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Adenosine induces a calcium-dependent glomerular contraction
European Journal of Pharmacology, 134 (1987) 365-367
365
Elsevier F_.JP152SC
Adenosine induces a calcium-dependent glomerular contraction J.M. L b p c z - N o v o a *, G . de Arriba, V. Barrio a n d D. R o d r i g u e z - P u y o l Renal Physiopathology Laboratory, Fundacibn Jim$nezx Diaz, Avda. Reyes Catblicos 2, 28040 Madrid, Spain
Received 9 October 1986, accepted 30 December1986
Glomemli isolated from rat kidney cortex were incubated with adenosine in the presence or absence of verapamil and calcium and their change in cross-sectional area was recorded. Adenosine induced a 10% decrease in glomerular cross-sectional area. This decrease was blocked by verapamil or a calcium-free medium. The results suggest that the effect of adenosine in the kidney could be due to glomerular constriction, and that this constriction depends on the entry of calcium into glomerular cells. Adenosine; Glomeruli; Verapamil; Calcium; (Rat)
1. Introduction
Adenosine, a metabolite produced in the normal as well as in the hypoxic kidney, shows a unique vasoactive behavior because it is a vasodilator in most vascular beds including even preparations of isolated renal artery, whereas it increases renal vascular resistances (Osswald, 1975). Furthermore, adenosine has been proposed as a possible endogenous regulator of renal blood flow (RBF) and glomerular filtration rate (GFR) (Spielman and Thomson, 1982). Previous studies from our laboratory had demonstrated that adenosine-induced renal vasoconstriction is not dependent on the activation or inhibition of the renin-angiotensin system but can be blocked by the intrarenal administration of the calcium channel blocker verapamil (Macias et al., 1985). Since renal glomerular contraction could modify RBF by increasing giomerular vascular resistance and G F R by decreasing the ultrafiltration coefficient
* To whom all correspondence should be addressed: Fundaci6n Jim6nez Diaz, Avda. Reyes Cat61icos 2, 28040 Madrid, Spain.
(Kreisberg et al., 1985; Schor et al., 1981) the present study was performed to find whether adenosine can induce isolated glomerular contraction and whether this contraction is dependent on extracellular calcium.
2. Materials and methods
Renal glomeruli were isolated from 150-200 g Wistar rats according to a previously published sieving technique (Rodriguez-Puyol et al., 1986) that yields a nearly pure glomeruli suspension with a tubular contamination (assessed in each run) lower than 3%. Aliquots (500 #1) of freshly prepared glomeruli were suspended in Tris-HC1 buffer (in mM: Tris 20, NaC1 100, KC1 10, sodium acetate 10, glucose 5, p H 7.45) containing about 500 glomeruli were placed in polystyrene tubes. CaC12 (final concentration 2.5 mM) was added to each tube except to the 'calcium-free' tubes and adenosine (10 -4 M, Sigma) was then added. Another set of tubes had verapamil (Knoll, 10 -5 M) added in addition to adenosine. A third series of tubes, with no calcium added, also contained EGTA (2 mM). A
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
366
control series was set up with glomeruli incubated with the buffer plus calcium without any drug added. Immediately before the addition of the drugs and after 20 min of incubation at room temperature, 100 btl samples of the glomeruli suspension were placed on temperature-equilibrated glass slides and 3-5 microphotographs were taken in a maximum of 2 min. Each glomerular crosssectional area visible was measured using a planimetric computerized technique (Cardio 80, Kontron Medical,) as previously described (L6pezNovoa et al., in press). The actual cross-sectional area was corrected for microscope power and photographic enlargement. Results are expressed as means _+ S.E.M. Comparisons were performed using an analysis of variance. A value of P < 0.05 was considered as statistically significant.
3. Results
The results are shown in table 1. The initial glomerular cross-sectional area was similar in all the experimental protocols. In control experiment, glomeruli incubated with buffer did not show any change in cross-sectional area. Adenosine induced a 10% reduction in glomerular cross-sectional area. Both verapamil and incubation in a calcium-free plus E G T A medium completely blocked the glomerular contraction induced by adenosine.
