Membrane transport of mitoxantrone by L1210 leukemia cells

Transport of radiolabeled mitoxantrone, a new antineoplastic agent, was studied using cultured mouse L1210 leukemia cells. The initial velocity of influx remained linear for about 90 sec and was 110 pmoles/10(6) cells measured at 60 sec. The steady-state accumulation of about 480 pmoles/10(6) cells was not reached until 30 min. The unidirectional drug influx was linear from 0 to 1000 microM extracellular drug concentration. The initial uptake was relatively temperature independent between 37 degrees and 27 degrees, but accumulation at steady state was 17% lower at 27 degrees. None of six metabolic inhibitors had an appreciable effect on initial uptake. Efflux was initially exponential with a half-life of 2.8 min; this efflux and the residual drug concentration plateau were not affected by KCN or verapamil. Under steady-state conditions, about 86% of the cell-associated label was contained in parent drug and the remainder in an unidentified metabolite. These studies indicate that the mechanism of mitoxantrone uptake is passive diffusion. The efflux is not energy requiring, but there is considerable tight binding of the drug to cellular structures.     

Stability of melphalan solutions during preparation and storage

A stability-indicating high-performance liquid chromatographic assay has been used to investigate the stability of solutions of melphalan under conditions that pertain to setting up in vitro chemosensitivity assays. Melphalan (1 and 20 micrograms/mL) was found to be 30% more stable in 150 mM NaCl solution (normal saline) than in Dulbecco's phosphate-buffered saline. The drug stored at 20 micrograms/mL in normal saline at room temperature (21.5 degrees C) lost 5% of its activity (t0.95) in 1.5 h; at 5 degrees C the t0.95 was 20 h. From an Arrhenius plot of the results, the activation energy of the hydrolysis reaction in normal saline was 100.2 kJ/mol. Less than 5% of drug activity was lost when solutions of melphalan were (a) stored at -20 degrees C and -35 degrees C for 7 months, (b) frozen and thawed up to four times, or (c) filtered through various membranes. Drug degradation was not affected by storage-container material. The half-life of melphalan in a Roswell Park Memorial Institute medium containing 10% fetal bovine serum at 37 degrees C was 1.13 +/- 0.10 h; the presence of 0.3% agar had no effect. It is suggested that melphalan be handled at temperatures greater than 5 degrees C for a minimum of time, but solutions of the drug can be stored for at least 6 and possibly up to 12 months at -20 degrees C without significant deterioration taking place.     

Anatagonism of the cytocidal activity and uptake of melphalan by tamoxifen in human breast cancer cells in vitro

The effect of the antiestrogen tamoxifen on the cytocidal activity and uptake of melphalan in human breast cancer cells was investigated. A clonogenic assay was used to obtain dose-survival curves of estrogen receptor-positive MCF-7 cells and of estrogen receptor-negative Evsa T cells following treatment with melphalan and/or tamoxifen. Isobolograms derived from these dose-survival curves were concave downward, suggesting that the drug interaction was antagonistic. The effect of tamoxifen on melphalan uptake by breast cancer cells was evaluated at steady-state conditions. Thin-layer chromatography revealed that the intracellular level of free intact melphalan (mean ± S.E.) in control cells was 6.47 ± 1.21 fmoles/cell and that in cells treated with tamoxifen was 3.60 ± 0.35 fmoles/cell; this 44% reduction in cellular melphalan was statistically significant (P = 0.006). Thus, the antagonistic cytocidal effect of melphalan and tamoxifen against breast cancer cells appeared to be due to inhibition of melphalan uptake at the steady state by the antiestrogen. Further investigation revealed that tamoxifen inhibited unidirectional melphalan influx in human breast cancer cells both by the sodium-independent system L and by the sodium-dependent system ASC. Tamoxifen also appeared to stimulate melphalan efflux from human breast cancer cells. The first-order rate constant K for melphalan efflux from control cells was 0.085 ± 0.008 and that from cells treated with tamoxifen was 0.129 ± 0.005; the difference was highly significant (P < 0.001). Therefore, the antagonistic effect of tamoxifen on the uptake and cytocidal activity of melphalan in breast cancer cells appeared to be due to inhibition of melphalan influx and stimulation of drug efflux.     

