Calcium channel blockers inhibit bacterial chemotaxis

The effect of several Ca2+ channel blockers, which inhibit the voltage-dependent Ca2+ uptake in Bacillus subtilis, on chemotactic behaviour of the bacterium was studied. Nitrendipine, verapamil, LaCl3 and omega-conotoxin were tested and these blockers inhibited chemotactic behaviour in the bacterium toward L-alanine. Among these blockers, omega-conotoxin was the most effective inhibitor of chemotaxis. EGTA was also as effective as omega-conotoxin. In contrast, these blockers, did not inhibit the motility and the growth of the bacterium. These results suggest that internal Ca2+ plays an important role in the sensory system of bacterial chemotaxis.     

Effects of organic antagonists of Ca(2+), Na(+), and K(+) on chemotaxis and motility of escherichia coli

Various Ca(2+) antagonists used in animal research, many of them known to be Ca(2+) channel blockers, inhibited Escherichia coli chemotaxis (measured as entry of cells into a capillary containing attractant). The most effective of these, acting in the nanomolar range, was omega-conotoxin GVIA. The next most effective were gallopamil and verapamil. At concentrations around 100-fold higher than that needed for inhibition of chemotaxis, each of these antagonists inhibited motility (measured as entry of cells into a capillary lacking attractant). Various other Ca(2+) antagonists were less effective, though chemotaxis was almost always more sensitive to inhibition than was motility. Cells treated with each of these Ca(2+) antagonists swam with a running bias, i.e., tumbling was inhibited. Similarly, some Na(+) antagonists used in animal research inhibited bacterial chemotaxis. E. coli chemotaxis was inhibited by saxitoxin at concentrations above 10(-7) M, while more than 10(-4) M was needed to inhibit motility. Cells treated with saxitoxin swam with a tumbling bias. In the case of other Na(+) antagonists in animals, aconitine inhibited bacterial chemotaxis 10 times more effectively than it inhibited motility, and two others inhibited chemotaxis and motility at about the same concentration. In the case of K(+) antagonists used in animal research, 4-aminopyridine blocked E. coli chemotaxis between 10(-3) M and, totally, 10(-2) M, while motility was not affected at 10(-2) M; on the other hand, tetraethylammonium chloride failed to inhibit either chemotaxis or motility at 10(-2) M.     

Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content.

The calA, calC, and calD mutants of Escherichia coli are known to be sensitive to Ca2+ (R. N. Brey and B. P. Rosen, J. Bacteriol. 139:824-834, 1979). In the absence of any added stimuli for chemotaxis, both the calC and the calD mutants swam with a tumbly bias. Both the calC and the calD mutants were defective in chemotaxis as measured by computer analysis, use of swarm plates, and capillary assays. The calA mutant was only slightly defective in motility and only slightly impaired in chemotaxis. Chemotactically wild-type cells had an intra-cellular free-Ca2+ level of about 105 nM. The intracellular free-Ca2+ levels of the mutants, as determined by use of the fluorescent Ca2+ indicator dye fura-2 or fluo-3, were about 90, about 1,130, and about 410 nM for calA, calC, and calD, respectively. Lowering the intracellular free-Ca2+ levels in wild-type cells and in the tumbly cal mutants by use of Ca2+ chelators promoted running (smooth swimming). Overexpression of CheZ (which causes dephosphorylation of CheY-phosphate) in the wild type and in the tumbly cal mutants decreased the level of tumbliness (which is caused by CheY-phosphate). The calA mutant was 4- to 10-fold more resistant than the wild type to the inhibitory effect of omega-conotoxin on chemotaxis. omega-Conotoxin had no effect on Ca2+ extrusion by wild-type E. coli; that result suggests that omega-conotoxin affects Ca2+ transport at the point of entry instead of exit.     

Interactions between the presynaptically active neurotoxins alpha-latrotoxin and omega-conotoxin GVIA: studies on calcium fluxes and binding parameters in rat and chicken synaptosomes

