The activity of cAMP degradation enzyme, cAMP phosphodiesterase (cAMP PDE), in renal tubules is a critically important factor in determining cellular cAMP levels, particularly in response to hormones. In this study we examine the nephron distribution of cAMP PDE activity in the mouse, rat and rabbit kidney and important cellular regulators of cAMP PDE, namely calmodulin and adenosine triphosphate (ATP). We assayed total low Km cAMP PDE in microdissected tubule segments, using 10(-6) M (3H) cAMP as a substrate. Activities were expressed in fentomol cAMP hydrolyzed per minute per mm tubular length or per one glomerulus. The content of ATP was measured in outer medullary collecting duct and medullary thick ascending limb of Henle's loop with microbioluminescence assay using firefly luciferase. In mouse kidney, cAMP PDE was significantly higher in all tubular segments compared to glomerulus. Proximal convoluted tubule, proximal straight tubule, medullary thick ascending limb of Henle's loop (mTAL), and outer medullary collecting duct (OMCD) had intermediated activity. Greater cAMP PDE activity was detected in cortical ascending limb of Henle's loop (cTAL), cortical collecting duct and in distal convoluted tubule (DCT). The highest activity was found in connecting tubules. In rat, nephron distribution of cAMP PDE activities was similar to mouse, except that activity in glomeruli was higher than in mouse glomeruli. In rabbit, nephron distribution of cAMP PDE activities was different from those of mouse and rat. There was no single prominent segment with high cAMP PDE activity. DCT and cTAL showed low enzyme activity. Overall, the highest cAMP PDE activities were measured in the mouse and the lowest were measured in the rabbit nephrons, with those of rat nephron showing an intermediate activity. The maximum effective dose of the calmodulin antagonist, trifluoperazine (200 microM), inhibited cAMP PDE in all nephron segments from the rat kidney. However, there is no statistical significance of its inhibition among nephron segments. In OMCD and mTAL of the rat kidney, cAMP PDE activity was inhibited by ATP (5 mM to approximately 10 mM) which is far beyond the physiological concentartion of ATP in normal epithelial cell. Actual determinations of ATP in mTAL and OMCD were 0.1 mM and 0.17 mM, respectively. These observations show that distal segments of tubules have more active catabolism of cAMP than proximal segments. cAMP PDE in each nephron segment appear to be almost equally dependent on trifluoperazine-sensitive pathway that may reflect the Ca2+-calmodulin system. Cellular concentration of ATP might not be involved in the regulation of the total low Km cAMP PDE activity in rat mTAL and OMCD.

Signaling via release of Ca2+ from intracellular stores is mediated by several systems, including the inositol 1,4,5-trisphosphate (IP3) and cADP-ribose (cADPR) pathway. We recently discovered a high capacity for cADPR synthesis in rat glomeruli and cultured mesangial cells (MC). We sought to determine whether 1) cADPR synthesis in MC is regulated by cytokines and hormones, 2) ryanodine receptors (RyRs) are expressed in MC, and 3) Ca2+ is released through RyRs in response to cADPR. We found that ADP-ribosyl cyclase, a CD38-like enzyme that catalyzes cADPR synthesis, is upregulated in MC by tumor necrosis factor-alpha, interleukin-1beta, and all-trans retinoic acid (atRA). [3H]ryanodine binds to microsomal fractions from MC with high affinity in a Ca2+-dependent manner; binding is enhanced by specific RyR agonists and blocked by ruthenium red and cADPR. Western blot analysis confirmed the presence of RyR in MC. Release of 45Ca2+ from MC microsomes was stimulated by cADPR; release was blocked by ruthenium red and 8-bromo-cADPR. ADPR (non-cyclic) was without effect. In MC, TNF-alpha and atRA amplified the increment of cytoplasmic Ca2+ elicited by vasopressin. We conclude that MC possess elements of a novel ADP-ribosyl cyclase-->cADPR-->RyR-->Ca2+-release signaling pathway subject to regulation by proinflammatory cytokines and steroid superfamily hormones.

