Determination of folate transport pathways in cultured rat proximal tubule cells

Deficiency of the vitamin folic acid has recently been linked with increased incidence of neural tube defects and of cardiovascular disease, through elevated plasma homocysteine levels. The kidney has an important role in conserving folate to counteract development of deficiency. Urinary folate excretion is regulated by the degree of reabsorption of folate by the proximal tubule cell. To evaluate an in vitro model for studies of the regulation of urinary folate excretion, the present studies examined the transport of 5-methyltetrahydrofolate (5-CH3-H4PteGlu), the primary form of folate in the glomerular filtrate, by normal rat proximal tubule (RPT) cells in confluent monolayer cultures. Specific binding of 5-CH3-H4PteGlu to the apical membrane was saturable (K(D) = 27 nM), but intracellular transport was not saturated up to 100 nM concentrations. 5-CH3-H4PteGlu transport was decreased 50% by concentrations of folic acid that completely blocked 5-CH3-H4PteGlu binding by the apical folate receptor. Probenecid (10 mM), an anion exchange (reduced folate carrier) inhibitor, reduced 5CH3-H4PteGlu transport by 50% without significantly affecting binding. Aspirin (3 mM) did not alter 5-CH3-H4PteGlu transport, but significantly enhanced the inhibition due to probenecid. Similarly, indomethacin (5 microM) potentiated the inhibition of 5-CH3-H4PteGlu transport by probenecid. These data suggest that RPT cells take up 5-CH3-H4PteGlu by both the folate receptor and the reduced folate carrier, implying a role for both pathways in regulating urinary folate excretion.     

Parathyroid hormone responses of cyclic AMP-, serum- and phorbol ester-responsive reporter genes in osteoblast-like UMR-106 cells

In a systematic search for vanadocene complexes with sperm immobilizing activity as a new class of contraceptive agents, we identified V(eta 5-C5H5)2((C2H5)2 NCS2)(BF4) (=[VCp2(DeDtc)](BF4)) as the most potent and stable spermicidal compound. Here we report the detailed biologic and physicochemical characterization of this lead spermicidal compound by computer-assisted sperm analysis, electron paramagnetic resonance spectroscopy, electrochemistry, and X-ray crystallography. [VCp2(DeDtc)](BF4) crystallized in the monoclinic space group P2(1)/c, with unit cell dimensions a = 7.0877(4) A, b = 22.2881(14) A, c = 11.8021(7) A, beta = 94.107(1) degree, V = 1859.6(2) A3. The final structure of [VCp2(DeDtc)](BF4) had an R factor of 0.0581 for 3191 independent reflections. The two sulfur atoms of the dithiocarbamate and centroids of the cyclopentadienyl rings in this vanadocene complex with unique contraceptive potential occupy four tetrahedral--like coordination sites about the central metal atom.     

Transforming growth factor-beta-mediated proliferation of renal tubular epithelial cells involves pertussis toxin-sensitive G protein-and protein kinase C-dependent pathways

Salmon calcitonin (5 micrograms/kg body wt) was administered in an elasmobranch, Dasyatis akajei, to investigate the effects upon plasma calcium and inorganic phosphate. The hormone produced hypocalcemia and hyperphosphatemia in the stingray. It is concluded that calcitonin may have a role in calcium homeostasis by a mechanism different from that on bones.     

Parathyroid hormone-related protein in patients with primary breast cancer and eucalcemia

Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in breast cancer and other malignancies. We studied circulating PTHrP levels with three different immunoassays directed against different parts of the PTHrP molecule in 48 patients with breast cancer and eucalcemia. The methods used were: (a) a RIA with antibodies directed toward the midregion (63-78); (b) an immunofluorometric assay with two antibodies against 1-34 and 38-67; and (c) an immunoradiometric assay with antibodies against 1-40 and 1-72. Although most patients had PTHrP levels indistinguishable from normal when measured by all three methods, four patients had increased serum levels in the IFMA. PTHrP was detected by immunohistochemistry in tumors from nearly all patients. One patient with elevated PTHrP in plasma measured by IFMA showed intense staining of tumor by immunohistochemistry; the tumor was histologically graded as III (severe) and was the largest of all tumors in this patient group. The IFMA can identify increased serum PTHrP in some patients with breast cancer who are not hypercalcemic. This assay may be especially useful in screening patients for this tumor during a relative early phase of the disease.     

Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to beta-amyloid precursor protein secretion

Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans, trans-farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.     

The Helicobacter pylori fatty acid cis-9,10-methyleneoctadecanoic acid stimulates protein kinase C and increases DNA synthesis of gastric HM02 cells

Protein kinase C (PKC) has been implicated in the control of epithelial proliferative activity and in the process of malignant transformation. Helicobacter pylori (H.p.) infection is associated with increased gastric epithelial cell proliferation and has been linked with gastric carcinoma. In the present study, we report that the H.p. fatty acid cis-9,10-methyleneoctadecanoic acid (MOA) directly activates PKC (Ka 3.3 microM). The effect of MOA upon PKC activation was Ca2+ dependent but did not require phosphatidylserine as phospholipid cofactor. MOA increased the stimulatory effect of phosphatidylserine at low Ca2+ (1 microM) concentrations. These findings indicate that MOA interacts at the phospholipid- and the diacylglycerol-binding domain to elicit PKC activation. Treatment of gastric mucous cells HM02 caused translocation of PKC from the cytosol to the nuclear, mitochondrial and membrane fraction. Furthermore, MOA stimulated [3H]thymidine incorporation into the DNA of HM02 cells. Our results show that the H.p. fatty acid MOA activates PKC and increases DNA synthesis in gastric epithelial cells.     

Protein kinase C isozyme expression and down-modulation in growing, quiescent, and transformed renal proximal tubule epithelial cells

Renal alpha-protein kinase C (PKC) is rapidly down-modulated modulated in animals treated with the renal toxin and tumor promoter, folic acid (Dong et al., Cancer Res., 53: 4542-4549, 1993). To further explore the role of PKC isozymes in renal growth and carcinogenesis, we compared phorbol ester receptor and PKC isozyme content, distribution, and regulation in primary and oncogene-altered rat renal proximal tubule epithelial cells (RPTE) in culture. Immunoblot analysis and RNase protection assays indicated that RPTE expressed at least four PKC isozymes, alpha, delta, epsilon, and zeta. Total phorbol ester receptors were decreased in primary proliferating, E1A-immortalized, and SV40-transformed RPTE compared to primary quiescent RPTE. The decrease in PDBu binding was largely due to a specific decrease in alpha-PKC protein content to approximately 50% of the level in quiescent RPTE. Degradation rates and message levels were compared to determine the mechanism for the decrease in alpha-PKC. Whereas alpha-PKC message levels in quiescent and proliferating primary RPTE were comparable, alpha-PKC degradation was increased in proliferating cells. These results indicate that the decreased alpha-PKC content was due largely to increased turnover. Phorbol ester stimulated the rate of degradation, thus demonstrating a link between degradation rate and PKC activation. These results suggest that the increased basal degradation rate in proliferating and oncogene-altered cells reflects an increase in activity of PKC in these cells.     

Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.     

Effects of genistein and staurosporine on angiotensin II-induced DNA synthesis, protein synthesis and mitogen-activated protein kinase activation in vascular smooth muscle cells

This study examines the effects of protein kinase inhibitors and activator on angiotensin II-induced DNA synthesis and protein synthesis of rat aortic smooth muscle cells. In quiescent confluent cells, angiotensin II induced a concentration-dependent increase in thymidine incorporation and leucine incorporation. The tyrosine kinase inhibitor genistein caused an inhibition of the angiotensin II-induced DNA synthesis but not of the agent-induced protein synthesis. The protein kinase C inhibitors staurosporine and calphostin C caused an inhibition of the angiotensin II-induced protein synthesis but not of the agent-induced DNA synthesis. The protein kinase C activator phorbol 12-myristate 13-acetate stimulated protein synthesis. Angiotensin II stimulated mitogen-activated protein (MAP) kinases and the angiotensin II-induced MAP kinase activation was inhibited by genistein but not by staurosporine. These findings suggest that angiotensin II-induced DNA synthesis is at least partly mediated via protein-tyrosine phosphorylation and angiotensin II-induced protein synthesis is at least partly mediated by activation of protein kinase C. It seems likely that MAP kinase activation is involved in DNA synthesis but not in protein synthesis induced by angiotensin II.     