Melphalan resistance and photoaffinity labelling of P-glycoprotein in multidrug-resistant Chinese hamster ovary cells: reversal of resistance by cyclosporin A and hyperthermia.

The multidrug resistance phenotype is often associated with overexpression of P-glycoprotein, an energy-dependent efflux pump responsible for decreased intracellular accumulation of chemotherapeutic agents. The role of P-glycoprotein in the mechanism of cross-resistance to melphalan in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was investigated by photoaffinity labelling of P-glycoprotein using [3H]azidopine. We investigated whether the chemosensitiser cyclosporin A and hyperthermia, either used alone or combined, could reverse melphalan resistance and alter transport processes for [14C]melphalan in CH(R)C5 cells. Melphalan inhibited azidopine photolabelling of P-glycoprotein, implicating drug efflux mediated by P-glycoprotein in the mechanism of melphalan resistance in CH(R)C5 cells. Azidopine photolabelling also was inhibited by the chemosensitiser cyclosporin A, which binds to P-glycoprotein. Cyclosporin A alone reversed melphalan resistance in CH(R)C5 cells, but had no effect in drug-sensitive AuxB1 cells. Hyperthermia (40-45 degrees) alone increased melphalan cytotoxicity in both cell lines. When hyperthermia was combined with cyclosporin A, a large increase in melphalan cytotoxicity occurred, but only in CH(R)C5 cells. This effect increased with temperature and exposure time. Sensitisation to melphalan cytotoxicity by heat and cyclosporin A in CH(R)C5 cells appeared to be explained by altered drug transport processes. Lower accumulation of melphalan occurred in CH(R)C5 cells than in drug-sensitive cells. At 37 degrees, cyclosporin A increased drug accumulation in CH(R)C5 cells, but not in AuxB1 cells, by slowing drug efflux from cells. Heat alone increased both melphalan uptake and drug efflux for both cell lines. Our findings suggest that the combination of cyclosporin A and hyperthermia could be very useful in overcoming melphalan resistance by increasing intracellular drug accumulation in multidrug-resistant cells.     

Breath-synchronized plume-control inhaler for pulmonary delivery of fluticasone propionate

The objective of this study was to investigate the effects of experimental conditions for measuring the water vapor transmission rate (WVTR) of high-density polyethylene (HDPE) bottles using a steady-state sorption method. Bottles were filled with desiccant, closed with caps and heat induction sealed, and then stored in stability chambers at controlled temperature and relative humidity. Weight gain of the bottles was determined every 1 or 2 weeks until a linear weight gain profile was obtained. WVTR of the bottles was determined from the slope of the linear portion of the weight gain versus time profile. The effects of desiccants and temperature/humidity were studied. Results show that, with a sufficient amount of anhydrous calcium chloride in bottles, a negligibly low and sufficiently constant headspace humidity is maintained, and a steady-state permeation rate is achieved. For all 8 sizes of bottles used in this study, steady-state was achieved in 1 or 2 weeks after the experiment was started. This method provided reproducible WVTR data for HDPE bottles. Apparent moisture permeability of all 8 sizes of bottles was (2.3+/-0.3)x10(-7), (2.6+/-0.2)x10(-7), and (3.4+/-0.2)x10(-7)cm(2)/s at 25 degrees C, 30 degrees C, 40 degrees C, respectively. Moisture permeability determined from the current study was similar to data reported in the literature, indicating that the steady-state weight gain method can be used to obtain reliable WVTR of containers for pharmaceutical products.     

Taurine effects on the transition temperature in Arrhenius plots of ATP-dependent calcium ion uptake in rat retinal membrane preparations

The transition temperatures calculated from Arrhenius plots of ATP-dependent calcium ion uptake in rat retinal membrane preparations differed depending upon the presence or absence of exogenous taurine. At a constant calcium ion concentration of 10 microM and in the absence of taurine the transition temperature was 17.9 +/- 4.9 degrees, whereas in the presence of 20 mM taurine the transition temperature was raised to 25.4 +/- 0.8 degrees. Arrhenius plots of maximum velocities of calcium ion uptake (calculated from six calcium ion concentrations which varied from 5 to 300 microM) also demonstrated a taurine effect on the transition temperatures (13.5 degrees vs 25.5 degrees). In addition, taurine lowered the apparent activation energy for calcium ion uptake.     

