Peptidase inhibitors from the salivary glands of the cockroach Nauphoeta cinerea

Inhibitory activity against subtilisin, proteinase K, chymotrypsin and trypsin was detected in the salivary glands and saliva of the cockroach Nauphoeta cinerea (Blattoptera: Blaberidae). Fractionation of the salivary glands extract by affinity chromatography followed by reverse-phase HPLC yielded five subtilisin-inhibiting peptides with molecular masses ranging from 5 to 14 kDa. N-terminal sequences and subsequently full-length cDNAs of inhibitors designated NcPIa and NcPIb were obtained. The NcPIa cDNA contains 216 nucleotides and encodes a pre-peptide of 72 amino-acid residues of which 19 make up the signal peptide. The cDNA of NcPIb consists of 240 nucleotides and yields a putative secretory peptide of 80 amino-acid residues. Mature NcPIa (5906.6 Da, 53 residues) and NcPIb (6713.3 Da, 60 residues) are structurally similar (65.4% amino acid overlap) single-domain Kazal-type peptidase inhibitors. NcPIa with Arg in P1 position and typical Kazal motif VCGSD interacted stoichiometrically (1:1) with subtilisin and was slightly less active against proteinase K. NcPIb with Leu in P1 and modified Kazal motif ICGSD had similar activity on subtilisin and no on proteinase K but was active on chymotrypsin.     

Le ionophore A23187

The acrosomic status of spermatozoa prepared for IVF has been evaluated by means of immunofluorescence test from Fenichel and Hsi using calcium A 23187 ionophore as inductor of acrosome reaction (AR). The spontaneous AR remains slight, even after 6 hour-incubation in Menezo B2 (6,8+2,7%). The response to ionophore, moderate before (11,2+9%), frankly increases after a 6h-capacitation (28,9+8,3%) in a group of 25 IVF couples (tubal indication, normal semen, positive fertilization). Nevertheless, it remains slight or null in 4 cases of unexplained repeated failure of fertilization. The response to ionophore A 23187 allows to explore the kinetics of capacitation of spermatozoa and their ability to perform AR. Its significance in terms of fecondance remains to be precised.     

The salivary glands of the cockroach Nauphoeta cinerea (Olivier)

The innervation of the salivary gland of the cockroach Nauphoeta cinerea (Olivier) has been investigated with the use of light and scanning electron microscopy. Light microscopy of methylene blue stained glands reveals the presence of a dual innervation arising from the ventral nerve cord and the stomodeal nervous system; the principal innervation is that from the ventral nerve cord which passes to the gland via the reservoir ducts. Branches of these nerves form a plexus on the acinar surface, the axons of which exhibit swelling at irregular intervals. The presence of this surface plexus and the axonal swellings was confirmed by scanning electron microscopy both in normal glands and in those in which the basal lamina had been removed by means of an HCl-collagenase digestion method. No acinar plexus was seen to be formed by branches of the stomatogastric nerve that were associated with the gland. However, other branches of this nerve were clearly connected with a complex network of multipolar neurones on the surfaces of the anterior regions of both salivary reservoirs.     

The effect of the ionophore A23187 on the ultrastructure and electrophysiological properties of frog skeletal muscle

Summary The divalent cation ionophore A23187 has three major effects on the thin cutaneous pectoris muscle of frog: (1) The membrane potential is depolarized, an action that is found only when the [Ca²⁺] of the bathing saline is very low. (2) It causes an increase in resting tension and the development of contraction. This action is produced at both normal and low values of [Ca²⁺]_o and is, therefore, independent of Ca²⁺ entry and of changes in E_m. The ionophore is believed to act primarily by releasing Ca²⁺ from intracellular stores. (3) It causes major ultrastructural damage to the muscle filaments. It is believed that this damage is the result of the action of A23187 on the sarcoplasmic reticulum and the elevation of [Ca²⁺]_i and we suggest that the action of this ionophore may serve as a useful model for the study of certain myopathies.     

