Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease characterized by massive enlargement of fluid-filled cysts in the kidney. However, there is no effective therapy yet for this disease. To examine whether ginkgolide B, a natural compound, inhibits cyst development, a Madin-Darby canine kidney (MDCK) cyst model, an embryonic kidney cyst model, and a PKD mouse model were used. Interestingly, ginkgolide B significantly inhibited MDCK cyst formation dose dependently, with up to 69% reduction by 2 μM ginkgolide B. Ginkgolide B also significantly inhibited cyst enlargement in the MDCK cyst model, embryonic kidney cyst model, and PKD mouse model. To determine the underlying mechanisms, the effect of ginkgolide B on MDCK cell viability, proliferation, apoptosis, chloride transporter CFTR activity, and intracellular signaling pathways were also studied. Ginkgolide B did not affect cell viability, proliferation, and expression and activity of the chloride transporter CFTR that mediates cyst fluid secretion. Ginkgolide B induced cyst cell differentiation and altered the Ras/MAPK signaling pathway. Taken together, our results demonstrate that ginkgolide B inhibits renal cyst formation and enlargement, suggesting that ginkgolide B might be developed into a novel candidate drug for ADPKD.     

Characterization of subclones of Madin-Darby canine kidney renal epithelial cell line

Heterogeneity in Madin-Darby canine kidney (MDCK) epithelial cells has been reported, however, its details have not been well described. In the present study, we show that subclones obtained from a MDCK cell line could be divided into two morphologically and biochemically distinct cell types with different hormonal responsiveness. Clones of the first type, motile clones, which had extended and flattened cytoplasm, were devoid of carbonic anhydrase activity. Clones of the second type, nonmotile clones, formed colonies of cuboidal cells and showed carbonic anhydrase activity. Motile clones synthesized cAMP in response to arginine vasopressin, prostaglandin E1, and isoproterenol but not glucagon. In contrast, nonmotile clones responded to all of these hormones. These findings suggest MDCK cells have multiple cellular origins. The motile clones have characteristics similar to the principal cells of the collecting system, whereas the nonmotile clones may be derived from the thick ascending limb or the intercalated cell. Our studies also demonstrate a significant influence of culture condition on MDCK cellular behavior (carbonic anhydrase activity, Na+/K+-ATPase activity and vasopressin responsiveness). Therefore, physiologic and biochemical experiments with MDCK cells must be interpreted with reservations about cellular heterogeneity as well as differences induced by culture conditions.     

Novel Madin Darby Canine Kidney cell clones exhibit unique phenotypes in response to morphogens

Summary Novel Madin Darby Canine Kidney cell clones were isolated. These cell clones exhibit differential responsiveness to inducers of tubule or cyst formation in collagen gel culture: hepatocyte growth factor or inducers of intracellular cAMP formation, respectively. In gel culture, clone OR93.22.D6 forms cysts and responds with morphological transformation to both hepatocyte growth factor and prostaglandin E₁, and is most typical of a previously described cell type except for its higher transepithelial electrical resistance. OR55.25.II20 forms tubules in culture, is unresponsive to hepatocyte growth factor, and forms prostaglandin-induced spherical cysts. OR55.28.V2 forms dense cell spheres under control conditions, is induced to form tubules by hepatocyte growth factor, and is unresponsive to prostaglandin. OR55.29 forms only cysts, and is the only clone to form domes in monolayer culture. Tubule formation induced by hepatocyte growth factor, in all clones except OR55.25.II20, is blocked by a neutralizing antibody. In defined medium, without hepatocyte growth factor or prostaglandin, OR55.25.II20 forms spontaneous tubules. This finding indicates that a tubulogenic serum factor is not responsible for the observed phenotype. Increasing prostaglandin concentrations lead to inhibition of tubule formation and increased cyst formation. This observation suggests that induction of intracellular cAMP formation negatively regulates tubule formation in these cells, and implies that cystogenesis may represent a “default pathway” for impaired tubulogenesis. These observations demonstrate that some facets of renal tubulogenesis may be independent of hepatocyte growth factor, and that care must be exercised when comparing biological studies utilizing different clones.     