Inhibition of demecolcin-induced DNA synthesis by inhibitors of phospholipase C and protein kinase C

PURPOSE OF STUDY: Interleukin-2 (IL-2) is a potent activator of lymphocytes, but its effectiveness as an anti-cancer agent is compromised by several adverse side effects including pulmonary edema. One explanation for the pulmonary toxicity of IL-2 is that activated lymphocytes directly induce the pulmonary vascular endothelium to become more leaky. METHODS: To test this hypothesis the number of total lymphocytes, gamma delta T cells, and CD2-positive cells (alpha beta T cells and natural killer cells) in peripheral blood and lung lymph of sheep were compared before and after IL-2 infusion. Hemodynamic and lymph dynamic changes were also evaluated. RESULTS: IL-2 decreased mean aortic pressure, increased cardiac output, lowered systemic vascular resistance, and doubled lung lymph flow (P < or = 0.05), but had no effect on plasma or lymph oncotic pressure. The lymph protein concentration and the lymph-to-plasma protein concentration ratio were not different after IL-2 infusion. IL-2 had no effect on the number of total lymphocytes, gamma delta T cells, or CD2-positive cells in the peripheral blood. In contrast, the number of total lymphocytes, gamma delta T cells, and CD2-positive cells in lung lymph decreased significantly (P < or = 0.05). CONCLUSIONS: The lymphocyte populations decreased more than could be explained by the increase in lymph flow, demonstrating that lung lymphocytes were not reduced simply by dilution. These results imply that the pulmonary edema associated with IL-2 is not caused by activated lymphocytes.     

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.     

Cholecystokinin stimulates formation of shc-grb2 complex in rat pancreatic acinar cells through a protein kinase C-dependent mechanism

Cholecystokinin (CCK) has recently been shown to activate the mitogen-activated protein kinase (MAPK) cascade (Ras-Raf-MAPK kinase-MAPK) in pancreatic acini. The mechanism by which the Gq protein-coupled CCK receptor activates Ras, however, is currently unknown. Growth factor receptors are known to activate Ras by means of adaptor proteins that bind to phosphotyrosine domains. We therefore compared the effects of CCK and epidermal growth factor (EGF) on Tyr phosphorylation of the adaptor proteins Shc and its association with Grb2 and the guanine nucleotide exchange factor SOS. Three major isoforms of Shc (p46, p52, p66) were detected in isolated rat pancreatic acini with p52 Shc being the predominant form. CCK and EGF increased tyrosyl phosphorylation of Shc (251 and 337% of control, respectively). CCK-stimulated tyrosyl phosphorylation of Shc as well as Shc-Grb2 complex formation was significant at 2.5 min, maximal at 5 min, and persisted for at least 30 min. Finally, SOS was found to be associated with Grb2 as assessed by probing of anti-Grb2 immunoprecipitates with anti-SOS. Since MAPK in pancreatic acini is activated via protein kinase C (PKC), we studied the effect of phorbol esters on Shc phosphorylation and found 12-O-tetradecanoylphorbol-13-acetate to be as potent as CCK. Furthermore, GF-109203X, a PKC inhibitor, abolished the effect of 12-O-tetradecanoylphorbol-13-acetate and also the effect of CCK but not the effect of EGF on Shc tyrosyl phosphorylation. CCK-induced tyrosyl phosphorylation of Shc was found to be phosphatidylinositol 3-kinase-independent, and CCK did not cause EGF receptor activation. These results suggest that formation of an Shc-Grb2-SOS complex via a PKC-dependent mechanism may provide the link between Gq protein-coupled CCK receptor stimulation and Ras activation in these cells.     

Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol

Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs.     

