Glomerular filtration rate (GFR) is maintained constant, at least in part, by balance between relaxation and contraction of renal glomeruli cells: smooth-muscle like cells i.e. mesangial cells and podocytes (1-3). The changes of tension of these cells are influenced by several factors e.g. angiotensin II, vasopressin, purinoceptors and ß-adrenergic agonists (4). Cyclic AMP has been generally recognised as second messenger of these factors (5). Elevation of intracellular concentration of cAMP affects tension of smooth muscle and mesangial cells (6). The signalling mechanism by which cAMP induces relaxation of mesangial cells is not fully understood. Changes of cells tension induced by cAMP are assumed to operate via activation of cAMP-dependent protein kinase A; PKA (7). Two major types of mammalian PKA (PKA-I and PKA-II) are described and can be separated by DEAE anion exchange chromatography. Type I is predominantly cytoplasmic, whereas type II PKA associates with specific cellular structures and organelles (8). The holoenzyme consists of two catalytic (C) subunits that are held in an inactive state by association with regulatory (R) subunit dimmer. Activation of PKA occurs upon binding of cAMP to each regulatory subunit, where the binding takes place on asymmetric sites (designated A and B) in positive cooperation fashion (9-11).

The vascular effects of cAMP-elevating agents are mediated via activation of PKA. Most of reports describe the role of cAMP in relaxation of vascular smooth muscle cells (5, 12). However, it should be noted that there are also reports where correlation between PKA activation and relaxation has not been found (13, 14). Moreover, there is very little information about role of PKA-I/PKA-II and specific function of A and B sites in the regulation of vascular and glomerular tension.

Therefore, we measured the changes of glomerular inulin space (GIS), as a marker of contraction/relaxation of glomerulus, in the presence of phosphodiesterase resistant cAMP analogues: (Sp) 8-Cl-cAMPS, (Rp) 8-Cl-cAMPS. The experimental approach with cAMP analogues that bind preferentially to A site (N6-benzoyl-cAMP) and B site (8-aminobutyloamino-cAMP) of PKA-I and PKA-II alone or with combination with A site of PKA-I and B site of PKA-II activator (8-piperidino-cAMP) allows achieving the role of specific sites in PKA-mediated changes of cells contractility.

Our finding suggests that activation of PKA-I or PKA-II leads to contraction of isolated glomeruli and cooperation between A and B site plays a key role in this process.


MATERIAL AND METHODS
Animals

Experiments were performed on male Wistar rats (weighing 220-250 g). The rats were housed in an animal care facility at Medical University of Gdansk and fed a standard rat chow and had free access to tap water. Experiments were approved by the local Ethics Committee for Animal Research.

Isolation of glomeruli

Animals were decapitated under ether anaesthesia and kidney were removed and placed in ice-cold phosphate buffered saline (PBS), pH 7.4 containing (mM): 137 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.5 KH2PO4, 0.9 CaCl2, 0.49 MgCl2 and 5.6 glucose. Glomeruli were isolated by gradual sieving technique (15). Briefly, renal capsule was removed and the cortex was minced with a razor blade to a paste-like consistency and strained through a steel sieve (pore size 250 µm). The mash which passed through this sieve was suspended in ice-cold PBS. Then, the suspension passed through two consecutive steel sieves (120 and 70 µm). The glomeruli retained on the top of the 70 µm sieve were washed off with ice-cold PBS. Glomeruli were resuspended in ice-cold PBS buffer. Tubular contamination was less than 5% as assessed under the light microscope. Entire procedure took no more than 1 hour.

Determination of glomerular inulin space

Glomerular inulin space (GIS) was measured according to the previously described method (16, 17). About 2000 glomeruli were suspended in 200 µl ice-cold PBS containing 1% bovine serum albumin. Samples were preincubated with 0.5 µCi [3H] inulin at 37°C in shaking water bath for 30 min. Incubation was continued with cAMP analogues for indicated time and concentration according to experimental protocol.

