Substance P induces histamine release from human pulmonary mast cells.

Substance P elicits histamine release from human skin and rodent mast cells. Since neuropeptide-mediated reflexes may be important in asthma, we examined the ability of substance P to stimulate human mast cells obtained at bronchoalveolar lavage (BAL). BAL samples were obtained at routine bronchoscopy from 35 non-preselected patients. Histamine release experiments were performed in a standard manner using substance P and the calcium ionophore A23187. Both substance P (50 μM) and A23187 caused histamine release (median 26.7%, range 6.2–62.8% and 32.1%, 7.7–56.8% respectively) which was significantly greater (P < 0.0001) than the spontaneous release (median 15.6%, range 4.1–33.4%), i.e. that in the absence of any stimulus. Substance P induced histamine release was via an energy dependent process and was blocked by preincubation with antimycin A. A significant correlation was observed between substance P induced release and spontaneous release but was not observed with A23187 induced release. Mast cell counts correlated significantly with substance P induced release but not with spontaneous or A23187 induced release. The substance P induced histamine secretion was elicited at similar concentrations to those used with rodent and human skin mast cells. Asthma is associated with increased numbers of mast cells which have both increased spontaneous and stimulated secretory responses. Thus, in vivo, the bronchoconstrictor action of substance P may in part result from activation of mast cells in the bronchial lumen.

MODULATORY ACTION OF HELICOBACTER-PYLORI ON HISTAMINE-RELEASE FROM MAST-CELLS AND BASOPHILS IN-VITRO

Helicobacter pylori is important in the aetiology of peptic ulceration. Despite inducing an inflammatory response in the mucosa, the organism persists, suggesting that it has efficient protective mechanisms. Some bacterial and viral products modulate histamine secretion from inflammatory cells. Therefore, this study examined the modulatory effects of H. pylori preparations on histamine release from rat peritoneal mast cells and human basophils. Eleven clinical isolates of H. pylori were prepared in different ways: as whole washed bacteria, washed sonicated bacteria, and formalin-killed bacteria, and as outer-membrane and lipopolysaccharide (LPS) extracts. Histamine release from mast cells or basophils was not elicited by any of these bacterial preparations alone. However, when mixed with various secretory stimulants, the bacterial preparations caused inhibition of histamine release from rat mast cells (calcium ionophore A23187, compound 48/80, concanavalin A, anti-rat IgE) and human basophils (A23187, N-formyl Met-Leu-Phe). The degree of inhibition ranged from 48 % to 97 %. These results indicate that H. pylori exerts an inhibitory effect on cells of the immune system that contributes to its persistence within the gastric mucosa.

Calcium oscillations in interstitial cells of the rabbit urethra.

Measurements were made (using fast confocal microscopy) of intracellular Ca2+ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca2+ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with D-myo-inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release using 100 microm 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca2+, and frequency was directly proportional to the external Ca2+ concentration. Frequency of Ca2+ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca2+ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca2+ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca2+ concentration and that conversion of the primary oscillation to a propagated Ca2+ wave depends upon IP3-induced Ca2+ release.

Laser-engineered dissolving microneedles for active transdermal delivery of nadroparin calcium

There is an urgent need to replace the injection currently used for low molecular weight heparin (LMWH) multidose therapy with a non- or minimally invasive delivery approach. In this study, laser-engineered dissolving microneedle (DMN) arrays fabricated from aqueous blends of 15% w/w poly(methylvinylether-co-maleic anhydride) were used for the first time in active transdermal delivery of the LMWH nadroparin calcium (NC). Importantly, an array loading of 630 IU of NC was achieved without compromising the array mechanical strength or drug bioactivity. Application of NC-DMNs to dermatomed human skin (DHS) using the single-step 'poke and release' approach allowed permeation of approximately 10.6% of the total NC load over a 48-h study period. The cumulative amount of NC that permeated DHS at 24 h and 48 h attained 12.28 ± 4.23 IU/cm and 164.84 ± 8.47 IU/cm , respectively. Skin permeation of NC could be modulated by controlling the DMN array variables, such as MN length and array density as well as application force to meet various clinical requirements including adjustment for body mass and renal function. NC-loaded DMN offers great potential as a relatively low-cost functional delivery system for enhanced transdermal delivery of LMWH and other macromolecules. © 2012 Elsevier B.V. All rights reserved.