TABLE 1 Effect of adenosine on isolated glomeruli cross-sectional area. Glomeruli isolated from rat kidney cortex were incubated with adenine and with or without verapamil and calcium. Glomerular cross-sectional area is expressed in m2 x l 0 -8. At least 3 experimental runs were performed. Data are means + S.E.M. In parentheses: number of glomeruli measured.
Control Adenosine Adenosine + verapamil Adenosine+ EDTA (no Ca)
Basal
Experimental
P
1.60 + 0.04 (50) 1.61 _+0.03 (75) 1.60 _+0.03 (81) 1.59_+0.04 (50)
1.59 + 0.04 (59) 1.45 + 0.03 (83) 1.58 _+0.03 (82) 1.51_+0.03 (51)
> 0.6 < 0.0005 > 0.4 > 0.3
4. Discussion
There is increasing experimental evidence for a role of glomerular contraction as a physiological regulator of G F R and RPF. Isolated glomeruli as well as cultured mesangial cells can be contracted with several hormones and substances e.g. angiotensin II, vasopressin, P A R etc., with vasoactive properties (Kreisberg et al., 1985). The mechanism by which adenosine increases vascular resistances in the kidney and decreases G F R is not known. It has been demonstrated that adenosine does not contract but relaxes renal artery preparations (Osswald, 1975). We have assessed the hypothesis that at least a part of the increase in renal vascular resistance induced by adenosine could be mediated by glomerular contraction. Our results demonstrate that adenosine effectively decreases glomerular cross-sectional area, taking into account that the maximal glomerular cross-sectional area reduction reported in the literature is about 15%. In our hands, adenosine induced contraction with an efficacy similar to that of angiotensin II or PAF (Lrpez-Novoa et al., in press). The glomerular contraction mediated by adenosine seems to be dependent on the stimulation of extracellular calcium entry into the glomerular contractile cells, since it could be blocked by verapamil or by incubation in a calcium-free plus E G T A medium. The in vivo reduction of RBF by adenosine can be also prevented by verapamil infusion into the renal artery (Macias et al., 1985). Both electrophysiological and contraction studies suggest an interaction between adenosine and calcium fluxes in vascular smooth muscle (Bellardinelli et al., 1979). Also, direct measurements of calcium fluxes have demonstrated that adenosine, at the doses used in the present experiment, reduced the uptake of Ca by quiescent or stimulated guinea-pig atria, porcine artery strips or cultured vascular smooth muscle cells (Fenton et al., 1972). It has been also suggested that adenosine could enhance the intracellular binding of calcium (Fenton et al., 1972). Both the reduction of extracellular calcium uptake and the increase of intracellular Ca binding will reduce cytosolic Ca concentration, thus explaining the vasorelaxant effects of adenosine. Interestingly, the effect of
367
adenosine on the calcium fluxes in the contractile cells of the glomeruli seems to be the opposite. It must be noted that, in addition to its vascular actions, adenosine induces a decrease of renin release from the kidney (Spielman and Thomson, 1982; Macias et al., 1985), an action that is associated to an increase rather than to a decrease of the cytosolic Ca concentration in the juxtaglomerular apparatus cells (Park and Marvin, 1978). Although the present results obtained in vitro cannot be applied directly to in vivo circumstances, the preparation used in the present experiment could be useful for assessing the effect of adenosine without the possible interference of renal nerves or circulating hormones. The results thus suggest that one of the mechanisms involved in the reduction of G F R and RPF by adenosine could be glomerular contraction, probably induced by the contraction of mesangial cells. Mesangial cell contraction would decrease the effective ultrafiltration surface, and subsequently the ultrafiltration coefficient and G F R (Schor et al., 1981). This contraction seems to be mediated by an increase of extracellular Ca entry into the contractile mesangial cells.
Acknowledgements This work was partially supported by grants from FIS, CAICYT and I~go Alvarez de Toledo Fundation. V. Barrio was a fellow of the I~go Alvarez de Toledo Fundation. We gratefully acknowledge the technical assistance of the Medical Media Department.