BACKGROUNDMesangial cell proliferation is a characteristic feature of IgA nephropathy and many other forms of glomerulonephritis. Recent clinical studies have shown that dietary fish oil supplementation retards renal disease progression in patients with IgA nephropathy. The mechanism by which this effect occurs is unknown.METHODSThe anti-Thy 1.1 (ATS) model of mesangial proliferative glomerulonephritis was employed to test the hypothesis that dietary fish oil supplementation reduces mesangial cell proliferation following acute injury. Subcultured rat mesangial cells were used to determine the in vitro effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the primary components of fish oil, on proliferation.RESULTSFollowing antithymocyte serum (ATS) administration, proteinuria was significantly decreased in animals treated with fish oil compared with sesame oil-treated controls. In ATS rats given fish oil, there was less mesangial cell and matrix expansion, mesangiolysis, or basement membrane disruption (delta% = -40%). ATS rats receiving fish oil had less glomerular cell proliferation (PCNA-delta% = -50%) and a reduction of alpha-smooth muscle actin expression (delta% = -27%) by mesangial cells. In subcultured rat mesangial cells, DHA, but not EPA, significantly inhibited proliferation.CONCLUSIONSFish oil inhibits mesangial cell activation and proliferation in ATS glomerulonephritis, reduces proteinuria, and decreases histologic evidence of glomerular damage. In vitro, the antiproliferative effects of fish oil are more likely related to the action of DHA. We suggest that orally administered fish oil, or purified DHA, may have a suppressive effect in acute phases or relapses of glomerulopathies by inhibiting activation and proliferation of mesangial cells.

We investigated whether ADP-ribosyl cyclase (ADPR-cyclase) in rat vascular smooth muscle cells (VSMCs) has enzymatic properties that differ from the well-characterized CD38-antigen ADPR-cyclase, expressed in HL-60 cells. ADPR-cyclase from VSMCs, but not CD38 ADPR-cyclase from HL-60 cells, was inhibited by gangliosides (10 micromol/L) GT(1B), GD(1), and GM(3). Preincubation of membranes from CD38 HL-60 cells, but not from VSMCs, with anti-CD38 antibodies increased ADPR-cyclase activity; CD38 antigen was detected both in VSMCs and in HL-60 cells. ADPR-cyclase in VSMC membranes was more sensitive than CD38 HL-60 ADPR-cyclase to inactivation by N-endoglycosidase F and to thermal inactivation at 45 degrees C. The specific activity of ADPR-cyclase in membranes from VSMCs was >20-fold higher than in membranes from CD38 HL-60 cells. Most importantly, VSMC ADPR-cyclase was inhibited by Zn(2+) and Cu(2+) ions; the inhibition by Zn(2+) was dose dependent, noncompetitive, and reversible by EDTA. In contrast, Zn(2+) stimulated the activity of CD38 HL-60 ADPR-cyclase and other known types of ADPR-cyclases. Retinoids act either via the nuclear receptor retinoic acid receptor or retinoid X receptor, including all-trans retinoic acid (atRA), and panagonist 9-cis-retinoic acid-upregulated VSMC ADPR-cyclase; the stimulatory effect of atRA was blocked by actinomycin D and cycloheximide. 1,25(OH)(2)-Vitamin D(3) (calciferol) stimulated VSMC ADPR-cyclase dose dependently at subnanomolar concentrations (ED(50) congruent with 56 pmol/L). Oral administration of atRA to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+60%) and, to a lesser degree, in myocardium of left ventricle (+18%), but atRA had no effect on ADPR-cyclases in lungs, spleen, intestinal smooth muscle, skeletal muscle, liver, or testis. Administration of 3,5,3'-triiodothyronine (T(3)) to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+89%), but not in liver or brain. We conclude the following: (1) ADPR-cyclase in VSMCs has enzymatic properties distinct from "classic" CD38 ADPR-cyclase, especially sensitivity to inhibition by Zn(2+) and Cu(2+); (2) ADPR-cyclase in VSMCs is upregulated by various retinoids, calcitriol, and T(3) in vitro; and (3) administration of atRA and T(3) increases ADPR-cyclase in aorta in vivo. We suggest that the cADPR signaling system plays an important role in the regulation of VSMC functions in response to steroid superfamily hormones.