Next, the suspension of glomeruli (200 µl) was transferred to a microtube containing ice-cold silicone oil (100 µl) and centrifuged at 5000 x g for 5 s. Supernatant (20 µl) and tip of microtube with glomerular pellet were placed in scintillation vial with 500 µl 0.3% Triton X-100. Afterwards, scintillation cocktail (2 ml) was added. Radioactivity of samples was measured in a liquid scintillation counter (LKB Wallace).

Glomerular inulin space (GIS) of single glomerulus was calculated as follows:



The number of glomeruli in suspension medium was counted with a light microscope at low magnification. Each GIS determination was carried out in quadruplicate samples.

Experimental Protocol

Glomeruli were incubated with non-specific and phosphodiesterase-resistant activator or inhibitor of protein kinase A i.e. (Sp) 8-Cl-cAMPS (0.1-100 µM) and (Rp) 8-Cl-cAMPS (0.1-100 µM), respectively (18). The selective activation of site A or B was done by using N6-benzoyl-cAMP or 8-aminobutyloamino-cAMP, respectively. Use of site-selective analogue pairs allows investigating the potential cooperation between A and B site of PKA-I or PKA-II i.e.

• 8-aminobutyloamino-cAMP (B site of PKA-I/PKA-II activator) in combination with 8-piperidino-cAMP (A site of PKA-I and B site of PKA-II activator) to investigate cooperation in PKA-I
• N6-benzoyl-cAMP (A site of PKA-I/PKA-II activator) in combination with 8-piperidino-cAMP (A site of PKA-I and B site of PKA-II activator) to investigate cooperation in PKA-II.

Materials

(Sp) 8-Cl-cAMPS, (Rp) 8-Cl-cAMPS, N6-benzoyl-cAMP, 8-aminobutyloamino-cAMP and 8-piperidino-cAMP have been synthesised in Prof. Jastorff's laboratory. [3H]-inulin was obtained from Du Pont NEN Products (Boston, MA, USA). All other agents were purchased from POCh (Gliwice, Poland).

Statistic

The results are expressed as a percentage of basal GIS value (618±22 pl glomerulus-1). Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's test to determine significance. P values <0.05 ware consider to be significant.


RESULTS
The effects of (Sp) and (Rp) diastereomers of the phosphorothiate, non-site-specific analogues of cyclic AMP, on glomerular inulin space (GIS) are presented in Figure 1 and 2. Incubation of glomeruli with (Sp) 8-Cl-cAMPS (0.1-100 µM), a phosphodiesterase-resistant activator of protein kinase A, induced decrease of GIS, whereas (Rp) 8-Cl-cAMPS (0.1-100 µM), a phosphodiesterase-resistant inhibitor of protein kinase A was ineffective. Reduction of GIS was time-dependent (Fig.1) and concentration-dependent with maximal effect (about 22±1.5% of basal value) at 1 µM (Fig.2).

Fig. 1. Time-dependent effect of 1 µM of (Sp) 8-Cl-cAMPS and (Rp) 8-Cl-cAMPS on GIS. Each point is the mean of 5-6 experiments. Values are expressed as a mean±S.E. P<0.05 (Sp) 8-Cl-cAMPS vs. (Rp) 8-Cl-cAMPS.

Fig. 2. Concentration-dependent effect of (Sp) 8-Cl-cAMPS and (Rp) 8-Cl-cAMPS on GIS at 5th minute of incubation. Each point is the mean of 4-6 experiments. Values are expressed as a mean±S.E. P<0.05 (Sp) 8-Cl-cAMPS vs. (Rp) 8-Cl-cAMPS.

Figure 3 shows the effects of (Sp) 8-Cl-cAMPS on GIS in the presence of (Rp) 8-Cl-cAMPS. The protein kinase A antagonist i.e. (Rp) 8-Cl-cAMPS (50 µM) shifted the concentration-response curve of (Sp) 8-Cl-cAMPS to the right. Maximal effect of (Sp) 8-Cl-cAMPS was decreased from 22±2.0% to 7±1.5% of basal value.

Fig. 3. Changes of GIS in response to (Sp) 8-Cl-cAMPS in the presence of 50 µM (Rp) 8-Cl-cAMPS. Each point is the mean of 3 experiments. Values are expressed as a mean±S.E. P<0.05 (Sp) (Sp) 8-Cl-cAMPS + (Rp) 8-Cl-cAMPS vs. 8-Cl-cAMPS.