References Belardinelli, L., R. Rubio and R.M. Berne, 1979, Blockade of Ca 2+ dependent rat atrial slow action potentials by adenosine and lanthanum, Pfliigers Arch. 380, 19. Fenton, R.A., S.P. Bruttig, R. Rubio and R.M. Berne, 1982, Effect of adenosine on calcium uptake by intact and cultured vascular smooth muscle, Am. J. Physiol. 242, H797. Kreisberg, J.I., M. Venkatachalam and D. Troyer, 1985, Contractile properties of cultured glomerular mesangial cells, Am. J. Physiol. 249, F457. L6pez-Novoa, J.M., G. Arriba, A. Fernandez-Cruz, L. Hernando, and D. Rodriguez-Puyol, Atrial natriuretic peptide modulates isolated rat glomeruli contraction, in: Atrial Natriuretic Peptide, eds. B.M. Brenner and J.H. Laragh (Plenum Press, New York) (in press). Macias, J.F., C. Garcia-Iglesias, J.C. Santos and J.M. L6pezNovoa, 1985, Influence of plasma renin content, intrarenal angiotensin II, captopril and calcium channel blockers on the vasoconstriction and renin release promoted by adenosine in the kidney, J. Lab. Clin. Med. 106, 562. Osswald, H., 1975, Renal effects of adenosine and their inhibition by theophylline in dogs, Naunyn-Schmiedeb. Arch. Pharmacol. 288, 79. Park, C.S., and R.L. Marvin, 1978, Calcium in the control of renin release, Am. J. Physiol. 235, F22. Rodriguez-Puyol, D., G. Arriba, A. Blanchart, F.C. Santos, C. Caramelo, A. Fernandez-Cruz, L. Hernando and J.M. L6pez-Novoa, 1986, Lack of a direct regulatory effect of atrial natriuretic factor on prostaglandins and renin release by isolated rat glomernli, Biochem. Biophys. Res. Commun. 138, 496. Schor, N., I. Ichikawa and B.M. Brenner, 1981, Mechanisms of action of various hormones and vasoactive substances on glomerular ultrafiltration in the rat, Kidney Int. 20, 442. Spielman, W.S. and C.I. Thomson, 1982, A proposed role for adenosine in the regulation of renal hemodynamics and renin release, Am. J. Physiol. 242, F423.
365
Elsevier F_.JP152SC
Adenosine induces a calcium-dependent glomerular contraction J.M. L b p c z - N o v o a *, G . de Arriba, V. Barrio a n d D. R o d r i g u e z - P u y o l Renal Physiopathology Laboratory, Fundacibn Jim$nezx Diaz, Avda. Reyes Catblicos 2, 28040 Madrid, Spain
Received 9 October 1986, accepted 30 December1986
Glomemli isolated from rat kidney cortex were incubated with adenosine in the presence or absence of verapamil and calcium and their change in cross-sectional area was recorded. Adenosine induced a 10% decrease in glomerular cross-sectional area. This decrease was blocked by verapamil or a calcium-free medium. The results suggest that the effect of adenosine in the kidney could be due to glomerular constriction, and that this constriction depends on the entry of calcium into glomerular cells. Adenosine; Glomeruli; Verapamil; Calcium; (Rat)
1. Introduction
Adenosine, a metabolite produced in the normal as well as in the hypoxic kidney, shows a unique vasoactive behavior because it is a vasodilator in most vascular beds including even preparations of isolated renal artery, whereas it increases renal vascular resistances (Osswald, 1975). Furthermore, adenosine has been proposed as a possible endogenous regulator of renal blood flow (RBF) and glomerular filtration rate (GFR) (Spielman and Thomson, 1982). Previous studies from our laboratory had demonstrated that adenosine-induced renal vasoconstriction is not dependent on the activation or inhibition of the renin-angiotensin system but can be blocked by the intrarenal administration of the calcium channel blocker verapamil (Macias et al., 1985). Since renal glomerular contraction could modify RBF by increasing giomerular vascular resistance and G F R by decreasing the ultrafiltration coefficient
* To whom all correspondence should be addressed: Fundaci6n Jim6nez Diaz, Avda. Reyes Cat61icos 2, 28040 Madrid, Spain.
(Kreisberg et al., 1985; Schor et al., 1981) the present study was performed to find whether adenosine can induce isolated glomerular contraction and whether this contraction is dependent on extracellular calcium.