Investigations of recent years revealed that isozymes of cyclic-3', 5'-nucleotide phosphodiesterase (PDE) are a critically important component of the cyclic-3',5'-adenosine monophosphate (cAMP) protein kinase A (PKA) signaling pathway. The superfamily of cyclic-3', 5'-phosphodiesterase (PDE) isozymes consists of at least nine gene families (types): PDE1 to PDE9. Some PDE families are very diverse and consist of several subtypes and numerous PDE isoform-splice variants. PDE isozymes differ in molecular structure, catalytic properties, intracellular regulation and location, and sensitivity to selective inhibitors, as well as differential expression in various cell types. A number of type-specific "second-generation" PDE inhibitors have been developed. Current evidence indicates that PDE isozymes play a role in several pathobiologic processes in kidney cells. In rat mesangial cells, PDE3 and PDE4 compartmentalize cAMP signaling to the PDE3-linked cAMP-PKA pathway that modulates mitogenesis and PDE4-linked cAMP-PKA pathway that modulates generation of reactive oxygen species. Administration of selective PDE isozyme inhibitors in vivo suppresses proteinuria and pathologic changes in experimental anti-Thy-1.1 mesangial proliferative glomerulonephritis in rats. Increased activity of PDE5 (and perhaps also PDE9) in glomeruli and in cells of collecting ducts in sodium-retaining states, such as nephrotic syndrome, accounts for renal resistance to atriopeptin; diminished ability to excrete sodium can be corrected by administration of the selective PDE5 inhibitor zaprinast. Anomalously high PDE4 activity in collecting ducts is a basis of unresponsiveness to vasopressin in mice with hereditary nephrogenic diabetes insipidus. Apparently, PDE isozymes apparently also play an important role in the pathogenesis of acute renal failure of different origins. Administration of PDE isozyme-selective inhibitors suppresses some components of immune responses to allograft transplant and improves preservation and survival of transplanted organ. PDE isozymes are a target for action of numerous novel selective PDE inhibitors, which are key components in the design of novel "signal transduction" pharmacotherapies of kidney diseases.

Here we investigated whether cADPR and NAADP are synthesized in mitochondria. We found that ADPR-cyclase activity is present in mitochondria. In addition, we describe for the first time synthesis of NAADP in this intracellular organelle. ADPR-cyclase activities (VMAX) and NAADP synthesis in mitochondria were about 4-fold lower than that in plasma membranes. Otherwise, ADPR-cyclases in mitochondria and in plasma membranes have similar catalytic properties in terms of apparent Km for the substrate NGD and Ki values for inhibition by dithiotreitol, β-NAD, and nicotinamide. ADPR-cyclase in plasma membranes and to a lesser degree mitochondrial enzyme, was inhibited by Zn2+ and Cu2+; ADPR-cyclase from mitochondria was more stable upon thermal inactivation. CD38 antigen, determined by Western blot, was well-expressed in plasma membranes but was far less so (17-fold less) in mitochondria. The major difference between ADPR-cyclase activity in mitochondria and plasma membranes is that mitochondrial cyclase activity was increased by incubation with nonionic detergents. Conversely, the incubation with phosphatidylinositol-specific phosphodiesterase C (PI-PLC) released ADPR-cyclase activity from plasma membranes, but not from mitochondria. We conclude that ADPR-cyclase in mitochondria and in plasma membranes are both multifunctional enzymes with similar catalytic properties; however, the two ADPR-cyclases differ in the mode of anchoring to the membrane: by glycosylphosphoinositol anchor in plasma membranes and by hydrophobic interactions in mitochondria. In addition, synthesis of NAADP can also be found in intracellular organelles via mitochondria. We propose that independent mitochondrial cADPR and NAADP systems may have an intracrine signaling function that is not dependent on direct input by extracellular hormonal stimuli, but rather responds to changes of intermediary cellular metabolism.

