Comparison of the protein content of three different bovine secretory granule membrane types: a search for exocytosis-specific shared proteins.

A two-dimensional polyacrylamide gel analysis of three types of bovine exocytotic granule membranes has been undertaken. Great care was taken to purify the membranes of biochemical homogeneity with minimal contamination from other membrane sources. The goal was to identify proteins that were present in all three membrane types. Although a number of minor components were observed that co-migrated for two membrane types, no proteins were detected that were present in all three granule membranes. We therefore conclude that such exocytosis-specific proteins do not exist or that they represent less than 0.1% of the total membrane protein present in a given isolated membrane preparation.

Expansion of the inner membrane compartment and its relation to mitochondrial volume and ion transport.

Glutaraldehyde has been used to fix mitochondria undergoing rapid volume changes associated with energized ion transport under oscillatory state conditions and valinomycin-induced potassium uptake. Fixation was found to prevent structural changes which normally occur during ion accumulation or loss. By correlating packed volume measurements with electron microscopy, it is shown that changes in volume associated with ion movements reflect changes in the inner membrane compartment and that this compartment can be related to the sucrose inaccessible space. The method can therefore be used to accurately determine volume changes that arise from ion translocation.

Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.

Structural requirements of simple organic cations for recognition by multidrug-resistant cells.

We previously noted that a wide variety of drugs which are recognized by multidrug-resistant cells (MDR+) are positively charged. However, it remains unclear why and how such a large number of structurally different compounds can be distinguished by MDR+ cells. The majority of the diverse compounds subject to MDR are complex and thereby complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism. Using a series of simple aromatic (alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations differing in their lipophilicity by stepwise additions of single alkyl carbons, we demonstrate by growth inhibition studies that a single aromatic moiety and a critical degree of lipophilicity (log P > -1) are required for recognition of these simple organic cations by MDR+ cells. Thus, MDR+ cells are not cross-resistant to the nonaromatic guanidiniums but do show cross-resistance to those aromatic pyridiniums with chain lengths > four. Resistance ratios, as determined by comparison of 50% inhibitory doses in MDR- versus MDR+ cells, increase as a function of increasing chain lengths of these latter simple aromatic compounds. Resistance to pyridinium analogues in MDR+ cells is reversible by co-treatment with nontoxic doses of verapamil. Preliminary uptake data with radioactive analogues further implicate the MDR mechanism of lowered drug accumulation in accounting for resistance to the pyridinium homologues. Utilization of these simple organic cations provides a rational basis for better defining the physical chemical properties of more complex compounds processed by the MDR mechanism and suggests a strategy for designing chemotherapeutic agents with reduced susceptibility to MDR.

Subcellular fractions of the adrenal medulla. Comparison by two-dimensional polyacrylamide gel electrophoresis.

Subfractions of adrenal medullary homogenates were analyzed in two-dimensional polyacrylamide gels to assess the extent of protein homology. Chromaffin granule proteins were highly acidic, with the exception of the soluble form of the enzyme dopamine beta-hydroxylase (EC 1.14.17.1). The purified granule membrane proteins were more heterogeneous, but still predominantly acidic. The soluble and membrane forms of dopamine beta-hydroxylase behaved identically in this gel system. Lactoperoxidase-catalyzed iodination of intact granules revealed that most, but not all, granule membrane proteins are accessible at the cytoplasmic face. Prominent proteins of the purified adrenal medullary mitochondria showed little if any homology with purified granule membranes. The crude microsome fraction showed significant homology with purified granule membranes despite low levels of cross-contamination between the two fractions in marker enzyme analysis. Among proteins that could be identified, dopamine beta-hydroxylase was at a low level in the microsomes, while the granule membrane protein cytochrome b-561 appeared to be in both fractions. The pattern obtained from primary cultures of adrenal chromaffin cells was very complex, but prominent proteins from the subcellular fractions were seen without difficulty. Actin and tubulin were very prominent in the whole cell pattern. Radioiodination of the whole cells resulted in a number of spots being labelled, although the majority of the label appeared to be in only two proteins of molecular weight 70000 and isoelectric point 5.7.

Comparison of the effects of the ionophore salinomycin and adrenaline on the haemodynamics and work efficiency of the dog heart.

