Douša, Thomas P.

C-type natriuretic peptide (CNP) is a vasoactive and antimitogenic peptide that is structurally similar but genetically distinct from atrial natriuretic peptide. While first discovered in the brain, CNP has been shown to be produced by endothelial cells and may function in a paracrine and autocrine fashion in the control of vascular tone. Recently, CNP immunoreactivity and B-type natriuretic peptide receptors (NPR-B), for which CNP is a specific ligand, have been identified in the kidney. The present study was designed to determine whether renal epithelial cells produce and secrete CNP and whether CNP immunoreactivity is present in canine kidney. Opossum kidney (OK) cells that express proximal tubular cell characteristics were incubated for 6 h in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). CNP immunoreactivity was measured in the preincubation and 6-h conditioned media by radioimmunoassay (RIA) using a specific antibody to CNP-22. Furthermore the molecular form of this CNP-like protein was determined by reverse-phase high-performance liquid chromatography (HPLC), and intracellular localization of the CNP immunoreactivity was determined by immunohistochemical staining. CNP immunoreactivity was also determined in renal tissue from dogs subjected to saline or endothelin infusion. Six-hour incubation in DMEM resulted in accumulation of CNP immunoreactivity (baseline below detection level vs. 6 h = 117.3 +/- 8.3 pg/ml, P < 0.001). Intracellular CNP concentration determined after sonication was 1.9 +/- 0.2 micrograms/g protein, and immunohistochemical staining for CNP was markedly positive in the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of Ca2+ from intracellular stores is a widespread mechanism in regulation of cell function. Two hitherto unknown adenine diphosphonucleotides were recently identified, which trigger Ca2+ release from intracellular stores via channels that are distinct from the well-known receptor/channel controlled by inositol 1,4,5,-trisphosphate (IP3): cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Here we review synthesis of cADPR from beta-NAD, its hydrolysis to adenosine diphosphoribose (noncyclic) by cADPR glycohydrolase, as well as our knowledge about the metabolism of NAADP. The Ca2+ release triggered by cADPR, NAADP, or IP3 can be distinguished by the action of inhibitors and by desensitization studies. Evidence now emerges that cADPR synthesis from beta-NAD can be stimulated, at least in some cell types by all-trans-retinoic acid as a first messenger. We then review the properties of cADPR and NAADP as potential second messengers in the intracrine regulation of cell functions. Although their exact role in signaling sequences is not yet known, cADPR and NAADP are likely to play important intracellular regulatory functions, as extensively documented for the process of egg fertilization.

Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs that ultimately activates the ryanodine channel. We now report that certain long-chain acyl-CoA derivative metabolites (14-18 carbons in length), such as palmitoyl-CoA, greatly potentiate the effect of cADPR on Ca2+ release. Furthermore, in higher concentrations, palmitoyl-CoA and other closely related long-chain acyl-CoA derivatives trigger Ca2+ release apparently through the ryanodine channel in sea urchin egg homogenates. Palmitoyl-CoA-induced Ca2+ release was suppressed by ruthenium red, spermine, and the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide, which all prevent activation of the ryanodine channel, but not by heparin or thionicotinamide-NADP. In addition, cADPR was able to desensitize the sea urchin egg homogenates to the subsequent Ca2+ release induced by palmitoyl-CoA and vice versa. In contrast, neither inositol 1,4,5-trisphosphate (IP3) nor the newly identified Ca2+ release agonist nicotinate adenine dinucleotide phosphate was able to desensitize the homogenate to palmitoyl-CoA, indicating that palmitoyl-CoA probably acts selectively by activating the ryanodine channel, but, unlike cADPR, palmitoyl-CoA might act directly on this channel. Finally, we found that palmitoyl-CoA was able to counteract the inhibitory effect of Mg2+ and spermine, which, in physiological concentrations, suppress specifically the cADPR-induced Ca2+ release. We propose that palmitoyl-CoA, present in micromolar concentrations, may trigger Ca2+ release through the ryanodine channel and, in lower concentrations, may increase the sensitivity of the Ca2+ release system to cADPR. Thus palmitoyl-CoA may serve as a regulatory link between the intermediary metabolism and the cADPR-induced Ca2+ release signaling pathway.

We have previously shown that NAD+ inhibits renal Na(+)-Pi symport; however, the biochemical mechanism of NAD+ in this action is not clarified. We now propose that NAD+ acts indirectly by first being converted to cyclic ADP-ribose (cADPR), a potent stimulator of intracellular Ca2+ mobilization. In permeabilized opossum kidney (OK) cells, a cell line often employed as a model for study of proximal tubular epithelial transport, cADPR is synthesized from beta-NAD+ in a substrate concentration (0.01-1 mM) and time-dependent manner. That cADPR was generated from beta-NAD+ by OK cells was verified by coelution with authentic cADPR on anion exchange high-performance liquid chromatography and by homologous desensitization of the Ca2+ release bioassay to authentic cADPR. cADPR synthesized by permeabilized OK cells was not influenced by the addition of parathyroid hormone. The OK cell also contains the enzyme activity necessary to catalyze catabolism of cADPR. Identification of these two key enzyme activities of cADPR metabolism in OK cells is consistent with a possible role of cADPR in regulation of the Na(+)-Pi symporter by NAD+ in response to metabolic stimuli. d

Cyclic ADP-ribose (cADPR) is a potent mediator of Ca2+ mobilization from intracellular stores in sea urchin eggs. However, the regulation of the cADPR-induced Ca2+ release system is not yet fully elucidated. We now report that spermine and related polyamines, in physiological concentrations, were able to inhibit the Ca2+ release induced by cADPR in sea urchin egg homogenate bioassays, as measured using the Ca2+ indicator fluo 3, but had no effect on the Ca2+ release induced by D-myo-inositol 1,4,5-trisphosphate (IP3) or by nicotinate adenine dinucleotide phosphate (NAADP). Spermine was a more potent inhibitor of the cADPR-induced Ca2+ release than spermidine and putrescine. Spermine inhibited not only the release induced by cADPR but also the Ca2+ release induced by caffeine and ryanodine. Finally, pretreatment of the sea urchin egg homogenates with caffeine or Sr2+ and Ca2+ prevented the inhibitory effect of spermine on cADPR-induced Ca2+ release. We propose that polyamines, which are present in millimolar concentrations in fertilized eggs, are specific inhibitors of the ryanodine channel and perhaps may serve as endogenous regulators of the cADPR-induced Ca2+ release system.