BACKGROUND For treatment of phosphate (Pi) overload in various pathophysiological states, an agent that selectively increases renal Pi excretion would be of major value. Previously, we have shown that dopamine (DA) inhibits Na(+)-Pi cotransport in renal epithelia. However, the administration of DA or its immediate precursor L-DOPA increases DA in multiple tissues. Synthetic dipeptide gamma-L-glutamyl-L-DOPA (gludopa) can serve as an inactive precursor (pro-pro-drug) of DA. This study tested the hypothesis that, because of the unique colocalization of gamma-glutamyltransferase (gamma-GT), aromatic amino acid decarboxylase, Na(+)-Pi cotransporter, and Na(+)-L-DOPA cotransporter in brush border membrane (BBM) of proximal tubular cells, gludopa may elicit phosphaturia by action of DA generated within the kidney. METHODS Thyroparathyrectomized rats were given placebo, or gludopa, or gludopa + gamma-GT inhibitor acivicin. Urinary excretion of Pi, Ca2+, Na+, K+, DA, cAMP, and cGMP was determined, and Na(+)-Pi cotransport was measured in BBM prepared from kidneys of rats at the end of the experiment. RESULTS The administration of gludopa resulted in: (a) an inhibition of Na(+)-Pi cotransport, but not cotransport of Na(+)-proline and Na(+)-alanine in BBM; (b) an increase (+300%) of fractional excretion (FE) of Pi and a drop (-35%) of plasma Pi, whereas the plasma levels and FEs of Ca2+, Na+, and K+ were unchanged; (c) an increase in urinary excretion of cAMP. but not cGMP; (d) a 1000-fold increase of urinary excretion of DA, without a change in excretion of norepinephrine; and (e) an incubation of gludopa with BBM in vitro, which caused a release of L-DOPA, and the in vivo administration of acivicin, which blocked actions of gludopa to inhibit Na(+)-Pi cotransport and to increase urinary excretions of Pi and DA. CONCLUSIONS We conclude that colocalization of enzymes of biotransformation, BBM transporters, and the autocrine/paracrine DA system in cells of proximal tubules constitutes a cellular basis for the potent and specific phosphaturic action of gludopa.

We investigated the effect of glycolytic pathway intermediaries upon Ca(2+) release induced by cyclic ADP-ribose (cADPR), inositol 1',4', 5-trisphosphate (IP(3)), and nicotinate adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenate. Fructose 1,6, -diphosphate (FDP), at concentrations up to 8 mM, did not induce Ca(2+) release by itself in sea urchin egg homogenate. However, FDP potentiates Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine, caffeine, and palmitoyl-CoA. Furthermore, glucose 6-phosphate had similar effects. FDP also potentiates activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased approximately 3.5 times by addition of 4 mM FDP. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to FDP. The Ca(2+) release mediated by FDP in the presence of subthreshold concentrations of cADPR was inhibited by antagonists of the ryanodine channel, such as ruthenium red, and by the cADPR inhibitor 8-Br-cADPR. However, inhibition of Ca(2+) release induced by IP(3) or NAADP had no effect upon Ca(2+) release induced by FDP in the presence of low concentrations of cADPR. Furthermore, FDP had inhibitory effects upon Ca(2+) release induced by both IP(3) and NAADP. We propose that the state of cellular intermediary metabolism may regulate cellular Ca(2+) homeostases by switching preferential effects from one intracellular Ca(2+) release channel to another.

BACKGROUNDAlthough the existence of multiple isoforms of plasma membrane calcium ATPase (PMCA) is now well documented, their biological functions are not yet known. In this study, we set out to investigate the potential role of PMCA isoforms, previously identified in renal cortical tissue, in tubular reabsorption of calcium (Ca2+).METHODSWith use of reverse transcription-polymerase chain reaction analysis, we determined levels of mRNAs encoding isoforms of PMCA1 through PMCA4 in renal cortex, liver, and brain of rats with hypercalciuria induced by feeding with a low-phosphate diet (LPD) as compared with Ca2+-retaining rats that were fed a high-phosphate diet (HPD).RESULTSWe observed that in hypercalciuric LPD-fed rats, the mRNAs encoding isoforms PMCA2b and PMCA3(a + c) are significantly lower (Delta approximately-50%) than in HPD-fed hypocalciuric rats, whereas no changes in mRNAs encoding isoforms PMCA1b and PMCA4 were observed, and mRNA encoding calbindin 28 kDa was increased. On the other hand, the content of mRNAs encoding PMCA2b and PMCA3(a + c) in liver and brain, respectively, was not changed.CONCLUSIONThese findings are evidence that expression of PMCA isoforms in the kidney can be selectively modulated in response to pathophysiologic stimuli. The association of a decrease in mRNA encoding PMCA2b and PMCA3(a + c) with hypercalciuria suggests that the two PMCA isoforms may be operant in tubular reabsorption of Ca2+ and its regulation.