Reduced phospholipase A2 activity is not accompanied by reduced arachidonic acid release

Arachidonic acid release in cells highly over expressing cytosolic phospholipase A2 has been attributed to mitogen-activated protein kinase phosphorylation of cytosolic phospholipase A2 on serine-505. To investigate the role of cytosolic phospholipase A2 in cellular physiology, we attempted to inhibit cytosolic phospholipase A2 in the intact cell employing an antisense RNA strategy. Swiss 3T3 cells were stably transfected with an antisense cytosolic phospholipase A2 expression vector. A clone of cells with reduced immunodetectable cytosolic phospholipase A2, compared to a vector transfected cell line, was identified by Western blotting and a corresponding decrease in phospholipase A2 activity was confirmed by enzymatic assay in cell free extracts. However, arachidonic acid release from intact cells in response to agonists was not different between antisense and control cell lines. Thus, arachidonic acid release in intact cells with decreased cytosolic phospholipase A2 activity is likely to be modulated by rate limiting factors that are extrinsic to cytosolic phospholipase A2.

Release of β-galactosidase from poloxamine/α-cyclodextrin hydrogels

All mammals lose their ability to produce lactase (β-galactosidase), the enzyme that cleaves lactose into galactose and glucose, after weaning. The prevalence of lactase deficiency (LD) spans from 2 to 15% among northern Europeans, to nearly 100% among Asians. Following lactose consumption, people with LD often experience gastrointestinal symptoms such as abdominal pain, bowel distension, cramps and flatulence, or even systemic problems such as headache, loss of concentration and muscle pain. These symptoms vary depending on the amount of lactose ingested, type of food and degree of intolerance. Although those affected can avoid the uptake of dairy products, in doing so, they lose a readily available source of calcium and protein. In this work, gels obtained by complexation of Tetronic 90R4 with α-cyclodextrin loaded with β-galactosidase are proposed as a way to administer the enzyme immediately before or with the lactose-containing meal. Both molecules are biocompatible, can form gels in situ, and show sustained erosion kinetics in aqueous media. The complex was characterized by FTIR that evidenced an inclusion complex between the polyethylene oxide block and α-cyclodextrin. The release profiles of β-galactosidase from two different matrices (gels and tablets) of the in situ hydrogels have been obtained. The influence of the percentage of Tetronic in media of different pH was evaluated. No differences were observed regarding the release rate from the gel matrices at pH 6 (t50 = 105 min). However, in the case of the tablets, the kinetics were faster and they released a greater amount of 90R4 (25%, t50 = 40–50 min). Also, the amount of enzyme released was higher for mixtures with 25% Tetronic. Using suitable mathematical models, the corresponding kinetic parameters have been calculated. In all cases, the release data fit quite well to the Peppas–Sahlin model equation, indicating that the release of β-galactosidase is governed by a combination of diffusion and erosion processes. It has been observed that the diffusion mechanism prevails over erosion during the first 50 minutes, followed by continued release of the enzyme due to the disintegration of the matrix.

Effects of menthol on rat tail artery mediated by TRPM8 and voltage-gated calcium channels

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold sensing role in sensory neurons, it is expressed and functional in several non-neuronal tissues, including vasculature. We previously demonstrated that menthol causes vasoconstriction and vasodilatation in isolated arteries, depending on vascular tone. Here we investigated calcium's role in responses mediated by TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology and ratiometric Ca2+ recording. Isometric contraction studies examined actions of TRPM8 ligands in the presence/absence of L-type calcium channel blocker. Menthol (300 μM), a concentration typically used to induce TRPM8 currents, strongly inhibited L-type voltage-dependent Ca2+ current (L-ICa) in myocytes, especially it's sustained component, most relevant for depolarisation-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol. Menthol-induced increases in PE-induced vasoconstrictions were mediated predominantly by Ca2+-release from sarcoplasmic reticulum, since they were significantly inhibited by cyclopiazonic acid. Pre-incubation of vascular rings with a TRPM8 antagonist strongly inhibited menthol-induced increases in PE-induced vasoconstrictions, thus confirming specific role of TRPM8. Finally, two other common TRPM8 agonists, WS-12 and icilin, inhibited L-ICa. Thus, TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels, and largely obscure TRPM8-mediated vasoconstriction.

The influence of transforming growth factor-beta1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporin A and a calcium channel blocker

The purpose of this study was to determine whether the prevalence and severity of gingival overgrowth in renal transplant recipients concomitantly treated with cyclosporin and a calcium channel blocker was associated with functional polymorphisms within the signal sequence of the transforming growth factor-(TGF)beta1 gene.