Mechanistic aspects of uptake and sinusoidal efflux of dibromosulfophthalein in the isolated perfused rat liver.

Using the isolated perfused rat liver technique we examined whether the accumulation and sinusoidal efflux processes of the organic anion dibromosulfophthalein (DBSP) are dependent on the intracellular ATP content, chloride concentration in the perfusion medium as well as temperature of the medium and whether they are mediated by the same or by separate carrier mechanisms. The net sinusoidal efflux rate, being the resultant of sinusoidal efflux and re-uptake, was decreased more than 50% after lowering the medium temperature from 37 to 26 degrees indicating that the efflux process is carrier-mediated. The uptake rate was decreased only 18% after lowering the medium temperature to 26 degrees. Lowering of the hepatic ATP content for more than 80% clearly decreased the DBSP uptake rate but not the sinusoidal efflux rate. These observations indicate that these opposing transport steps probably are mediated by two separate carriers. Additional evidence for this hypothesis originated from the observation that sinusoidal efflux of DBSP was decreased about 30% whereas hepatic uptake of the substrate remained unaltered after replacing chloride in the perfusion medium with gluconate. In summary, we conclude that uptake and sinusoidal efflux of DBSP are mediated by two separate carrier systems that are influenced differently by ATP depletion, temperature lowering and presence of Cl-gradients.     

Validated HPLC method for determination of sennosides A and B in senna tablets

This study developed an efficient and reliable ion-pair liquid chromatographic method for quantitation of sennosides A and B in commercial senna tablets. Separation was conducted on a Hypersil C 18 column (250 x 4.6 mm, 5 microm) at a temperature of 40 degrees C, using a mixture of 0.1 M acetate buffer (pH 6.0) and acetonitrile (70:30, v/v) containing 5 mM tetrahexylammonium bromide as mobile phase. Sennosides A and B were completely separated from other constituents within 14 min. The developed method was validated. Both run-to-run repeatability (n=10) and day-to-day reproducibility (n=3) of peak area were below 0.4% RSD. Linearity of peak area was tested in the range 30-70 microg/ml (r>0.9997). Accuracy was assessed with recovery and the recoveries for sennosides A and B were 101.73+/-1.30% and 101.81+/-2.18% (n=3 x 6), respectively. Robustness of the analytical method was tested using a three-leveled Plackett-Burman design in which 11 factors were assessed with 23 experiments. Eight factors (column, concentration of ion pair reagent, % of organic modifier (acetonitrile), buffer pH, column temperature, flow rate, time constant and detection wavelength) were investigated in a specified range above and below the nominal method conditions. It was found that: (1) column and % acetonitrile affected significantly resolution and retention time, (2) column, % acetonitrile, column temperature, flow rate and time constant affected significantly the plate number of sennoside A, and (3) column and time constant affected significantly the tailing factor.     

Characterization of bradykinin B2 receptors on human IMR-90 lung fibroblasts: stimulation of 45Ca2+ efflux by D-Phe substituted bradykinin analogues.

[3H]Bradykinin binds to intact human IMR-90 fetal lung fibroblasts in a time and dose-dependent manner. Binding equilibrium was attained by 120 minutes at 4 degrees C. [3H]Bradykinin binding was saturable; Scatchard analysis of saturation binding data demonstrated a single binding site having a KD = 1.8 +/- 0.2 nM and a receptor concentration of 17.4 +/- 4.0 fmol/10(5) cells. The calculated value for KD(k-1/k1) from the association (k1 = 4.71 x 10(6) mol-1 min-1) and dissociation (k-1 = 1.13 x 10(-2) min-1) rate constants was 2.4 nM. The rank order of potency observed for bradykinin peptide agonists, bradykinin > Lys-bradykinin > Met,Lys-bradykinin > Ile,Ser-bradykinin > des-Arg9-bradykinin, is consistent with that of a bradykinin B2 receptor. Bradykinin stimulated efflux of 45Ca2+ from IMR-90 cells dose dependently with an EC50 = 331 +/- 50 pM. 45Ca2+ efflux was also demonstrated with Lys-bradykinin and Met-Lys-bradykinin but not by des-Arg10-kallidin (100 nM) or NKA (1 microM). Hoe-140 inhibited bradykinin-induced 45Ca2+ efflux (IC50 = 3 +/- 2 nM). D-Phe7-substituted bradykinin analogues stimulated 45Ca2+ efflux dose dependently and this stimulation of 45Ca2+ efflux was inhibited by Hoe-140. These results suggest that D-Phe7 substituted bradykinin analogues are agonists at the bradykinin B2 receptor in IMR-90 cells.     