We investigated the localization of mRNA encoding the calcium-sensing receptor (CaSR) along the rat nephron. For this purpose, we combined microdissection of nephron segments and RT-PCR techniques. The results indicate that mRNA encoding rat CaSR is present in rat glomeruli and distal segments (medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule and cortical collecting duct), whereas it was not detected in proximal convoluted tubules or proximal straight tubules. We also studied whether the CaSR transcription in kidney cortex was modified in response to low dietary phosphate. No significant changes were detected. Given the fact that a low-phosphate diet increased Ca2+ excretion by more than 50-fold, the results suggest that if the CaSR regulates Ca2+ reabsorption, it does so through receptor occupancy by Ca2+ rather than by changes in receptor expression.

To survey the presence of the four different isoforms of the plasma membrane calcium pump (PMCA) and their alternative splicing variants in the rat kidney, three major zones (cortex, outer medulla, and inner medulla) were macrodissected and probed for the presence of mRNA encoding these isoforms and their variants at the splicing site C by using reverse transcription-polymerase chain reaction (RTPCR). Both the cortex and the outer medulla showed PMCA 1b, 2b, 3(a and c), and 4b. Semiquantitative comparisons indicated that isoform 2b is more abundant in the cortex than in the outer medulla and conversely, that isoform 3 (a and c) is more abundant in the outer medulla than in the cortex. The inner medulla showed only mRNA for isoforms 1b and 4b. The nephron segments present in the cortex and outer medulla were microdissected and analyzed by RT-PCR. Isoforms 1 b, 2b, and 4b were found in all nephron segments but were found more frequently in tubular segments with high rates of Ca2+ reabsorption, suggesting that these isoforms may be involved in transepithelial transport. On the other hand, mRNA encoding isoform 3 (a and c) was most abundant in descending thin limb of Henle but was detected also in glomeruli and cortical thin ascending limb. Its distinct localization suggests that this isoform might have another function, such as in intracellular signalling.

We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate-adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels.

Some major pathobiologic processes in renal mesangial cells, elicited in response to immunoinflammatory stimuli, are modulated via cAMP-protein kinase A (PKA) signaling pathways; namely, generation of reactive oxygen metabolites (ROM) and accelerated proliferation of mesangial cells. We investigated the role of cAMP phosphodiesterase (PDE) isozymes in these regulatory mechanisms. Generation of ROM in cultured rat mesangial cells was inhibited by selective inhibitors of PDE4, rolipram and denbufylline, whereas PDES inhibitors, cilostamide and lixazinone, had no effect. Conversely, cilostamide or lixazinone suppressed mitogenic synthesis of DNA in mesangial cells, but 1 mu M rolipram or 1 mu M denbufylline showed no inhibitory effect. The efficacy of PDE isozyme inhibitors (IC50) to suppress [H-3]thymidine incorporation or ROM generation paralleled IC50 values for inhibition of cAMP PDE, incubation of mesangial cells with either rolipram alone or with cilostamide alone increased significantly in situ activity of PKA in mesangial cells, assessed by (-cAMP/+cAMP) PKA activity ratio, and the stimulatory effects were additive. Results indicate that in mesangial cells a cAMP pool that is metabolized by PDE4 activates PKA and thereby inhibits ROM generation; another cAMP pool that is metabolized by PDES activates another PKA (isozyme or pool) which suppresses proliferation of mesangial cells, We propose that in mesangial cells, a cAMP-PKA pathway that regulates mitogenesis is determined by activity of PDE3, whereas another cAMP-PKA pathway is directed by activity of PDE4 and controls ROM generation. Therefore, two PDE isozymes within one cell type compartmentalize distinct cAMP signaling pathways.

The molecular mechanism that initiates Ca2+ signaling in sea urchin egg fertilization has not yet been clarified. To determine whether sea urchin sperm may generate and possibly supply cyclic ADP-ribose (cADPR) as a Ca2+-releasing factor in the course of sea urchin egg fertilization, we determined cADPR content and the capacity for cADPR synthesis in sea urchin sperm. cADPR content was determined using the sea urchin egg homogenate Ca2+-release bioassay combined with high-performance liquid chromatography (HPLC). We found that sperm homogenates synthesized cADPR from beta-NAD but did not synthesize cADPR when alpha-NAD was the substrate. The identity of cADPR generated by sperm homogenates was verified by HPLC analysis, use of specific Ca2+-release antagonists, and homologous desensitization of the sea urchin egg homogenate Ca2+-release bioassay. The ambient content of cADPR was approximately 0.3 nmol cADPR/g wet wt sea urchin sperm. Our results show that sperm can synthesize cADPR and that they contain cADPR levels comparable to other tissues.