The effects of calcium ionophore A23187 on interstitial cell steroidogenesis

The present study was performed to evaluate the effects of calcium ionophore A23187 on adenosine 3',5'-monophosphate (cyclic AMP) and testosterone production in rat interstitial cells. Interstitial cells were incubated in Krebs-Ringer solution with varying amounts of luteinizing hormone, pregnenolone, or A23187. Cyclic AMP and testosterone were measured in the incubation medium after 4 h incubation. A23187 (0.01--10 microgram/ml) caused progressive increases of cyclic AMP formation (from 0.18 +/- 0.02 (S.E.) pmol/10(6) cells for the control of 0.42 +/- 0.02 pmol/10(6) cells, P less than 0.025), while testosterone production remained unaltered. When varying amounts of A23187 were added concomitantly with luteinizing hormone (5 IU/l), A23187 inhibited luteinizing hormone-induced steroidogenesis in a dose-dependent manner, but it had no effect on luteinizing hormone-induced cyclic AMP formation. When pregnenolone (10(-6) M) was added to the cells, testosterone formation increased from 1.50 +/- 0.22 to 8.46 +/- 1.65 ng/10(6) cells. A23187 (1 microgram/ml) had no discernable effect on the conversion of pregnenolone to testosterone. The main effect of increased cytosol calcium on steroidogenesis seems to be at the steps beyond adenylate cyclase-cyclic AMP. These results suggest that calcium is important for the conversion of cholesterol to pregnenolone, while the steps beyond pregnenolone are relatively independent of Ca2+.     

The effects of the ionophore A23187 on pattern formation in the alga Micrasterias

We have used the divalent cation ionophore A23187 to investigate the hypothesis that cytoplasmic localization of Ca2+ is responsible for localized growth in the alga Micrasterias. In a preliminary study we found that, of the major salts contained in the cell's medium, only CaCl2 was needed for normal development. In cells developing in the presence of A23187 and extracellular Ca2+, we postulated that the ionophore would induce a spatially uniform influx of Ca2+ that would overwhelm endogenous Ca2+ gradients. When developing cells were treated with A23187 and 2 mM CaCl2, we observed a delocalization of the cell's normal pattern of wall deposition. This effect was less pronounced when cells were exposed to A23187 and 2 mM MgCl2. These results support the hypothesis that localized regions of high Ca2+ concentration normally mediate localized expansion of tip-growing lobes in Micrasterias.     

The effects of the ionophore A-23187 on the rat vas deferens

The calcium ionophore A-23187 induced spontaneous, rhythmic contractions in the rat isolated vas deferens in a concentration-dependent manner. Contractions were blocked by phentolamine and were abolished following pretreatment with reserpine. In tissues preloaded with [3H]noradrenaline, A-23187 (10 microM) caused a time-dependent increase in the release of tritium. The findings suggest that A-23187-induced contractions in the rat vas deferens are secondary to the release of endogenous noradrenaline from the adrenergic nerves, as are contractions induced in this preparation by X-537A (another calcium ionophore) described earlier by other investigators.     

A nervous system specific protein potentiates the biological effects of the calcium ionophore A23187

The present study was undertaken to determine whether the nervous system specific protein S-100, whose function is, so far, unknown, could be involved in the regulation of neurotransmitter release from central nerve endings. Our results indicate that the exogenous protein was, by itself, unable to alter the spontaneous and the depolarization-induced release of neurotransmitters from rat brain synaptosomes. However, nanomolar concentrations of S-100 potentiated the effects of the Ca²⁺ ionophore A23187 on the release of putative transmitter amino acids and catecholamines. The action of the S-100 protein seems to be related to its ability to promote, in combination with the ionophore, a higher influx of Ca²⁺ into synaptosomes than that elicited by the ionophore alone.We hypothesize that the role of the S-100 present in nerve ending membranes might be that of facilitating the function of an endogenous, voltage-dependent Ca²⁺ ionophore.     

Calcium-dependent irreversible effect of ionophore A23187 on melanophores

The calcium ionophore, A231187, induces a Ca2+ -dependent movement (dispersion) of melanosomes within skin melanophores of the lizard, Anolis carolinensis, in vitro. The effects of A23187 are irreversible, since after repeated rinsing of the skins in the absence of the ionophore they will always darken in Ringer containing Ca2+ but will immediately lighten when transferred to Ca2+ -free Ringer. These results suggest that the ionophore is irreversibly localized to the melanophore membrane and that its melanosome-dispersing effect is continuously dependent upon extracellular calcium.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Fluorescence study of the divalent cation-transport mechanism of ionophore A23187 in phospholipid membranes

The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.     