Hydrolysis and protein binding of melphalan

Melphalan (30 microgram/ml) is completely hydrolyzed in water at 37 degrees after 8 hr. At lower temperatures, hydrolysis proceeds at slower rates. The presence of bovine serum albumin retards hydrolysis of melphalan (30 microgram/ml) in water. The melphalan hydrolysis rate is directely releated to the bovine serum albumin concentration. At 37 degrees, 8 g of bovine serum albumin/100 ml of water gives a recovery rate of melphalan similar to that of human plasma. In vitro alkylation of melphalan at 37 degrees with human plasma containing 30 microgram/ml, calculated by equilibrium dialysis, methanol extraction, and high-pressure liquid chromatographic analysis, is 30% after 8 hr.     

Augmentation of alpha1-adrenoceptor-mediated contraction by warming without increased phosphorylation of myosin in rat caudal arterial smooth muscle

We previously reported the relationship between alpha1-adrenoceptor-mediated contraction and phosphorylation of 20-kDa myosin light chain (LC20) in de-endothelialized rat caudal arterial smooth muscle at room temperature (Mita M, Walsh MP. Biochem J. 1997;327:669-674). We now describe the effect of increasing the temperature to 37 degrees C on this relationship. The EC50 value (76.6 +/- 18.2 nM) for cirazoline (alpha1-adrenergic agonist)-induced contraction of the strips at room temperature (23 degrees C) was significantly greater than that (14.5 +/- 1.9 nM) at 37 degrees C. The initial rate of the contraction to a sub-maximal concentration of cirazoline (0.3 microM) was similar at the two temperatures. However, cirazoline-induced maximal force at 37 degrees C was approximately 1.8 times that at room temperature. LC20 phosphorylation in response to cirazoline at room temperature and 37 degrees C closely matched the time courses of contraction, but values were not significantly different at the two temperatures: resting phosphorylation levels were 0.09 +/- 0.04 mol P(i)/mol LC20 at 37 degrees C and 0.22 +/- 0.06 mol P(i)/mol LC20 at room temperature; maximal cirazoline-stimulated LC20 phosphorylation levels were 0.58 +/- 0.08 mol P(i)/mol LC20 at room temperature and 0.49 +/- 0.05 mol P(i)/mol LC20 at 37 degrees C. We conclude, therefore, that the enhanced cirazoline-induced contraction at 37 degrees C is not due to increased LC20 phosphorylation.     

Relationship between the uptake, binding and pharmacological action of procaine in the isolated heart

1. The uptake, efflux and pharmacological actions of procaine hydrochloride were studied on isolated hearts of female guinea-pigs. The hearts were perfused with Krebs solution containing (14)C-procaine (0.1-500 mug/ml.) by the Langendorff technique at 37 degrees C, using a constant flow pump. Hearts and cardiac effluent were assayed for procaine by liquid scintillation spectrometry.2. Accumulation of procaine did not appear to involve active transport mechanisms for the following reasons. The rate of procaine uptake was most rapid at the highest perfusion concentration (500 mug/ml.), when it was three times faster than at the lowest concentration (0.1 mug/ml.); it was not affected by lowering the temperature to 3 degrees C. The ratio of the concentration of procaine in the heart to the concentration in the perfusing fluid decreased with increasing concentration in the perfusion fluid.3. When the efflux of (14)C-procaine from hearts previously perfused with procaine-containing Krebs solution for 10 min was compared with the efflux of (14)C-inulin, the patterns of efflux of both compounds were similar, and showed at least two exponential components. At the highest concentration of procaine (500 mug/ml.) the efflux of procaine was more rapid than that of inulin.4. A relationship was found between the pharmacological action of procaine, the rate of uptake and the level of procaine in the heart. When the procaine-containing perfusion fluid was changed to a procaine-free solution, the heart rate increased rapidly, and there was a rapid decline in the levels of procaine in the hearts.5. It is concluded that guinea-pig hearts accumulate procaine by a passive diffusion process, and that the pattern and rate of efflux indicate that the drug is loosely bound. If it is permissible to extrapolate from these findings to the antiarrhythmic effect in man, the short duration of its action may be due to loose binding rather than to rapid metabolic inactivation.     