Possible interactions between alpha-latrotoxin, an activator of synaptosomal calcium uptake, and omega-conotoxin GVIA, an inhibitor of voltage-sensitive calcium channels of the N-type, were investigated in rat and chicken synaptosomal preparations. While omega-conotoxin GVIA potently and effectively inhibited calcium uptake induced by elevated potassium in chick synaptosomes, little or no effect of omega-conotoxin GVIA was observed either in potassium-treated rat synaptosomes or in alpha-latrotoxin-exposed synaptosomes of either spaces. In contrast to the lack of effect of omega-conotoxin on stimulated calcium uptake in rat synaptosomes, cadmium effectively inhibited calcium uptake induced by either potassium or alpha-latrotoxin. Synaptosomal calcium transport induced by alpha-latrotoxin can be bidirectional, since alpha-latrotoxin also induced efflux of preaccumulated calcium. Competition experiments revealed that binding of 125I-labelled omega-conotoxin and 125I-labelled alpha-latrotoxin was similar in either chicken or rat synaptosomes. Neither alpha-latrotoxin nor omega-conotoxin competed with the binding of the other ligand in either species. The results reported here show that (1) elevated potassium evokes calcium uptake principally through N-channels in avian but not in rat synaptosomes; (2) alpha-latrotoxin-activated calcium fluxes are omega-conotoxin insensitive but cadmium sensitive; (3) the molecular acceptors for the two ligands are likely to be unrelated synaptic membrane constituents.     

Separate Binding and Functional Sites for ω co-Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.     

Identification of ω-Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

The involvement of calcium and protein kinase C in modulating agonist-stimulated chemotaxis of human neutrophils

Chemotaxis of human neutrophils in response to a gradient of the chemotactic peptide, fmet-leu-phe (FMLP), was measured by the under-agarose technique. The dose-response curve for FMLP was biphasic; low concentrations were stimulatory, and the response was reduced at higher concentrations. The response to FMLP was partially inhibited (about 50%) in the absence of extracellular Ca2 (EGTA added). NiCl2 dose-dependently inhibited FMLP-stimulated chemotaxis in the presence of extracellular Ca2+; the maximum inhibition obtainable with NiCl2 was similar to that with the absence of extracellular Ca2+. These results suggest that FMLP-stimulated chemotaxis is, at least partially, dependent on stimulation of Ca2+ influx. The phorbol ester, PMA, dose-dependently inhibited chemotaxis; the response was almost completely inhibited by 10 nM PMA. This result indicates that activation of protein kinase C inhibits chemotaxis. These results are discussed in relation to the physiological responses of neutrophils.     

Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.     

Different contributions of voltage-sensitive Ca2+ channels to histamine-induced catecholamine release and tyrosine hydroxylase activation in bovine adrenal chromaffin cells.

Histamine stimulates catecholamine release and tyrosine hydroxylase activity in a Ca(2+)-dependent manner in bovine adrenal chromaffin cells. The role of voltage-sensitive Ca2+ channels in these two responses has been investigated. Using an EC50 concentration of histamine, 1 microM, catecholamine release was enhanced by (+/-)BayK8644, and partially inhibited by nitrendipine and omega-agatoxin IVA, blockers of L- and P/Q-type Ca2+ channels. omega-Conotoxin GVIA gave small and variable inhibitory effects. With a maximal histamine concentration, 10 microM, similar results were obtained except that now omega-conotoxin GVIA reliably reduced release. In contrast, neither (+/-)BayK8644 nor any of the individual Ca2+ channel antagonists had any significant effect on tyrosine hydroxylase (TOH) activation induced by either an EC50 or a maximal concentration of histamine. When high concentrations of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA were combined with omega-conotoxin MVIIC (a non-selective blocker of N, P and Q channels) to block voltage-sensitive Ca2+ channels in these cells, release induced by K+ depolarization was completely blocked. Release caused by histamine, however, was substantially reduced but not abolished. The combination of antagonists also only partially inhibited TOH activation by histamine. The results show that the G protein-coupled receptor agonist histamine activates several different types of voltage-sensitive Ca2+ channels in chromaffin cells to mediate its cellular effects. Histamine may also activate additional pathways for Ca2+ entry. The results also suggest that the manner by which Ca2+ controls release and TOH activation once it has entered chromaffin cells through these channels are different.     

A monoclonal antibody to the beta subunit of the skeletal muscle dihydropyridine receptor immunoprecipitates the brain omega-conotoxin GVIA receptor.