Controlled release of a model antibacterial drug from a novel self-lubricating silicone biomaterial

Abstract There is considerable interest in developing medical devices that provide controlled delivery of biologically active agents, for example, to reduce the incidence of device-related infection. Silicone elastomers are one of the commonest biomaterials used in medical device production. However, they have a relatively high coefficient of friction and the resulting lack of lubricity can cause pain and tissue damage on device insertion and removal. Novel silicone cross-linking agents have recently been reported that produce inherently ‘self-lubricating’ silicone elastomers with very low coefficients of friction. In this study, the model antibacterial drug metronidazole has been incorporated into these self-lubricating silicone elastomers to produce a novel bioactive biomaterial. The in vitro release characteristics of the bioactive component were evaluated as a function of cross-linker composition and drug loading. Although conventional matrix-type release kinetics were observed for metronidazole from the silicone systems, it was also observed that increasing the concentration of the cross-linking agent responsible for the lubricious character (tetra(oleyloxy)silane) relative to that of the standard non-lubricious cross-linking agent (tetrapropoxysilane) produced an increase in the metronidazole flux rate by up to 65% for a specified drug loading. The results highlight the potential for developing lubricious silicone medical devices with enhanced drug release characteristics.

Release kinetics of oleyl alcohol from a self-lubricating silicone biomaterial

The development of self-lubricating silicone elastomeric biomaterials, prepared using the novel crosslinking agent tetra( oleyloxy) silane and having very low coefficients of friction, has recently been reported. In this study, the in vitro release characteristics of lubricious oleyl alcohol produced during the silicone curing reaction have been quantitatively evaluated for a range of tetra( propoxy) silane/tetra(oleyloxy) silane crosslinker compositions using a novel evaporative light scattering detection method in combination with high performance liquid chromatography. The mechanism of oleyl alcohol release was seen to deviate from a simple, matrix-controlled diffusion process and instead obeyed an anomalous transport mechanism. An explanation for the observed release behaviour has been proposed based on competitive reaction kinetics between the tetra( oleyloxy) silane and tetra( propoxy) silane substituents of the silicone crosslinking agents.

In vitro release of nonoxynol-9 from silicone matrix intravaginal rings

The controlled-release characteristics of matrix silicone intravaginal rings loaded with between 100 and 971 mg of nonoxynol-9 have been investigated with a view to developing a ring that may offer a new female-controlled method for the prevention of transmission of sexually transmitted diseases, particularly HIV. Intravaginal rings containing 253, 487 and 971 mg of nonoxynol-9 provided a daily release of 2 mg or more over the 8-day release period, the minimal amount of nonoxynol-9 considered to provide an effective vaginal concentration for the prevention of HIV. Furthermore, the maximum daily release of N9 was about 6 mg, an amount significantly smaller than that observed for other nonoxynol-9 products whose large daily doses may in fact increase the occurrence of HIV by causing epithelial damage to the vaginal tissue. The release mechanism of the liquid nonoxynol-9 from the intravaginal rings has also been investigated and compared to models describing the release of solid drugs from the rings. It has been demonstrated through release studies and surface microscopy that a drug depletion zone is not established in such liquid-loaded intravaginal ring systems, with implications for the release kinetics. (C) 2003 Elsevier B.V. All rights reserved.

Influence of silicone elastomer solubility and diffusivity on the in vitro release of drugs from intravaginal rings

The in vitro release characteristics of eight low-molecular-weight drugs (clindamycin, 17beta-estradiol, 17beta-estradiol-3-acetate, 17beta-estradiol diacetate, metronidazole, norethisterone, norethisterone acetate and oxybutynin) from silicone matrixtype intravaginal rings of various drug loadings have been evaluated under sink conditions. Through modelling of the release data using the Higuchi equation, and determination of the silicone solubility of the drugs, the apparent silicone elastomer diffusion coefficients of the drugs have been calculated. Furthermore, in an attempt to develop a quantitative model for predicting release rates of new drug substances from these vaginal ring devices, it has been observed that linear relationships exist between the log of the silicone solubility of the drug (mg ml(-1)) and the reciprocal of its melting point (K-1) (y = 3.558x - 9.620, R = 0.77), and also between the log of the diffusion coefficient (cm(2) s(-1)) and the molecular weight of the drug molecule (g mol(-1)) (y = - 0.0068x - 4.0738, R = 0.95). Given that the silicone solubility and silicone diffusion coefficient are the major parameters influencing the permeation of drugs through silicone elastomers, it is now possible to predict through use of the appropriate mathematical equations both matrix-type and reservoir-type intravaginal ring release rates simply from a knowledge of drug melting temperature and molecular weight. (C) 2003 Elsevier Science B.V. All rights reserved.