Comparative study on the stimulation of superoxide production in guinea-pig eosinophils by the calcium ionophore A23187

The effect of calcium and/or magnesium on O2- production by guinea-pig eosinophils stimulated by the calcium ionophore A23187 was studied in comparison to neutrophils. In the absence of calcium, A23187 did not stimulate O2- production in resting eosinophils and neutrophils, regardless of the presence of extracellular magnesium. The A23187-induced O2- production by both cells increased linearly with extracellular Ca2+ concentrations. However, the concentration of Ca2+ required for maximum O2- production in eosinophils was about 10-times lower than that required of neutrophils. The addition of Mg2+ strongly inhibited O2- production, especially in eosinophils at low Ca2+ concentrations. The intracellular Ca2+ concentration was lower in eosinophils than in neutrophils in the resting state, and the enhancement of the intracellular Ca2+ concentration in response to A23187 was much lower in eosinophils than in neutrophils. The activation of the NADPH-dependent O2(-)-forming enzyme (NADPH oxidase) in eosinophils depended on extracellular calcium, as observed in O2- production. However, the NADPH oxidase activity in the particulate fraction from A23187-stimulated eosinophils was only slightly affected by the addition of divalent cations or EDTA. The compound W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), a calmodulin antagonist, significantly inhibited O2- production by both cells. On the other hand, the compound H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a protein kinase C antagonist, was less effective on O2- production than was W-7. H-7 had little effect on O2- production of eosinophils. These findings suggest that NADPH oxidase might be activated by a smaller Ca2+ concentration through the calmodulin-dependent reaction. This was not observed with protein kinase C, at least in eosinophils.     

Differential effects of prostaglandins on macrophage activation induced by calcium ionophore A23187 or IFN-gamma.

Calcium ionophore A23187 can mimic IFN-gamma-induced macrophage activation for intracellular Leishmania killing and secretion of L-arginine-derived nitrite. Because the effects of ionophore are not restricted to calcium mobilization but also involve alterations of phospholipid metabolism, we have examined the role of PGE2 in the activation process. Macrophages exposed to A23187 or IFN-gamma in the presence of LPS and FCS secreted significant amounts of PGE2 independently of the presence of L-arginine in the incubation medium. The addition of the cyclooxygenase inhibitor indomethacin or omission of FCS abrogated PGE2 secretion but had little effect on nitrite production or intracellular killing. The addition of exogenous PGE2, of agents increasing PGE2 production such as arachidonic acid and colchicine, or of an analogue of cAMP, dibutyryl cAMP inhibited A23187 + LPS-induced activation whereas that mediated by IFN-gamma + LPS remained unimpaired. Our results indicate that PGE2 can modulate activation induced by A23187 but not by IFN-gamma, probably by a process involving cAMP. Conceivably, ionophore can mimic IFN-gamma for the induction of activation but lacks the capacity to help maintain the activated state because of its inability to desensitize macrophages to negative regulation by PGE2, as suggested previously for IFN-gamma-dependent activation.     

On the myoepithelium of human salivary glands. An immunocytochemical study

This study investigated the immunocytochemical characteristics of normal myoepithelial cells (MECs) of human major and minor salivary glands using the LSAB method. Other human exocrine glands were used as controls. Immunoreactivity of MECs was observed exclusively with fully differentiated smooth muscle antibodies (a-SMA; SMMS-1; CALP; hCD) and with epithelial markers (cytokeratins) Ck14 and Ck17. This epithelial-muscular immunophenotype was similarly expressed in the MECs of other human exocrine glands used as control. In the salivary MECs, we did not observe evidence for neuroectodermic phenotype (S-100 protein, GFAP, NSE). On the contrary, positivity was observed for S-100 protein in Mecs of control glands (mammary, bronchial and sweat glands). Immunoreaction for extracellular matrix markers (fibronectin, laminin and collagen IV) and vimentin always were negative. Our data show that in normal "non transformed" MECs of the salivary glands the smooth muscle phenotype is in a state of complete differentiation, while the epithelial phenotype expresses only Ck14 and Ck17. This double and simultaneous immunoreactivity represents a differential marker of MECs from the epithelial basal cell (EBCs).