Pressure-induced expression of heat shock protein 70 mRNA in adult rat heart is coupled both to protein kinase A-dependent and protein kinase C-dependent systems

BACKGROUND: Production of heat shock protein 70 (HSP70) in the heart is induced by hemodynamic stress, but its intracellular signal transduction system has not been elucidated well. OBJECTIVE: To investigate the hypothesis that protein kinase A (PKA)-dependent and protein kinase C (PKC)dependent systems are involved in the pressure-induced expression of HSP70 mRNA in perfused adult rat heart METHODS: Isolated tetrodotoxin-arrested Sprague-Dawley rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. Aortic pressure in rats of the pressure-overloaded group was elevated from 60 to 120 mmHg for 2-120 min. cAMP contents and rates of synthesis of protein were measured by radioimmunoassay and the incorporation of [14C]-phenylalanine into total heart protein, respectively. Expression of HSP70 mRNA was determined by Northern blot analysis. RESULTS: Elevation of aortic pressure significantly increased cAMP content after 2 min of perfusion (by 41%), significantly increased rates of synthesis of protein during the second hour of perfusion (by 41%), and induced expression of HSP70 mRNA maximally after 60 min of perfusion (2.7-fold the control value). Exposure to glucagon, forskolin or 1 -methyl-3-isobutylxanthine mimicked increases in these parameters caused by elevation of aortic pressure. Administration of a selective PKA inhibitor, H-89, significantly prevented induction of increases in expression of HSP70 mRNA and rates of synthesis of protein by a high pressure overload and exposure to agents that increase cAMP content. Furthermore, administration of phorbol ester induced expression of HSP70 mRNA. Administration of a PKC inhibitor, calphostin C, significantly prevented induction of increases in expression of HSP70 mRNA by a pressure overload and by exposure to phorbol ester. CONCLUSIONS: These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.     

Activation of mitogen-activated protein kinase pathways by Mycoplasma fermentans membrane lipoproteins in murine macrophages: involvement in cytokine synthesis

We have investigated aspects of ion selectivity in K+ channels by functional expression of wild-type and mutant heteromultimeric G protein-coupled inward-rectifier K+ (GIRK) channels in Xenopus oocytes. Within the K+ channel pore (P) region signature sequence, a large number of point mutations in GIRK1 and GIRK4 subunits have been made at a key tyrosine residue--the "signature" tyrosine of the GYG. Studies of mutant GIRK1/GIRK4 heteromultimers reveal that the GIRK1 and GIRK4 subunits contribute asymmetrically to K+ selectivity. The signature tyrosine of GIRK1 can be mutated to many different residues while retaining selectivity; in contrast, the analogous position in GIRK4 must be tyrosine for maximum selectivity. Other residues of the P region also contribute to selectivity, and studies with GIRK1/GIRK4 chimeras reveal that an intact, heteromultimeric P region is necessary and sufficient for optimal K+ selectivity. We propose that the GIRK1 and GIRK4 P regions play roles similar to the two P regions of an emerging family of K+ channels whose subunits each have two P regions connected in tandem. We find different consequences between similar mutations in inward-rectifier and voltage-gated K+ channels, which suggests that the pore structures and selectivity mechanisms in the two classes of channel may not be identical. We confirm that GIRK4 subunits alone can form functional channels in oocytes, but we find that these channels are measurably permeable to Na2+ and Ca2+.     

Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways

The intracellular signal transduction pathways utilized by the HIV-1-derived protein, Tat, in the activation of human central nervous system-derived endothelial cells (CNS-ECs) were examined using specific enzymatic assays. Tat induced an increase in interleukin 6 (IL-6) mRNA within 1 hr of treatment. This biological effect of Tat involved activation of both protein kinase C (PK-C) and cAMP-dependent protein kinase (PK-A) in CNS-ECs. Tat at 10 ng/ml induced a sharp, transient increase in membrane PK-C activity within 30 sec of incubation, and reached maximum levels at 2 min, declining to control values within 10 min. Tat also induced a sharp increase in intracellular cAMP levels and PK-A activity in these cells, with the PK-A activity reaching a maximum at 10 min and slowly declining to control values in 4 hr of incubation. Activation of PK-A was dependent on a Tat-induced increase in membrane PK-C activity as demonstrated by calphostin C (a PK-C inhibitor) abolishing this effect. Incubation of cells with the cyclooxygenase inhibitor indomethacin did not affect Tat-induced activation of PK-A, indicating that prostacyclins are not involved in this process. Tat-induced increase in IL-6 mRNA was abolished in the presence on PK-A inhibitor H-89, demonstrating that activation of PK-A is necessary and sufficient for the increase in IL-6 production by these cells. Both the Tat-induced increase in intracellular cAMP and IL-6 mRNA levels in CNS-ECs may play a role in altering the blood-brain barrier and thereby inducing pathology often observed in AIDS dementia.