Kinetic analysis about the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) by isolated rat hepatocytes

The purpose of the present study was to investigate the bidirectional transport of 1-anilino-8-naphthalene sulfonate (ANS) using isolated rat hepatocytes. The initial uptake rate of ANS by isolated hepatocytes was determined. The uptake process of ANS was saturable, with a Km of 29.1 +/- 3.2 microM and Vmax of 2.9 +/- 0.1 mmol/min/mg protein. Subsequently, the initial efflux rate of ANS from isolated hepatocytes was determined by resuspending preloaded cells to 3.0% (w/v) BSA buffer. The efflux process for total ANS revealed a little saturability. The mean value of the efflux clearance was 2.2 +/- 0.1 microL/min/mg protein. The efflux rate of ANS from hepatocytes was markedly decreased at 4 degrees C, indicating that the apparent efflux of ANS might not be attributed to the release of ANS bound to the cell surface, but to the efflux of ANS from intracellular space. The efflux clearance was furthermore corrected for the unbound intracellular ANS concentration on the basis of its binding parameters to cytosol. The relation between efflux rate and unbound ANS concentration was fitted well to the Michaelis-Menten equation with a saturable and a nonsaturable components. The Vmax and Km values were 0.54 mmol/min/mg protein, and 10.0 microM, respectively. Based on the comparison of the ratios of Vmax to Km (Vmax/Km) corresponding to the transport clearance, the influx clearance was two times higher than the efflux clearance. Together with our preliminary studies that ATP suppression in hepatocytes substantially inhibited ANS influx rate, we concluded that the hepatic uptake of ANS is actively taken up into hepatocytes via the carrier mediated transport system.     

Irreversible inhibition of epithelial sodium channels by ultraviolet irradiation.

1 The effects of u.v. irradiation at 254 nm and 350 nm on sodium transport across frog skin epithelium have been investigated. 2 Irradiation at 254 nm but not at 350 nm produces a dose-dependent, functionally selective blockade of sodium transport. The effect is apparently due to the irreversible closure of apical sodium channels. 3 The amiloride-sensitive conductance was directly related to sodium transport as measured by short circuit current (SCC) both in normal and irradiated tissues, although both conductance and current were reduced in irradiated tissues. 4 The sensitivity of epithelia to irradiation at 254 nm was defined from the rate constants for the decline in SCC during three 2 min periods of irradiation at 1850 microW cm-2. The rate constant for the initial 2 min irradiation was 0.093 +/- 0.008 min-1. 5 Lowering the sodium concentration to 5.5 mM from 110 mM increased the rate constant to 0.141 +/- 0.014 min-1, consistent with the view that more functional sodium channels exist at lowered sodium concentration. 6 Lowering the temperature to 7 degrees C from 23 degrees C reduced the rate constant to 0.032 +/- 0.007 min-1 suggesting that blockade of channels is not due to a direct interaction with photons. 7 Using a variety of experimental protocols we were unable to demonstrate that bromamiloride or iodoamiloride can act as photoligands for sodium channels in the epithelium of Rana temporaria. This is in contrast to earlier reports with other epithelia.     

Receptor-mediated stimulation of taurocholate efflux from the rat hepatocyte and the ex vivo perfused rat liver

The peptide hormone, arginine-vasopressin[( Arg8]vasopressin, AVP), stimulates efflux of the bile salts taurocholate and glycocholate from the rat hepatocyte in suspension via its association with the V1 receptor on the hepatic cell membrane. At a concentration ratio of 5:1 (antagonist to hormone), the V1 vasopressin antagonist, (dCH2)5Tyr(Me)AVP, inhibits the vasopressin induced efflux of taurocholate by approximately 82%, and of glycocholate, by approximately 85%. In contrast, the V2 antagonist (d(CH2)5[D-Ile2,Ala4]AVP, does not interfere with the stimulation of taurocholate and glycocholate efflux by vasopressin. In the isolated perfused rat liver, vasopressin (5 X 10(-10) M) causes an immediate increase of 55 +/- 12% over baseline in [14C]taurocholate secretion and a corresponding increase in bile flow. A more gradual and prolonged increase in [14C]taurocholate secretion, reflecting an increased biliary concentration of [14C]taurocholate, is observed beginning 6 min after vasopressin, reaching a plateau of 23 +/- 12% over baseline by 14 min and returning to baseline by 30 min. The mean rate of 14C secretion during the 30 min following administration of vasopressin (non-steady state) is increased by 14.3 +/- 6.4% over pre-infusion steady-state baseline (P less than 0.05). Prior administration of the V1 receptor antagonist d(CH2)5Tyr(Me)AVP attenuates these effects of vasopressin. The combination of these in vitro and in vivo findings suggest that vasopressin may play a role in regulating bile salt efflux. Furthermore, these studies in the isolated hepatocyte and the intact liver may provide a unique approach for defining biochemical changes associated with bile salt transport from the hepatic cell.     