Results of experimental approach, which allows to specific activation of A or B sites, are presented in Figure 4. The specific activation of sites was obtained by incubation of the glomeruli with N6-benzoyl-cAMP or 8-aminobutyloamino-cAMP, respectively. As shown in Figure 4, N6-benzoyl-cAMP, similarly to (Sp) 8-Cl-cAMPS, induced concentration-dependent decrease of GIS with maximum at 0.1 µM. However, 8-aminobutyloamino-cAMP at concentration range of 0.1-100 µM had no effect. Next, we investigated the effects of simultaneous activation of A and B sites of PKA-I or PKA-II on GIS. To investigate the role of PKA-I we used 8-aminobutyloamino-cAMP as a priming analogue, since it binds with comparable affinity to A site of both PKA-I and PKA-II in subinhibitory concentration (1 nM) in combination with various concentration of 8-piperidino-cAMP e.g. A site of PKA-I and B site of PKA-II activator. Significant reduction of GIS about 7 ± 1.7% was observed at 1 pM with maximum at 1 nM. Similarly, to investigate the role of PKA-II we used N6-benzoyl-cAMP as a priming analogue (comparable affinity to B site of both PKA-I and PKA-II) at 5 nM in combination with various concentration of 8-piperidino-cAMP. We observed reduction of GIS about 14 ± 2% at 1 pM with maximum at 10 pM.

Fig. 4. Concentration-dependent effects of N6-benzoyl-cAMP, 8-aminobutyloamino-cAMP and 8-piperidino-cAMP in the presence of N6-benzoyl-cAMP at 1 nM or 8-aminobutyloamino-cAMP at 5 nM on GIS. Each point is the mean of 4-6 experiments (incubation time -5 minutes). Values are expressed as a mean±S.E. P<0.05 analogue vs. basal value.

DISCUSSION
In the present experiments we have checked the involvement of PKA in the regulation of glomerular contractility. We have used cAMP analogues which effectively penetrate cell membranes and are resistant to phosphodiesterase (18), to investigate the role of PKA in regulation of glomerular tension. Contraction/relaxation were evaluated based on changes of extracellular volume (3H-inulin space, GIS) of isolated decapsulated rat glomeruli, where most part of the extracellular space is intracapillary (19). The validity of this method was endorsed by previous study (20, 21). It has been shown that decrease of GIS reflects glomerular contraction i.e. angiotensin II-induced decrease of GIS about 10% is equivalent to about 4% decrease in glomerular diameter (16).

Results of our experiments show, for the first time, that (Sp) 8-Cl-cAMPS activator of PKA induces concentration-dependent contraction of isolated rat renal glomeruli. (Rp) 8-Cl-cAMPS inhibitor of PKA markedly reduced this effect. These observations suggest that PKA may be involved in cAMP analogues induced contraction of isolated glomeruli. The contraction of glomeruli was mimicked by N6-benzoyl-cAMP, activator of PKA that preferentially binds to A site. However, 8-aminobutyloamino-cAMP, activator of PKA that preferentially binds to B site did not cause glomerular contraction. These results suggest that A site plays a key role in PKA-mediated glomerular contraction. Biochemical data suggest that A site is masked so cAMP and its analogues bind first to B site. Subsequent conformational changes make A site more accessible for ligands. Binding to A site mediates dissociate holoenzyme to R and C subunit. Unanchored C subunit catalyzes phosphorylation of cytoplasmic and nuclear proteins (8, 10). Based on present findings we suggest that, at least in isolated rat renal glomeruli, binding to A site without prior binding to B site may activate PKA. Moreover, activation of only B site is insufficient to activate PKA. However, it should be noted that contraction of glomeruli is enhanced, at least 1000-fold for PKA-I, when both sites are activated. This strongly supports the concept that there is cooperation between A and B site in PKA-mediated contraction of glomeruli.