Serotonin (5-HT) was recently reported to inhibit cAMP generation in oppossum (OK) cells. We thus investigated the effects of 5-HT upon the Na(+)-Pi cotransport in cultured OK cells and its interactions with dopamine. Incubation of OK cells with 1 nM-10 microM 5-HT resulted in dose-dependent stimulation of Na(+)-Pi contransport (ED50 approximately equal to 8 nM) and also counteracted inhibition of Na(+)-Pi cotransport elicited by dopamine. Pre-incubation with 5-HT decreased cAMP accumulation elicited by forskolin or dopamine and pre-treatment with pertussis toxin abolished both the inhibitory effect of 5-HT upon cAMP levels and stimulation of Na(+)-Pi cotransport. Incubation of OK cells with the 5-HT precursor 5-hydroxytryptophan resulted in time- and dose-dependent accumulation of 5-HT in the medium that also elicited an increase in Na(+)-Pi cotransport. Both the effects of 5-HT and dopamine on Na(+)-Pi cotransport were prevented by carbidopa. The stimulatory effect of 5-HT was specific for the Na(+)-Pi cotransport system since no effects were observed on Na(+)-alanine cotransport. The results indicate that 5-HT stimulates Na(+)-Pi cotransport at least in part via inhibition of cAMP accumulation. We propose that 5-HT and dopamine have opposite actions as paracrine/autocrine regulators of Na(+)-Pi cotransport via opposite effects upon cAMP formation.

In previous studies we observed that inhibition of cyclic 3′,5′-nucleotide phosphodiesterase (PDE) isozymes, namely isozyme PDE3, suppresses proliferation of rat renal glomerular mesangial cells in vitro and in vivo. To determine whether activation of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway coupled to specific PDE isozymes modulates accelerated proliferation of renal epithelial cells, we investigated the effect of selective PDE isozyme inhibition on renal epithelial cell proliferation induced in rats by injection of folic acid (FA). In extracts from suspensions of renal cortical tubules, cAMP was metabolized predominantly by isozyme PDE4; activity of PDE3 was about three times lower. The increase in proliferative activity of renal cortical tissue from FA-injected rats, evaluated by immunostaining with Mib-1 antibody, was limited to tubular epithelial cells. Administration of the PDE3 inhibitors cilostazol or cilostamide together with the PDE4 inhibitor rolipram blocked mitogenic synthesis of DNA, as determined by (3H)-thymidine incorporation into renal cortical DNA, in FA-treated rats. FA injection caused an increase of more than 10-fold in proliferating cell nuclear antigen (PCNA) in renal cortical tissue; administration of the potent PDE3 inhibitor lixazinone or, to a lesser degree, cilostazol suppressed these high PCNA levels, whereas rolipram alone had no effect. The results indicate that FA-stimulated in vivo proliferation of renal tubular epithelial cells is down-regulated by activation of a cAMPPKA signaling pathway linked to PDE3 isozymes. These observations are consistent with the notion that negative crosstalk between cAMP signaling and mitogenstimulated signaling pathways regulates mitogenesis of renal cells of different terminal differentiation, including tubular epithelial cells.