Inhibition by fendiline of the transient outward current in rat ventricular cardiomyocytes.

The effects of fendiline on the transient outward current (Ito) were investigated in rat ventricular cardiomyocytes. Extracellularly applied fendiline reduced peak and steady-state current amplitude of Ito; the inactivation of Ito was accelerated by the drug, which reflects onset of block. The described effects were concentration dependent: half-maximal effects were achieved at approximately 3 microM fendiline. Intracellularly applied fendiline (3 microM) did not affect Ito within 5 min. The steady-state current amplitude of Ito was more efficiently suppressed by the drug at 22 +/- 1 degrees C than at 36 +/- 1 degrees C. The recovery of Ito was analyzed by the application of twin depolarizing voltage pulses, interrupted by variable pulse intervals. In the presence of fendiline, recovery of Ito was about twofold slower than that under control conditions, independent of the drug concentration used, which reflects offset from block. Concentration-dependent onset but concentration-independent offset of block suggest that the described time constants correspond to voltage-dependent net binding and unbinding, respectively, of fendiline at its receptor sites. It is proposed that fendiline binds extracellularly at positive potentials to Ito channels in their open state and dissociates from the channels at rest.     

Membrane transport,sulfhydryl levels and DNA cross-linking in Chinese hamster ovary cell mutants sensitive and resistant to melphalan

The mechanism of resistance to the alkylating agent melphalan was investigated in drug-sensitive and -resistant mutants of Chinese hamster ovary cells. Melphalan-resistant cells (Mel^R6), selected by a single exposure to melphalan, were 4.5-fold more resistant to drug than sensitive AUXB1 parental cells. Colchicine-resistant cells (CH^RC5), which are cross-resistant to melphalan, were 15-fold more resistant than wild type cells. The kinetic parameters for drug influx were not significantly different in sensitive and resistant cells. The steady-state drug level in both Mel^R6 and CH^RC5 cells was approximately 25 and 35% lower respectively than that in sensitive cells and this difference was accounted for by a more rapid rate of drug efflux from the resistant mutants. However, the level of drug resistance could not be explained entirely by this difference in drug transport.Sulfyddryl group levels were elevated in both Mel^R6 and CH^RC5 cells relative to sensitive cells and these differences were statistically significant (P < 0.001). Furthermore, DNA interstrand cross-link formation was significantly lower in resistant cells than in sensitive cells. A similar rate of repair of DNA interstrand cross-links was observed in sensitive and resistant cells with the possible exception of a slower rate of repair in Mel^R6 cells. A higher level of DNA-protein cross-link activity, which may represent a mechanism for drug inactivation was observed in Mel^R6 cells.These studies suggest that resistance to melphalan in Mel^R6 and CH^RC5 Chinese hamster ovary cell mutants is multifactorial involving lowered steady-state drug levels, enhanced drug efflux, elevated levels of sulfhydryl groups and decreased DNA interstrand cross-linking.     