Autosomal dominant polycystic kidney disease: Disrupted pathways and potential therapeutic interventions

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic inherited renal cystic disease that occurs in different races worldwide. It is characterized by the development of a multitude of renal cysts, which leads to massive enlargement of the kidney and often to renal failure in adulthood. ADPKD is caused by a mutation in PKD1 or PKD2 genes encoding the proteins polycystin-1 and polycystin-2, respectively. Recent studies showed that cyst formation and growth result from deregulation of multiple cellular pathways like proliferation, apoptosis, metabolic processes, cell polarity, and immune defense. In ADPKD, intracellular cyclic adenosine monophosphate (cAMP) promotes cyst enlargement by stimulating cell proliferation and transepithelial fluid secretion. Several interventions affecting many of these defective signaling pathways have been effective in animal models and some are currently being tested in clinical trials. Moreover, the stem cell therapy can improve nephropathies and according to studies were done in this field, can be considered as a hopeful therapeutic approach in future for PKD. This study provides an in-depth review of the relevant molecular pathways associated with the pathogenesis of ADPKD and their implications in development of potential therapeutic strategies.     

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.     

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase‐4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.     

alpha 1- and beta 2-adrenergic receptor expression in the Madin-Darby canine kidney epithelial cell line

The Madin-Darby canine kidney (MDCK) cell line, derived from distal tubule/collecting duct, expresses differentiated properties of renal tubule epithelium in culture. We studied the expression of adrenergic receptors in MDCK to examine the role of catecholamines in the regulation of renal function. Radioligand-binding studies demonstrated, on the basis of receptor affinities of subtype-selective adrenergic agonists and antagonists, that MDCK cells have both alpha 1- and beta 2- adrenergic receptors. To determine whether these receptor types were expressed by the same cell, we developed a number of clonal MDCK cell lines. The clonal lines had stable but unique morphologies reflecting heterogeneity in the parent cell line. Some clones expressed only beta 2-adrenergic receptors and were nonmotile, whereas others expressed both alpha 1- and beta 2-receptors and demonstrated motility on the culture substrate at low cell densities. In one clone, alpha- and beta- receptor expression was stable for more than 50 passages. Catecholamine agonists increased phosphatidylinositol turnover by activating alpha- adrenergic receptors and cellular cyclic adenosine monophosphate accumulation by activating beta-adrenergic receptors. Guanine nucleotide decreased the affinity of isoproterenol for the beta 2- receptor but did not alter the affinity of epinephrine for the alpha 1- receptor. These results show that alpha 1- and beta 2-receptors can be expressed by a single renal tubular cell and that the two receptors behave as distinct entities in terms of cellular response and receptor regulation. Heterogeneity of adrenergic receptor expression in MDCK clones may reflect properties of different types of renal tubule cells.     

Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Prostaglandin E₁(PGE₁), one of the components in the hormone-supplemented, serum-free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K-1), is required for both long-term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE₁, requirement for long-term growth in Medium K-1. These variants will be useful in identifying the molecular events initiated by PGE₁ which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE₁ independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K-1 and in serum-supplemented medium; the growth rate was lower in Medium K-1 lacking PGE₁. In contrast, PGE₁ independent clone 1 grew at an equivalent rate in Medium K-1 minus PGE₁, and in serum-supplemented medium. When PGE₁ was added to K-1 minus PGE₁, less growth of PGE₁ independent clone 1 was observed. A similar observation was made with one other PGE₁ independent clone which was studied. A hormone deletion study indicated that PGE₁ independent clone 1 still retained growth responses to the other four supplements in Medium K-1 (insulin, transferrin, T₃, and hydrocortisone). The molecular alterations associated with loss of the PGE₁ requirement for long-term growth were examined. At confluency, all of the PGE₁ independent clones studied had higher intracellular cyclic AMP levels following PGE₁ treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE₂ for the PGE₁ receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE₁ independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE₁ on dome formation by the variant cells was also examined. The frequency of dome formation by PGE₁ independent clone 1 was enhanced in a dosage-dependent manner, like normal MDCK cells. This observation suggests that PGE₁ affects MDCK cell growth and dome formation by different mechanisms.     

Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.     

Induction of differentiation in v-Ha-ras-transformed MDCK cells by prostaglandin E2 and 8-bromo-cyclic AMP is associated with a decrease in steady-state level of inositol 1,4,5-trisphosphate.

We used Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells as a model to study possible signal transduction mechanisms underlying the induction of glucagon responsiveness by the differentiation inducers prostaglandin E2 (PGE2) and 8-bromo-cyclic (8-Br-cAMP) AMP and the inhibition of induction by phorbol ester or a serum factor. The steady-state level of inositol 1,4,5-trisphosphate (IP3) was higher in Ha-ras-transformed MDCK cells than in parental MDCK cells. In contrast, the steady-state level of intracellular cAMP of transformed cells was similar to that of normal cells. PGE2 and 8-Br-cAMP increased cAMP content but decreased IP3 levels in a concentration-dependent fashion after 5 days of treatment. We examined the time course for effects of PGE2 and 8-Br-cAMP and found that there was a lag period of 8 to 16 h between elevation of cAMP after the addition of 8-Br-cAMP or PGE2 and the decrease of IP3 levels. Another lag period of 2 days existed before the induction of differentiation. Both the reduction of IP3 levels and the induction of glucagon responsiveness were blocked by phorbol-12-myristate-13-acetate or serum, suggesting that a decrease in the IP3 level might be causally involved in induction of differentiation in transformed MDCK cells. However, induction of differentiation was not due to changes in the expression or guanine nucleotide-binding properties of p21 protein. It is likely that cAMP has a direct regulatory effect on the phospholipid signaling pathway. We conclude that perturbation of the inositol phosphate signaling pathway may be responsible for the induction of differentiation by PGE2 and 8-Br-cAMP in transformed MDCK cells.     

Requirement of focal adhesion kinase in branching tubulogenesis

We previously demonstrated that α3β1 integrins are essential to hepatocyte growth factor (HGF)-independent branching tubulogenesis in Mardin-Darby Canine Kidney (MDCK) cells. However, the involvement of integrin downstream signaling molecules remains unclear. In the present study, we successfully isolated cell lines possessing different tubulogenic potentials from the MDCK cells; cyst clones (CA4, CA6) forming cystic structures when cultured in 0.3% type I collagen gel and mass clones (M610, M611, M612) forming aggregated masses. Cyst clones maintained cystic structure in 0.1% collagen gel, whereas mass clones spontaneously developed into tubules. Both clones exhibited various morphologies when cultured on a dish: cyst clones formed aggregated islands, while mass clones were more scattered and exhibited higher migration capacity. Among several focal adhesion machinery proteins examined, only the expression and phosphorylation level of focal adhesion kinase (FAK) in mass clones was higher than in cyst clones, while other proteins showed no obvious differences. However, overexpression of wild type FAK in CA6 cells did not facilitate branching tubule formation in 0.1% collagen gel. Targeted decrease in the expression level of FAK in M610 cells with the application of antisense cDNA resulted in a marked reduction of branching tubule formation in 0.1% collagen gel and showed a down-regulation of fibronectin assembly, which is known to promote tubulogenesis. In contrast, overexpression of wild type FAK in CA6 cells had no effect on fibronectin assembly. Taken together, our data demonstrates that FAK is required, but not sufficient for HGF-independent branching tubulogenesis in MDCK cells.     