All-trans-retinoic acid (atRA) is a regulator of cellular growth and differentiation. We investigated whether atRA can upregulate Na(+)-dependent cotransporters in opossum kidney (OK) cells and thus increase uptake from tubular fluid of several solutes needed for growth during early stages of ontogenesis. In OK cells, incubation with atRA for 24 h increased the Na+ gradient-dependent cotransports of phosphate, L-proline, L-glutamic acid, and SO(4)2- by a similar degree (approximately 40%) that was prevented by pretreatment with actinomycin D. In contrast, activities of other Na(+)-dependent transporters, Na(+)-K(+)-adenosinetriphosphatase, gamma-glutamyltranspeptidase, and leucine aminopeptidase, were unchanged by atRA. Cell proliferation determined by [3H]thymidine incorporation was not increased by atRA. The stimulatory effects of atRA and phosphate deprivation on Na(+)-Pi cotransport demonstrated additivity, whereas the combination of atRA and 3,5,3'-triiodothyronine did not. atRA stimulated Na(+)-Pi cotransport in LLC-PK1 cells with an analogous time course and to a similar extent as observed in OK cells. We conclude that atRA stimulates several Na(+)-dependent cotransporters via a genomic mechanism and may represent a synchronous adaptation to nutritional requirements of early phases of ontogenesis.

Cyclic ADP ribose (cADPR) has been shown to trigger Ca2+ release from intracellular stores through ryanodine receptor/channel. In our previous study we observed that all-trans-retinoic acid stimulates cADPR synthesis by ADP ribose cyclase (ADPR cyclase) in cultured epithelial cells. We have now investigated whether cADPR may play a signaling role in action of beta-estradiol (E2), an archetypal steroid superfamily hormone, upon its major target organ, uterus, in vivo. Administration of E2 to gonadectomized rats (0.2 mg/kg per day for 7 days) resulted in an approximately Delta + 300% increase of ADPR cyclase activity in extracts from uterus, but in liver, brain, or skeletal muscle ADPR cyclase was unchanged. Most of the E2-stimulated uterine ADPR cyclase was associated with membranes. The higher ADPR cyclase activity in response to E2 was due to the increase of VMAX without change in Km. Simultaneous administration of estrogen antagonist tamoxifen (8 mg/kg per day) with E2 (0.2 mg/kg per day) prevented an increase in ADPR cyclase. In uterine extracts from E2-treated rats, the rate of cADPR inactivation by cADPR hydrolase and the activity of NADase was increased, but to a much lesser degree than activity of ADPR cyclase. Our results indicate that E2, via action to its nuclear receptors in vivo, increases ADPR cyclase activity in uterus. We propose that some of the estrogen effects, and by extension the effects of other steroid superfamily hormones, upon specialized cellular functions and upon hormone-induced gene expression in target cells, are mediated by cADPR-Ca2+ release pathway.

Activation of cAMP signaling pathway was shown to inhibit some pathobiologic processes in mesangial cells (MC). We investigated whether adrenomedullin (ADM), a potent agonist of adenylate cyclase, is synthesized in MC and whether it can, via cAMP, suppress the generation of reactive oxygen metabolites (ROM) and proliferation of cells in glomeruli. With the use of an immunohistologic technique ADM was detected in mesangial and microvascular areas of rat glomeruli. MC grown in primary culture synthesized ADM, and the synthesis was stimulated by TNF alpha and IL-1 beta but not by PDGF and EGF. ADM inhibited ROM generation in MC dose-dependently and caused in situ activation of protein kinase A (PKA). In macrophages (cell line J774) ROM generation was about four times higher than in MC and was inhibited by ADM in a similar way as in MC. The rate of MC proliferation, measured by [3H]-incorporation, and the activity of mitogen-activated protein kinase (MAPK) stimulated by PDGF and EGF were dose-dependently inhibited by ADM; the maximum inhibition (at 10 nM ADM) was about -80%. Mitogenesis of MC and MAPK activity when stimulated to a similar extent by endothelin (ET-1) was inhibited by ADM to a significantly (P < 0.01) lesser degree (-30%). Further, ADM inhibited PDF-stimulated mitogenesis and activation of MAPK in cultured vascular smooth muscle cells (VSMC). The inhibition of PDGF-activated MAPK by ADM in VSMC was reversed by the protein kinase A (PKA) inhibitor, H89. Taken together, results indicate the adrenomedullin (ADM) generated in mesangial cells (MC) can suppress, via activation of the cAMP-protein kinase A (PKA) signaling pathway, reactive oxygen metabolites (ROM) generation in MC and infiltrating macrophages as well as mitogen-activated protein kinase (MAPK)-mediated mitogenesis in MC and vascular smooth muscle cells (VSMC). We suggest that introglomerular ADM may serve as a cytoprotective autoacoid that suppresses pathobiologic processes evoked by immuno-inflammatory injury of glomeruli.