Demonstration of a [K+,Cl-]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoic acids: regulation of cell swelling and distinction from the bumetanide-sensitive [Na+,K+,Cl-]-cotransport system

A screening of several families of compounds on NEM-stimulated K+ efflux in human red cells allowed us to select a [(dihydroindenyl)oxy] alkanoic acid (DIOA) as the first potent inhibitor of this K+ flux (IC50 of 10(-5) M) without side effects on the bumetanide-sensitive [Na+,K+,Cl-]-cotransport system. Incubation of human red cells in hypotonic media (179 mosm) increased cell volume (by 18-20%) and provoked the appearance of a DIOA-sensitive K+ efflux of 4.48 +/- 0.83 mmol.(liter of cells X hr)-1 (mean +/- SD of nine experiments). This DIOA-sensitive K+ efflux exhibited a Michaelian-like dependence on the Cl- concentration of the incubation media (freely equilibrated with intracellular Cl-) with an apparent dissociation constant of 39.6 +/- 14.7 mM and a maximal rate of 4.7 +/- 0.9 mmol.(liter of cells X hr)-1 (mean +/- SD of five experiments). The chloride effect was mediated by intracellular and not by extracellular Cl-, as expected for an outward [K+,Cl-]-cotransport. The above properties of DIOA-sensitive K+ efflux clearly confirm that human red cells have a [K+,Cl-]-cotransport system that regulates cell swelling. The regulatory response to hypotonic media was also strongly depressed by cytochalasin B at a concentration of 1 mM, suggesting that the activating signal is probably transduced by the cytoskeleton.     

Effects of the anti-platelet agent cilostazol on peripheral vascular disease in patients with diabetes mellitus.

Effects of cilostazol (OPC-13013, CAS 73963-72-1), a selective inhibitor of platelet cAMP-phosphodiesterase, on peripheral vascular disease in diabetes mellitus were studied. Cilostazol in a dose of 200 to 300 mg/d was administered to 5 diabetic patients with arteriosclerosis obliterans. Skin temperature of the finger and the toe, which reflects blood flow to the tissue, was selected as an objective index of cilostazol effects and measured by infra-red thermography at a constant temperature of 26 degrees C. Before administration, digital skin temperatures were low in 9 limbs of 5 patients. 200 mg/d of cilostazol significantly (p less than 0.001) increased the digital skin temperatures of 8 limbs, the increase (mean +/- SD) ranging from 29.9 +/- 1.4 degrees C to 33.2 degrees C +/- 1.2 degrees C for the average skin temperatures and from 28.7 +/- 2.1 degrees C to 33.1 +/- 1.5 degrees C for the lowest ones. An increase in the dose to 300 mg/d resulted in further elevation of skin temperatures of the digits. Cilostazol constantly elicited an increase in blood flow to the digits within the range of its therapeutic dose. This effect was observed about 1 month after initiation of administration and persisted while administration was continued. The measurement of digital skin temperatures by infrared thermography provided a noninvasive means to individualize the dosage of cilostazol and to monitor the cilostazol effect and patient complicance during long-term administration. It is concluded that cilostazol exerts a potent and steady vasodilatory effect on peripheral circulation in patients with diabetes mellitus.     

Effect of hypothermia on the intestinal absorption of uracil and L-dopa in the rat

The in situ rat gut technique was used to explore the effects of hypothermia on the intestinal absorption of L-dopa and uracil. A hypothermic state was induced when male Sprague-Dawley rats, weighing between 300 and 370 g, were exposed to an atmosphere of helox (helium:oxygen, 80:20) at 0-4 degrees C. After 4 to 5 h, the rectal temperatures were decreased from 37 to 20 degrees C. The rewarming process for hypothermic animals undergoing anesthesia appears to be prolonged. The animals were prepared for surgery using ether as anesthetic after a rectal temperature of 20 degrees C was attained. Hypothermia showed a significant influence on decreasing L-dopa and uracil disappearances from the intestinal lumen. About 40 percent reduction of the rates of disappearance was observed with a 10 degrees C reduction of the rectal temperature. Additionally, a rapid drop of the rate of drug disappearance was observed in the beginning stage of rectal temperature decrease (31-36 degrees C) as well as in the hypothermic state with rectal temperatures < 26 degrees C. There appears to be a thermal stable region for absorption between 26 and 30 degrees C. Water efflux was studied in both normothermic and hypothermic animals. Normothermic rats showed greater average cumulative water efflux over a period of 30 min (71 and 61% for L-dopa and uracil, respectively) in comparison with that of hypothermic rats (34 and 41% for L-dopa and uracil, respectively). The cold-treated animals showed decreased rates of disappearance associated with the decreased water efflux. Therefore, the reduction of the rates of drug disappearance from the intestinal lumen caused by hypothermia may be partially related to the decrease of water efflux during hypothermia.