PGE1-independent MDCK cells have elevated intracellular cyclic AMP but retain the growth stimulatory effects of glucagon and epidermal growth factor in serum-free medium

Prostaglandin E1 (PGE1), a component in the hormone-supplemented, serum-free medium for the Madin Darby canine kidney (MDCK) cell line, has been proposed to increase MDCK cell growth by increasing intracellular cyclic AMP levels. The association between increased intracellular cyclic AMP and the growth stimulatory effect of PGE1 has been examined in normal MDCK cells and in PGE1-independent variants of MDCK. These variant cells have lost the PGE1 requirement for long term growth in defined medium. Normal MDCK cells had almost twofold higher intracellular cyclic AMP levels during growth in Medium K-1 (9.0 pmol/mg protein) than in Medium K-1 minus PGE1. Furthermore, PGE1-independent clone 1 had higher intracellular cyclic AMP levels in Medium K-1 minus PGE1 than normal MDCK cells in Medium K-1. This latter observation suggests that the PGE1 requirement for MDCK cell growth is associated with the low intracellular cyclic AMP levels of this cell line. An involvement of cyclic AMP in the growth response to PGE1 is supported by these observations, as well as by the growth stimulatory effects of other agents that affect cyclic AMP metabolism in MDCK cells. These agents include glucagon, isobutyl methylxanthine (IBMX), and dibutyryl cyclic AMP. The growth of PGE1-independent clone 1 was inhibited rather than stimulated by PGE1. Similarly, PGE1-independent cell growth was inhibited by IBMX and dibutyryl cyclic AMP. However, the growth response to one agent which increases cyclic AMP (glucagon) was retained in PGE1-independent clone 1. This result suggests that the effect of glucagon is not associated with increases in intracellular cyclic AMP. The growth stimulatory effect of epidermal growth factor (EGF) on normal MDCK cells was also studied. Although EGF does not act via a cyclic AMP-mediated mechanism, EGF increased normal MDCK cell growth and substituted for PGE1 in Medium K-1. Thus, EGF and PGE1 could possibly affect similar growth-related functions in MDCK cells, although by different pathways. This possibility was examined further, using PGE1-independent clone 1. EGF, like glucagon, was still growth stimulatory to the PGE1-independent cells. Consequently, the biochemical pathways by which EGF and PGE1 increase MDCK cell growth probably do not converge.     

Renal epithelial fluid secretion and cyst growth: the role of cyclic AMP

Transepithelial fluid secretion has been postulated to account for the accumulation of fluid within hereditary and acquired renal cysts, but no such mechanism has been demonstrated in human kidney epithelium. It is shown here that transepithelial fluid secretion was stimulated by prostaglandin E1 (PGE1), forskolin, 8-Br-cyclic AMP, and 1-methyl-3-isobutylxanthine in polarized monolayers of established renal cell lines (MDCK and rat glomerular epithelial cells) and in monolayer cultures derived from the cyst walls of human autosomal dominant polycystic kidney disease and from epithelial cells of normal human renal cortex. Treatment with cyclic AMP agonists caused the same cells, when dispersed within a gel matrix of type I collagen (Vitrogen), to proliferate and form spherical fluid-filled monolayered cysts. Our findings suggest that increased intracellular cyclic AMP levels may have a critical role in the formation and expansion of hereditary and acquired renal cysts.     