It is generally held that vascular effects of cAMP-elevating agents are mediated via activation of PKA. Most of reports describe the role of cAMP in relaxation of vascular smooth muscle cells (5, 12). However, it should be noted that there are also reports where correlation between PKA activation and vasorelaxation has not been found (13, 14). Moreover, it has been shown that catecholamines cause mesangial cells to change their shape in association with elevation of intracellular cAMP (22). Prostaglandin E1 induced contraction of rabbit aorta and this is accompanied by increase of cAMP accumulation and activity of PKA (23). The observed different effects of vascular cells due to cAMP accumulation/PKA activation may be resulted from involvement of different isoforms of PKA. Described isoforms of PKA-I and PKA-II are compartmentalised in cell (24-26). PKA-II is found in the cell particulate fraction, often near its protein substrate (25). Therefore, its activation ensures rapid phosphorylation of specific substrate. PKA-I and PKA-II are anchored to cellular compartments by a family of proteins, the A-kinase anchor proteins (AKAPs) (8). AKAPs control the intracellular localization of PKA and coordinate signalling complexes by recruiting multiple signalling enzymes near potential substrates. The phosphatases and phosphodiesterases are examples of AKAPs which may affect PKA action (27, 28). The anchored phosphatases and phosphodiesterases may allow for shorter-acting phosphorylation effects and control the local cAMP level, respectively. Thus, compartmentalization of different enzymes on the same site may lead to bidirectional signaling of physiological processes. It is possible that in our present experiments contraction of glomeruli is due to direct or indirect effect of PKA/phosphatases on phosphorylation of myosin light chain kinase or other unknown proteins involved in contraction of glomerular cells. However, we can not answer the question which isoform of PKA is involved in contraction of glomeruli. Further studies with more specific analogues are needed.

Finally, we can not exclude that, in addition to activation of PKA, there are also other intra- or extracellular systems involved in cAMP analogues-induced contraction of glomeruli. For example, it has been shown that cAMP-stimulating agents i.e. isoproterenol and dopamine enhance activity of calcium-activated potassium channels in myocytes by activation cGMP-dependent protein kinase; PKG. This effect was abolished by inhibitors of PKG but not by inhibitors of PKA (29). In our experiments, inhibitors of PKA affect the effect of PKA activators and this support the hypothesis that activation of PKA in glomeruli leads to its contraction.

In summary, we have shown that PKA activators which effectively penetrate cell membranes and are resistant to phosphodiesterase induce glomerular contraction. There appears that activation of PKA by cAMP analogues may lead to reduction of filtration surface with subsequent decrease of glomerular filtration rate. On a more general note, additional insightful work on role of PKA in the regulation on glomerulus contractility is need.

Acknowledgements: The study was supported by the State Committee for Scientific Research (grant no. 6P05A 08721, SA) and Medical University of Gdansk (grant no W-97, MJ).