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.

We investigated the suitability of alpha-bromophosphonoacetic acid (alpha-BrPAA) to act as a possible irreversible inhibitor of Na(+)-dependent transport of Pi across renal brush-border membrane (BBM). When added directly into the Pi uptake medium, alpha-BrPAA causes specific, competitive [apparent inhibition constant (Ki) = 0.33 mM; no change in maximum velocity (Vmax)], and reversible (by washing) inhibition of Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by BBM vesicles (BBMV). Next, BBMV were preincubated with 5 mM alpha-BrPAA in alkaline (pH 9) medium for 30 min, then twice washed by 1:100 dilution and recentrifugation, and tested for transport and other properties. This preincubation of BBMV with alpha-BrPAA in alkaline medium resulted in a different type of inhibition [lower Vmax; no change in Michaelis constant (Km)] of the Na+ gradient-dependent uptake of 32Pi, whereas the uptakes of D-[3H]glucose and other solutes were not altered. This inhibition of Pi transport was not reversed by dilution and washing of BBMV. The BBMV Na(+)-dependent binding of [14C]phosphonoformic acid, but not of [3H]phlorizin, was decreased; activities of BBM marker enzymes were not changed. Results suggest that alpha-BrPAA binds onto the same locus on luminal surface of BBM on which Pi and Na+ bind and inhibits Na(+)-Pi cotransporter similar to phosphonoformic acid. Furthermore, after a 30-min incubation in alkaline medium, alpha-BrPAA apparently forms a more stable association with BBM in the vicinity of the Na(+)-Pi cotransporter. We thus suggest that alpha-BrPAA acts under these conditions as an apparently irreversible inhibitor of Na(+)-Pi cotransporter in BBM.(ABSTRACT TRUNCATED AT 250 WORDS)

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-α, interleukin-1β, 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 of45Ca2+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-α 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.

We have studied localization of mRNAs coding isozymes of rat plasma membrane Ca(2+)-adenosinetriphosphatase pump (rPMCA) in the rat kidney, with use of reverse transcription (RT) with subsequent amplification by polymerase chain reaction (PCR). When zones of the kidney were separated by macrodissection, a large amount of mRNA coding isozyme rPMCA1 was found in all zones; mRNA for isozyme rPMCA2 was abundant in cortex and in outer medulla, and mRNA for isozyme rPMCA3 was prominent in outer medulla. The mRNAs were analyzed in microdissected cortical nephron segments by use of RT-PCR approach described previously [T. Moriyama, H. R. Murphy, B. M. Martin, and A. Garcia-Perez. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1470-F1474, 1990]. We detected mRNA for isozyme rPMCA2 in microdissected distal convoluted tubules (DCT) and in cortical thick ascending limbs (CTAL) and, less consistently, also in proximal convoluted tubule and in glomeruli. The mRNA for isozyme rPMCA1 was abundant in glomeruli but was absent in all examined cortical tubular segments. Our results document that mRNAs for all three major isozymes of rPMCA are present and show a unique distribution in the three major zones of rat renal parenchyma. Specific mRNA coding for rPMCA2 was detected in cortical tubules, namely in CTAL and DCT, whereas mRNA coding isozyme rPMCA1 was found in glomeruli. We suggest that isozyme rPMCA2 might be specifically related to epithelial cells and their function, whereas rPMCA1 is probably a component of nonepithelial cells including these in glomeruli.

We studied cyclic 3',5'-nucleotide phosphodiesterase (PDE) isozymes and their role in adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP metabolism in a rat inner medullary collecting duct (IMCD) cell line. The homogenized and fractionated IMCD cells of cAMP-PDE and all of cGMP-PDE activity were found in the cytosol. The majority of cytosolic cAMP-PDE (greater than 50%) was isozyme PDE-IV; the Ca(2+)-calmodulin-sensitive PDE-I was present only in cytosol. Preincubation of IMCD cells with PDE-IV inhibitor rolipram markedly (5x) enhanced levels of cAMP both basal and in the presence of [Arg8]vasopressin (AVP). Cilostamide (for PDE-III) or vinpocetine had no effect, whereas PDE-I inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MeoM-IBMX) enhanced AVP-dependent cAMP levels. Exposure of IMCD cells to 2 microM ionomycin decreased both basal and AVP-stimulated cAMP. Depletion of Ca2+ by preincubation of IMCD cells in the Ca(2+)-free medium with ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid markedly enhanced the stimulatory response of cAMP to AVP, and addition of 8-MeoM-IBMX further enhanced the AVP response. The levels of cGMP, basal or in response to atriopeptin (ANP), were not affected by PDE-V inhibitor zaprinast, but both inhibitors of PDE-I, 8-MeoM-IBMX and vinpocetine, increased basal cGMP, and 8-MeoM-IBMX also increased cGMP levels enhanced by ANP. The depletion of Ca2+ from IMCD cells alone had no effect on cGMP levels, but effects of 8-MeoM-IBMX and vinpocetine on the ANP-stimulated cGMP levels were enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)