Y, neurotensin, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, somatostatin) immunohistochemistry and ultrastructure of renal nerves. Histochemistry 89, 1-9. SAID, S. I. & MUTT, V. (1970). Polypeptide with broad biological activity: isolation from small intestine. Science 169, 1217-1218. SEAMON, K. B. & DALY, J. W. (1983). Forskolin, cyclic AMP and cellular physiology. Trends in Pharmacological Sciences 2, 120-123.