The microenvironmental determinants for kidney epithelial cyst morphogenesis

Although epithelial morphogenesis is tightly controlled by intrinsic genetic programs, the microenvironment in which epithelial cells proliferate and differentiate also contributes to the morphogenetic process. The roles of the physical microenvironment in epithelial morphogenesis, however, have not been well dissected. In this study, we assessed the impact of the microenvironment on epithelial cyst formation, which often marks the beginning or end step of morphogenesis of epithelial tissues and the pathological characteristic of some diseases. Previous studies have demonstrated that Madin-Darby canine kidney (MDCK) epithelial cells form cysts when grown in a three-dimensional (3D) extracellullar matrix (ECM) environment. We have now further demonstrated that the presence of ECM in the 3D scaffold is required for the formation of properly polarized cysts. Also, we have found that the full interface of epithelial cells with the ECM environment (in-3D) is not essential for cyst formation, since partial contact (on-3D) is sufficient to induce cystogenesis. In addition, we have defined the minimal ECM environment or the physical threshold for cystogenesis under the on-3D condition. Only above the threshold can the morphological cues from the ECM environment induce cyst formation. Moreover, cyst formation under the on-3D condition described in this study defines a novel and more feasible model to analyze in vitro morphogenesis. Finally, we have found that, during cystogenesis, MDCK cells generate basal microprotrusions and produce vesicle-like structures to the basal extracellular space, which are specific to and correlated with cyst formation. For the first time, we have systematically and quantitatively elucidated the microenvironmental determinants for epithelial cystogenesis.     

Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase activity and a diminished effect of PGE1 on differentiated function

Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain-sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.     

Calcium-mediated mechanisms of cystic expansion

In this review, we will discuss several well-accepted signaling pathways toward calcium-mediated mechanisms of cystic expansion. The second messenger calcium ion has contributed to a vast diversity of signal transduction pathways. We will dissect calcium signaling as a possible mechanism that contributes to renal cyst formation. Because cytosolic calcium also regulates an array of signaling pathways, we will first discuss cilia-induced calcium fluxes, followed by Wnt signaling that has attributed to much-discussed planar cell polarity. We will then look at the relationship between cytosolic calcium and cAMP as one of the most important aspects of cyst progression. The signaling of cAMP on MAPK and mTOR will also be discussed. We infer that while cilia-induced calcium fluxes may be the initial signaling messenger for various cellular pathways, no single signaling mediator or pathway is implicated exclusively in the progression of the cystic expansion. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Hemicyst formation stimulated by cyclic AMP in dog kidney cell line MDCK.

Certain epithelial cell lines have morphologic, physiologic, biochemical and pharmacologic characteristics of transporting epithelia from intact organs. In this paper we show that dibutyryl cyclic AMP, 5' AMP, adenosine and cyclic AMP phosphodiesterase inhibitors stimulate hemicyst formation by the dog kidney cell line MDCK. It is suggested that this effect is explained by elevation of intracellular cyclic AMP levels by means of an exogenous non-metabolizable source of cyclic AMP, phosphodiesterase inhibition or adenyl cyclase stimulation. Since hemicyst formation is in part due to transepithelial fluid transport, these findings raise the possibility that this fraction might be modulated by cAMP in an established cell line. We believe that cultured epithelial cells may provide an exploitable model system to investigate at the cellular and subcellular levels, the mechanism by which cyclic AMP modifies water and solute movements across epithelia.     

Molecular cloning of cAMP-dependent protein kinase catalytic subunit isoforms from the lone star tick, Amblyomma americanum (L.)

The salivary glands of ixodid ticks are central to tick feeding and to survival during off-host periods. They produce and secrete a number of molecules critical to maintaining the complex host-vector interface and to maintaining osmotic balance. We have previously shown that a cyclic AMP-dependent protein kinase (cAPK) is involved in the mechanism of salivary gland secretion. We have now cloned cDNAs encoding three isoforms of the catalytic subunit (cAPK-C) of the cAPK from Amblyomma americanum, which are probably produced from alternative RNA processing of a single cAPK-C gene. The cDNAs contain unique N-termini of variable lengths that are linked to a common region containing the alpha A helix, catalytic core, and a C-terminal tail. The common region is highly similar to both insect and vertebrate cAPK-Cs. We have examined mRNA profiles in whole ticks and in isolated salivary glands throughout feeding and find that a single cAPK-C isoform is expressed in the salivary glands of both unfed and feeding females.