Antibodies against the subunits of the dihydropyridine-sensitive L-type calcium channel of skeletal muscle were tested for their ability to immunoprecipitate the high affinity (Kd = 0.13 nM) 125I-omega-conotoxin GVIA receptor from rabbit brain membranes. Monoclonal antibody VD2(1) against the beta subunit of the dihydropyridine receptor from skeletal muscle specifically immunoprecipitated up to 86% of the 125I-omega-conotoxin receptor solubilized from brain membranes whereas specific antibodies against the alpha 1, alpha 2, and gamma subunits did not precipitate the brain receptor. Purified skeletal muscle dihydropyridine receptor inhibited the immunoprecipitation of the brain omega-conotoxin receptor by monoclonal antibody VD2(1). The dihydropyridine receptor from rabbit brain membranes was also precipitated by monoclonal antibody VD2(1). However, neither the neuronal ryanodine receptor nor the sodium channel was precipitated by monoclonal antibody VD2(1). The omega-conotoxin receptor immunoprecipitated by monoclonal antibody VD2(1) showed high affinity 125I-omega-conotoxin binding, which was inhibited by unlabeled omega-contoxin and by CaCl2 but not by nitrendipine or by diltiazem. An antibody against the beta subunit of the skeletal muscle dihydropyridine receptor stained 58- and 78-kDa proteins on immunoblot of the omega-conotoxin receptor, partially purified through heparin-agarose chromatography and VD2(1)-Sepharose chromatography. These results suggest that the brain omega-conotoxin-sensitive calcium channel contains a component homologous to the beta subunit of the dihydropyridine-sensitive calcium channel of skeletal muscle and brain.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Effect of morphine analogues on chemotaxis in Escherichia coli.

Pretreatment of Escherichia coli w3110 with levorphanol, a morphine analogue, reduced chemotaxis to serine, aspartic acid and galactose. This decreased chemotaxis was not due to decreased viability or motility. Pretreatment with 1.1 mM-levorphanol for 1 h, followed by washing to remove the drug prior to determination of chemotaxis, inhibited chemotaxis to each of the attractants by at least 80%. Pretreatment with dextrorphan, the enantiomorph of levorphanol, or levallorphan, the N-allyl analogue of levorphanol, resulted in a similar inhibition of chemotaxis. Reversal of the inhibition produced by pretreatment with levorphanol required a period of growth of at least one generation time.     

THE REGULATORY ROLE OF DIVALENT CATIONS IN HUMAN GRANULOCYTE CHEMOTAXIS : Evidence for an Association between Calcium Exchanges and Microtubule Assembly

Optimal human granulocyte chemotaxis has been shown to require both calcium and magnesium. Exposure of granulocytes to three different chemotactic factors (C5a, kallikrein, and dialyzable transfer factor) yielded a rapid calcium release, depressed calcium uptake, and was associated with a shift of calcium out of the cytoplasm and into a granule fraction. Colchicine, sodium azide, and cytochalasin B, in concentrations that inhibited chemotaxis, also inhibited calcium release while low concentrations of cytochalasin B, which enhanced chemotaxis, also enhanced calcium release. Microtubule assembly was visualized both in cells suspended in C5a without a chemotactic gradient and in cells actively migrating through a Micropore filter. The data suggest microtubule assembly is regulated, at least, in part, by the level of cytoplasmic calcium. It is proposed that asymmetric assembly of microtubules may be instrumental in imparting the net vector of motion during chemotaxis.     

Inhibition of human neutrophil chemotaxis by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine

The protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine (C-I), inhibits superoxide release from human neutrophils (PMN) stimulated with phorbol myristate acetate or synthetic diacylglycerol, without inhibiting superoxide release from PMN stimulated with the chemoattractants C5a or N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). In this study, we investigated the effect of C-I on human PMN chemotaxis to C5a, f-Met-Leu-Phe, leukotriene B4 (LTB4), and fluoresceinated N-formyl-methionyl-leucyl-phenylalanine-lysine (f-Met-Leu-Phe-Lys-FITC). PMN, preincubated for 5 min at 37 degrees C with 0 to 200 microM C-I, were tested for their migratory responses to the chemoattractants. C-I (greater than or equal to 1 microM) significantly inhibited PMN chemotaxis to f-Met-Leu-Phe, f-Met-Leu-Phe-Lys-FITC, and C5a without affecting random migration. Maximal inhibition of chemotaxis to these attractants occurred with greater than or equal to 50 microM C-I, at which chemotaxis was inhibited by 80 to 95%. The C-I inhibition was reversible. In contrast, 200 microM C-I did not inhibit the number of PMN migrating to LTB4, although, the leading front of PMN migration to LTB4 was inhibited by C-I. C-I inhibited PMN orientation to C5a and f-Met-Leu-Phe without affecting orientation to LTB4. C-I did not inhibit the binding of radiolabeled f-Met-Leu-Phe or f-Met-Leu-Phe-Lys-FITC to PMN. These findings suggest that the chemotactic responses of PMN to f-Met-Leu-Phe and C5a involve a protein kinase-dependent reaction which is inhibited by C-I.     

Chemotaxis by Pseudomonas aeruginosa.

Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa.     

Differential control of tyrosine hydroxylase activation and catecholamine secretion by voltage-operated Ca2+ channels in bovine chromaffin cells

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.     

Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.