The positive inotropic, chronotropic, pressor, and coronary vasodilative effects of infused adrenaline (1 microgram X kg-1 X min-1) were compared with those of an intravenous injection of the carboxylic ionophore salinomycin (150 micrograms X kg-1) in 10 dogs anaesthetised with pentobarbital. At doses normalised to produce a doubling of left ventricular dP/dtmax both drugs produced pronounced increases in blood pressure, cardiac output, and plasma catecholamine concentration and a small increase in heart rate. After 10 minutes of adrenaline infusion coronary artery blood flow doubled whereas salinomycin produced a sixfold increase, reflecting its specific coronary vasodilative properties. The increases in cardiac output and pressor, chronotropic, and inotropic actions of salinomycin were related to the release of endogenous catecholamines into the plasma by the ionophore, whereas the increase in coronary blood flow indicated a non-adrenergic relaxation of the coronary blood vessels. Calculated values of left ventricular hydraulic work appreciably increased with both drugs, but left ventricular oxygen consumption was much higher during adrenaline infusion than the peak effect obtained with salinomycin. Accordingly, the mechanical efficiency of the left ventricle was slightly decreased by adrenaline and doubled by salinomycin. Because of its favourable haemodynamic profile, salinomycin has potential as a drug for increasing cardiac output, blood pressure, and left ventricular force of contraction and for improving the myocardial blood perfusion and mechanical efficiency of the heart.

Relevance of the chemical charge of rhodamine dyes to multiple drug resistance.

Previously, we have shown that multiple drug resistant (MDR) Friend leukemia cells (FLC) are cross-resistant to the positively-charged dye, Rhodamine 123 (Rho 123), and that this resistance can be reversed by verapamil (VER). In the present study we used two zwitterionic rhodamine analogs, Rhodamine 116 and Rhodamine 110, and another positively-charged analog, Rhodamine 6G, to determine whether drug accumulation, resistance and modulation were affected by changes in the charge of these compounds. While there was no differential sensitivity between sensitive and resistant FLC to zwitterionic rhodamines, there was marked differential toxicity between these cell types for the positively-charged analogs. The IC50 values were 1000- and 100-fold greater in resistant than in sensitive cells for Rho 123 and Rho 6G respectively. Intracellular drug accumulation was significantly higher in sensitive as compared to resistant cells for both Rho 123 and Rho 6G, but little difference in drug uptake between these two cell types was observed for Rho 110 and Rho 116. It was also found that the intracellular to extracellular ratio of the positively-charged compounds was greater than unity in both sensitive and resistant cells whereas for the zwitterionic analogs this ratio was less than 1. Furthermore, this ratio of drug uptake was found to be significantly higher for Rho 6G than for Rho 123, which correlated with the high oil:water partition coefficient of Rho 6G (115.6). In MDR cells, verapamil increased Rho 123 and Rho 6G accumulation by 9.4- and 8.6-fold respectively. In addition, IC50 values in resistant cells were reduced greater than 100-fold for Rho 6G and greater than 1000-fold for Rho 123 in the presence of 10 micrograms/ml of verapamil. In contrast, less than 2-fold reduction of IC50 values for both of the zwitterionic analogs could be obtained under the same conditions. These results indicate that the chemical charge of rhodamines plays an important role in their differential accumulation, cytotoxicity and sensitivity to modulators such as verapamil, in sensitive and multi-drug resistant cells. The data also suggest that increased lipophilicity of the positively-charged rhodamines may increase their ability to accumulate in, and subsequently kill, MDR cells.

Improvement of cardiac performance by carboxylic ionophore monensin in greyhound and mongrel dogs.

The effects of the cardiotonic ionophore monensin on coronary blood flow, cardiac output, left ventricular (LV) O2 consumption, LV contractility and arterial blood pressure were measured in greyhounds and compared with those in mongrel dogs under pentobarbital anaesthesia. Following intravenous injection of 100 micrograms/kg monensin, all parameters rose, the greyhounds showing a relatively greater response in peak values of coronary blood flow, LV contractility and cardiac output. The increase in the calculated LV mechanical work was proportionately greater than the corresponding increase in LV O2 consumption, hence the derived index, external mechanical efficiency, rose rapidly from an initial value of 0.165 to 0.285 (73%) in greyhounds; mongrel dogs showed a more modest rise from 0.094 to 0.120 (28%) during the same interval. The difference in the responses in the two breeds of dogs are attributed mainly to larger heart size and exercise training in greyhounds as compared with untrained mongrel dogs.

Diets high in polyunsaturated oils decrease lymphocyte membrane anisotropy and B-cell mitogenesis.

Balb/c mice were fed semipurified diets containing 40% of total calories as lipids: olive, safflower, or fish oil. After 2 weeks of feeding, splenic mononuclear cells were isolated and assayed for steady-state fluorescence anisotropy by means of the probe diphenylhexatriene. Splenocytes were also incubated with the mitogens concanavalin A, phytohemagglutinin, or lipopolysaccharide; subsequent uptake of tritiated thymidine was measured as an index of proliferation. No differences were observed in the membrane anisotropy and mitogenesis of mice fed the olive oil diet (high in monounsaturated fatty acids) and the control group. However, membrane anisotropy was significantly lowered in mice fed the safflower or fish oil diets (high in polyunsaturated fatty acids), and the mitogenic response to lipopolysaccharide was suppressed. Mitogenic responses to concanavalin A and phytohemagglutinin were not significantly affected by the type of lipid in the diet.