Characterization of the histamine releasing effect of neurotensin in the rat heart

Bolus injections of neurotensin (NT) in the rat perfused heart elicited a transient, dose-dependent histamine release. The histamine releasing effect of NT appears to be independent of the heart rate and coronary perfusion pressure and it was not influenced by atropine, propanolol, prazosin, methysergide, ketanserin, indomethacin, morphine, lidocaine or by removal of the atria. However, it was potentiated by adenosine, inhibited by sub-stimulatory concentrations of NT and the mast cell membrane stabilizing drug cromoglycate but was unaltered by the calcium antagonist verapamil. The absence of calcium in the heart perfusate suppressed the histamine releasing effect of NT. These results suggest that the histamine releasing effect of NT in the rat heart results from a direct effect on ventricular mast cells and is calcium-dependent.     

Effect of Some Calcium Antagonists on Muscarinic Receptor-Mediated Cyclic GMP Formation

Several calcium antagonists were screened for their effect on muscarinic acetylcholine receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Mn2+, Ni2+, and verapamil rapidly antagonized the response noncompetitively, with the following order of potency: verapamil greater than Mn2+ greater than Ni2+. The effects of Mn2+ and Ni2+, but not those of verapamil, were largely reversed by increasing extracellular calcium concentration. Additional effects of these agents included displacement of [3H]quinuclidinyl benzilate binding by verapamil and elevation of cyclic GMP levels by Mn2+ and Ni2+ in the absence of agonists. These results are in support of the hypothesis that receptor-mediated cyclic GMP formation by these cells is dependent upon entry of calcium into the cell and demonstrate the complexity of the effects of calcium antagonists.     

Functional Expression of P-Glycoprotein in an Immortalised Cell Line of Rat Brain Endothelial Cells, RBE4

Abstract: The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [³H]colchicine and [³H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt¹]-[Val²]-cyclosporin) at 50 or 100 µ M concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive,Ca2+-activated K+ conductance

In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^– symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl^– backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl^– backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (V _m) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10^–4 m HCTZ. It was significantly enhanced by reducing luminal Cl^– concentration to 30 mm; it was abolished by SCN^–, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl^– and (probably) Ca²⁺ conductances and, indirectly, a Ca²⁺-sensitive, apamin inhibitable K⁺ conductance: since the intracellular Cl^– activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl^– conductance depolarizes V _mand enhances Cl^– backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca²⁺ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl^– conductance opened by HCTZ has been estimated and the Cl^– backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl^– leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.This research was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome, Italy. We are very grateful to prof. G. Meyer and dr. G. Bottà for helpful discussion and criticism.     

Toxicologic and pharmacokinetic study of low doses of verapamil combined with doxorubicin

The effect of a chronic treatment with low oral doses of verapamil, a calcium channel blocker commonly employed in cardiovascular therapy, on doxorubicin toxicity, was evaluated in CD1 mice. Verapamil, administered at a dosage corresponding to a typical cardiovascular posology in humans, significantly increased doxorubicin toxicity. In particular the mortality was significantly higher and earlier and histological analysis revealed an increase in the severity of lesions in the liver, kidney and small bowel of verapamil pretreated animals. The pharmacokinetic profiles revealed that verapamil treated group had higher doxorubicin peak plasma and tissue levels and AUCs.This study shows that verapamil, administered at low doses, dramatically increases doxorubicin toxicity, probably through an interaction between the two drugs, both P-glycoprotein substrates, on the protein expressed in normal tissues, and suggests caution in the use of the calcium channel blocker for cardiovascular pathologies in patients who have to be treated with antineoplastic agents, substrates of P-glycoprotein.     

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(−)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 – 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(−)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2–6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(−)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5–6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.     

Effect of temperature on the expression of P-glycoprotein in the zebra mussel, Dreissena polymorpha

The multixenobiotic resistance (MXR) system is a defence mechanism which protects living organisms by actively extruding xenobiotics from cells. Membrane transporters responsible for this phenotype can be induced by numerous compounds, but also by physical stressors such as temperature. In order to better understand the contribution of temperature in MXR expression, the MXR system was studied in the freshwater bivalve, Dreissena polymorpha. Results showed that the zebra mussel possesses a gene closely related to the ATP-binding cassette (ABC) subfamily B, which is expressed as a 120-kDa protein. Moreover, gill cells can transport the model substrate Rhodamine B in a verapamil-sensitive manner, which is a good indication of MXR protein activity such as P-glycoprotein (Pgp). When bivalves were slowly acclimated to temperatures ranging from 4 to 20 °C (maximum temperature variation, 2–3 °C/day), no significant change in MXR protein expression could be immunochemically measured. Furthermore, the MXR transport activity did not show significant variation in this temperature range. Conversely, incubation of zebra mussel specimens in 20 μM Dacthal dramatically enhanced both protein expression and transport activity, thus indicating that temperature is not a major determinant in zebra mussel gill Pgp expression.     

Fibronectin and laminin enhance engraftibility of cultured hematopoietic stem cells

To test the hypothesis that extracellular matrix (ECM) components maintain stem cell property, murine bone marrow (BM) cells were expanded in fibronectin and laminin coated plate in the presence of cytokines. We observed significant phenotypic and functional improvement of expanded cells. In 10 days, 800-fold expansion of colony-forming unit-granulocyte erythrocyte monocyte megakaryocyte (CFU-GEMM) was observed in the cultured cells. No apparent activation of cell cycle was observed, but CD29 and very late antigen-4 (VLA-4) expression was increased, as compared to the normal BM cells. A fraction of the expanded cells became verapamil sensitive, suggesting upregulation of multi-drug resistant gene(s), as found in the primitive hematopoietic stem cells (HSCs). Competitive repopulation assay confirmed that HSCs compartment was amplified during culture. Overall, our study clearly demonstrated that ex vivo culture of murine HSCs in the presence of fibronectin and laminin resulted in expansion of primitive stem cells and improvement in the marrow engraftibility.     

Verapamil, a Ca channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli

Verapamil is used clinically as a Ca²⁺ channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia. Here we study the effect of verapamil on the bacterium Escherichia coli. The drug was shown to inhibit cell division at growth sub inhibitory concentrations, independently of the SOS response. We show verapamil is a membrane active drug, with similar effects to dibucaine, a local anesthetic. Thus, both verapamil and dibucaine abolish the proton motive force and decrease the intracellular ATP concentration. This is accompanied by induction of degP expression, as a result of the activation of the RpoE (SigmaE) extra-cytoplasmic stress response, and activation of the psp operon. Such effects of verapamil, as a membrane active compound, could explain its general toxicity in eukaryotic cells.