REFERENCES

1. Dworkin LD, Ichikawa I, Brenner BM. Hormonal modulation of glomerular function. Am J Physiol 1983; 244: F95-F104.
2. Mene P, Simonson MS, Dunn MJ. Physiology of the mesangial cell. Physiol Rev 1989; 69: 1347-1424.
3. Pavenstadt H, Kriz W, Kretzler M. Cell biology of the glomerular podocyte. Physiol Rev 2003; 83: 253-307.
4. Navar LG, Inscho EW, Majid DSA, Imig JD, Harrison-Bernard LM, Michell KD. Paracrine regulation of the renal microcirculation. Physiol Rev 1996; 76: 425-536.
5. Hardman JG. Cyclic nucleotides and regulation of vascular smooth muscle. J Cardiovasc Pharmacol 1984; 6: 5639-5645.
6. Stockand JD, Sansom SC. Glomerular mesangial cells: electrophysiology and regulation of contraction. Physiol Rev 1998; 78, 723-744.
7. Haynes J, Robinson J, Saunders L, Taylor AE, Strada SJ. Role of cAMP-dependent protein kinase in cAMP-mediated vasodilation. Am J Physiol 1992; 262: H511-H516.
8. Francis SH, Corbin JD. Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol 199; 56: 237-272.
9. Ogreid D, Doskeland SO, Miller JP. Evidence that cyclic nucleotides activating rabbit muscle protein kinase I interact with both types of cAMP binding sites associated with the enzyme. J Biol Chem 1983; 258: 1041-1049.
10. Beebe SJ, Holloway R, Rannels SR, Corbin JD. Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes. J Biol Chem 1984; 259: 3539-3547.
11. Skalhegg BS, Landmark BF, Doskeland SO, Hansson V, Lea T, Jahnsen T. Cyclic AMP-dependent protein kinase type I mediates the inhibitory effects of 3', 5'-cyclic adenosine monophosphate on cell replication in human T lymphocytes. J Biol Chem 1992; 267: 15707-15714.
12. Kotlikoff MI, Kamm KE. Molecular mechanism of b-adrenergic relaxation of airway smooth muscle. Ann Rev Physiol 1996; 58: 115-141.
13. Hei YJ, Macdonell KL, Mcneill JH, Diamond J. Lack of correlation between activation of cyclic AMP-dependent protein kinase and inhibition of contraction of rat vas deferens by cyclic AMP analogs. Mol Pharmacol 1991; 39: 233-238.
14. MacDonell KL, Diamond J. Activation of cAMP-dependent protein kinase in rat aorta by cAMP analogs is not correlated with relaxation. J Vasc Res 1994; 31: 121-130.
15. Misra RP. Isolation of glomeruli from mammalian kidneys by graded sieving. Am J Clin Pathol 1972; 62: 135-139.
16. Fujiwara Y, Kitamura E, Ueda N, Fukunaga M, Orita Y, Kamada J. Mechanism of action of angiotensin II on isolated rat glomeruli. Kidney Int 1989; 36: 985- 991.
17. Jankowski M, Szczepañska-Konkel M, Kalinowski L, Angielski S. Cyclic GMP-dependent relaxation of isolated rat renal glomeruli induced by extracellular ATP. J Physiol 2001; 530.1: 123-130.
18. Yokozaki H, Tortora G, Pepe S, Maronde E, Genieser HG, Jastorff B, Cho-Chung YS. Unhydrolyzable analogues of adenosine 3':5'-monophosphate demonstrating growth inhibition and differentiation in human cancer cells. Cancer Res 1992; 52: 2504-2508.
19. Savin VJ, Terreros DA. Filtration in single isolated mammalian glomeruli. Kidney Int 1981; 20: 188-197.
20. Fujiwara Y, Kikkawa R, Kitamura E, Takama T, Shigeta Y. Angiotensin II effects upon glomerular intracapillary volume in the rat. Renal Physiol 1984; 7: 344-348.
21. Kikkawa R, Kitamura E, Fujiwara Y, Arimura T, Haneda M, Shigeta Y. Impaired contractile responsiveness of diabetic glomeruli to angiotensin II: a possible indication of mesangial dysfunction in diabetes mellitus. Biochem Biophys Res Commun 1986; 136: 1185-1190.
22. Kreisberg J, Venkatachalam MA, Patel PY. Cyclic AMP-associated shape change in mesangial cells and its reversal by prostaglandin E2. Kidney Int 1984; 25: 874-879.
23. Vegesna RV, Diamond J. Activation of cyclic AMP dependent protein kinase in rabbit aortic rings by prostaglandin E1 and forskolin is accompanied by contraction and relaxation, respectively. Proc West Pharmacol Soc 1986; 29: 39-43.
24. Hohl CM, Li QA. Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients. Circ Res 1991; 69: 1369-1379.
25. Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 1995; 268: 247-251.
26. Zaccolo M, Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 2002; 295: 1711-1715.
27. Marx SO, Reiken S, Hisamatsu Y, Gaburjakova M, Gaburjakova J, Yang YM, Rosemblit N, Marks AR. Phosphorylation-dependent regulation of ryanodine receptors: a novel role for leucine/isoleucine zippers. J Cell Biol 2001; 153: 699-708.
28. Taske E, Aandahl EM. Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev 2003; 84: 137-167.
29. White RE, Kryman JP, El-Mowafy AM, Han G, Carrier GO. cAMP-dependent vasodilatators cross-activate the cGMP-dependent protein kinase to stimulate BKCa channel activity in coronary artery smooth muscle cells. Circ Res 2000; 86: 897-905.