Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.

Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor

Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C/hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein/LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors.

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Muscarinic stimulation of synaptic activity by protein kinase C is inhibited by adenosine in cultured hippocampal neurons

We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1–43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular Ca2+ signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular Ca2+ concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular Ca2+ concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4β-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.

Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10−8 M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using 35S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [35S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent Kd values for [35S]PSK-α binding were found to be 1 × 10−9 M for the high-affinity type and 1 × 10−7 M for the low-affinity type, with maximal numbers of binding sites of 1 × 104 sites per cell and 1 × 105 sites per cell, respectively. Competition studies with [35S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca2+ permeation properties suggest an important function in Ca2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca2+-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control.

Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein.

Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein-expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity.

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.

Peptide mimicry of the meningococcal group C capsular polysaccharide.

Sequence analysis of the variable regions of the heavy and light chains of the anti-idiotypic antibody 6F9, which mimics the meningococcal group C capsular polysaccharide (MCP), was performed. The immunogenic site on 6F9 responsible for inducing an anti-MCP antibody response was determined by means of sequence and computer model analysis of these data. Complementarity-determining region 3 (CDR3) was found to be unique in that the sequence tract YRY was exposed on the surface. A synthetic peptide spanning the CDR3 domain was synthesized and complexed to proteosomes (meningococcal group B outer membrane protein). Immunizations of BALB/c mice with the peptide-proteosome complex resulted in a significant anti-MCP antibody response. Immunized mice were protected against infection with a lethal dose of Neisseria meningitidis serogroup C.