2. Materials and methods
Renal glomeruli were isolated from 150-200 g Wistar rats according to a previously published sieving technique (Rodriguez-Puyol et al., 1986) that yields a nearly pure glomeruli suspension with a tubular contamination (assessed in each run) lower than 3%. Aliquots (500 #1) of freshly prepared glomeruli were suspended in Tris-HC1 buffer (in mM: Tris 20, NaC1 100, KC1 10, sodium acetate 10, glucose 5, p H 7.45) containing about 500 glomeruli were placed in polystyrene tubes. CaC12 (final concentration 2.5 mM) was added to each tube except to the 'calcium-free' tubes and adenosine (10 -4 M, Sigma) was then added. Another set of tubes had verapamil (Knoll, 10 -5 M) added in addition to adenosine. A third series of tubes, with no calcium added, also contained EGTA (2 mM). A
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
366
control series was set up with glomeruli incubated with the buffer plus calcium without any drug added. Immediately before the addition of the drugs and after 20 min of incubation at room temperature, 100 btl samples of the glomeruli suspension were placed on temperature-equilibrated glass slides and 3-5 microphotographs were taken in a maximum of 2 min. Each glomerular crosssectional area visible was measured using a planimetric computerized technique (Cardio 80, Kontron Medical,) as previously described (L6pezNovoa et al., in press). The actual cross-sectional area was corrected for microscope power and photographic enlargement. Results are expressed as means _+ S.E.M. Comparisons were performed using an analysis of variance. A value of P < 0.05 was considered as statistically significant.
3. Results
The results are shown in table 1. The initial glomerular cross-sectional area was similar in all the experimental protocols. In control experiment, glomeruli incubated with buffer did not show any change in cross-sectional area. Adenosine induced a 10% reduction in glomerular cross-sectional area. Both verapamil and incubation in a calcium-free plus E G T A medium completely blocked the glomerular contraction induced by adenosine.
TABLE 1 Effect of adenosine on isolated glomeruli cross-sectional area. Glomeruli isolated from rat kidney cortex were incubated with adenine and with or without verapamil and calcium. Glomerular cross-sectional area is expressed in m2 x l 0 -8. At least 3 experimental runs were performed. Data are means + S.E.M. In parentheses: number of glomeruli measured.
Control Adenosine Adenosine + verapamil Adenosine+ EDTA (no Ca)
Basal
Experimental
P
1.60 + 0.04 (50) 1.61 _+0.03 (75) 1.60 _+0.03 (81) 1.59_+0.04 (50)
1.59 + 0.04 (59) 1.45 + 0.03 (83) 1.58 _+0.03 (82) 1.51_+0.03 (51)
> 0.6 < 0.0005 > 0.4 > 0.3
4. Discussion
There is increasing experimental evidence for a role of glomerular contraction as a physiological regulator of G F R and RPF. Isolated glomeruli as well as cultured mesangial cells can be contracted with several hormones and substances e.g. angiotensin II, vasopressin, P A R etc., with vasoactive properties (Kreisberg et al., 1985). The mechanism by which adenosine increases vascular resistances in the kidney and decreases G F R is not known. It has been demonstrated that adenosine does not contract but relaxes renal artery preparations (Osswald, 1975). We have assessed the hypothesis that at least a part of the increase in renal vascular resistance induced by adenosine could be mediated by glomerular contraction. Our results demonstrate that adenosine effectively decreases glomerular cross-sectional area, taking into account that the maximal glomerular cross-sectional area reduction reported in the literature is about 15%. In our hands, adenosine induced contraction with an efficacy similar to that of angiotensin II or PAF (Lrpez-Novoa et al., in press). The glomerular contraction mediated by adenosine seems to be dependent on the stimulation of extracellular calcium entry into the glomerular contractile cells, since it could be blocked by verapamil or by incubation in a calcium-free plus E G T A medium. The in vivo reduction of RBF by adenosine can be also prevented by verapamil infusion into the renal artery (Macias et al., 1985). Both electrophysiological and contraction studies suggest an interaction between adenosine and calcium fluxes in vascular smooth muscle (Bellardinelli et al., 1979). Also, direct measurements of calcium fluxes have demonstrated that adenosine, at the doses used in the present experiment, reduced the uptake of Ca by quiescent or stimulated guinea-pig atria, porcine artery strips or cultured vascular smooth muscle cells (Fenton et al., 1972). It has been also suggested that adenosine could enhance the intracellular binding of calcium (Fenton et al., 1972). Both the reduction of extracellular calcium uptake and the increase of intracellular Ca binding will reduce cytosolic Ca concentration, thus explaining the vasorelaxant effects of adenosine. Interestingly, the effect of
367
adenosine on the calcium fluxes in the contractile cells of the glomeruli seems to be the opposite. It must be noted that, in addition to its vascular actions, adenosine induces a decrease of renin release from the kidney (Spielman and Thomson, 1982; Macias et al., 1985), an action that is associated to an increase rather than to a decrease of the cytosolic Ca concentration in the juxtaglomerular apparatus cells (Park and Marvin, 1978). Although the present results obtained in vitro cannot be applied directly to in vivo circumstances, the preparation used in the present experiment could be useful for assessing the effect of adenosine without the possible interference of renal nerves or circulating hormones. The results thus suggest that one of the mechanisms involved in the reduction of G F R and RPF by adenosine could be glomerular contraction, probably induced by the contraction of mesangial cells. Mesangial cell contraction would decrease the effective ultrafiltration surface, and subsequently the ultrafiltration coefficient and G F R (Schor et al., 1981). This contraction seems to be mediated by an increase of extracellular Ca entry into the contractile mesangial cells.
Acknowledgements This work was partially supported by grants from FIS, CAICYT and I~go Alvarez de Toledo Fundation. V. Barrio was a fellow of the I~go Alvarez de Toledo Fundation. We gratefully acknowledge the technical assistance of the Medical Media Department.
References Belardinelli, L., R. Rubio and R.M. Berne, 1979, Blockade of Ca 2+ dependent rat atrial slow action potentials by adenosine and lanthanum, Pfliigers Arch. 380, 19. Fenton, R.A., S.P. Bruttig, R. Rubio and R.M. Berne, 1982, Effect of adenosine on calcium uptake by intact and cultured vascular smooth muscle, Am. J. Physiol. 242, H797. Kreisberg, J.I., M. Venkatachalam and D. Troyer, 1985, Contractile properties of cultured glomerular mesangial cells, Am. J. Physiol. 249, F457. L6pez-Novoa, J.M., G. Arriba, A. Fernandez-Cruz, L. Hernando, and D. Rodriguez-Puyol, Atrial natriuretic peptide modulates isolated rat glomeruli contraction, in: Atrial Natriuretic Peptide, eds. B.M. Brenner and J.H. Laragh (Plenum Press, New York) (in press). Macias, J.F., C. Garcia-Iglesias, J.C. Santos and J.M. L6pezNovoa, 1985, Influence of plasma renin content, intrarenal angiotensin II, captopril and calcium channel blockers on the vasoconstriction and renin release promoted by adenosine in the kidney, J. Lab. Clin. Med. 106, 562. Osswald, H., 1975, Renal effects of adenosine and their inhibition by theophylline in dogs, Naunyn-Schmiedeb. Arch. Pharmacol. 288, 79. Park, C.S., and R.L. Marvin, 1978, Calcium in the control of renin release, Am. J. Physiol. 235, F22. Rodriguez-Puyol, D., G. Arriba, A. Blanchart, F.C. Santos, C. Caramelo, A. Fernandez-Cruz, L. Hernando and J.M. L6pez-Novoa, 1986, Lack of a direct regulatory effect of atrial natriuretic factor on prostaglandins and renin release by isolated rat glomernli, Biochem. Biophys. Res. Commun. 138, 496. Schor, N., I. Ichikawa and B.M. Brenner, 1981, Mechanisms of action of various hormones and vasoactive substances on glomerular ultrafiltration in the rat, Kidney Int. 20, 442. Spielman, W.S. and C.I. Thomson, 1982, A proposed role for adenosine in the regulation of renal hemodynamics and renin release, Am. J. Physiol. 242, F423.