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Experiments were carried out to test the hypothesis that the differences between the surfaces of erythrocytes from normal and sickle cell patients are reflected in the degree of attachment to the capillary lining. An assay was used that measured the number of 51Cr- labeled erythrocytes (normal or sickle) attaching to a monolayer of endothelium cultured from calf aortas. Under these conditions, erythrocytes from sickle cell patients adhered better to the endothelium than did those from normal patients. The results suggested that the enhanced adhesion of the sickle cells to the endothelium may be partially responsible for the increased blockage of capillaries that produce the symptoms in sickle cell anemia.

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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 Ba 2÷ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na ÷ but not Ca 2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca 2÷ 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 Ca ~+ into the cell. We propose that the monensin-stimulated increase in intracellular Na + levels causes an increase in the availability of intracellular Ca 2+ 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 Ca 2+ that enters the cell as a result of acetylcholine stimulation.The key role of Ca ~+ in stimulus-secretion coupling in secretory cells was first recognized by Douglas and Rubin (1). The subsequent availability of divalent cation ionophores, e.g. A23187, lasalocid (X-537A), ionomycin, prompted an extensive series of investigations of the ability of these agents to promote cellular secretion by admitting extracellular Ca 2+ into cells (2-11).A number of recent studies indicate that a rise in the cytoplasmic Na + concentration also stimulates the secretory activity of cells (12)(13)(14)(15). The Na ÷ may dislodge a small fraction of the relatively large store of intracellularly sequestered Ca ~÷ so that it becomes available to the presumptive intracellular Ca 2+ receptors, calmodulin or troponin C (16). Alternately, increased intracellular Na + may interfere with the ability of the intracellular Ca2+-sequestering mechanisms to intercept Ca 2÷ that is brought into the cytosol by secretagogue-initiated processes. Ouabain, which alters the egress of intracellular Na + by a specific inhibition of the Na+-K+-ATPase (Na ÷ pump), elevates intracellular Na ÷ levels and thereby stimulates secretion of catecholamines, acetylcholine, and insulin (17)(18)(...

Rhythmic, spontaneously pulsating cardiac cells cultured from newborn rats are imediately stmulated to beat faster by addition of a number of tubulin-binding agents but not by their non-tubulin-binding an .The tubulinbinding agents tested incde vinbastine, crie, navelbine, two analogs of vinblastine (S12362 and S12363), nocodazode, colchicne, and podophylotoxin. In addi to binding tubulin, all ofthe above agents also depolymerize microtubules. Tubulin-binding agents such as colchicine, the vinca alkaloids, and benzimidazole derivatives have been extremely useful in demonstrating the large number of diverse cellular functions that depend either directly or indirectly upon the proper functioning of microtubules. These include a number of processes that involve movement-i.e., movement of cilia and flagella, movement of chromosomes, and intracellular movement of vesicles and organelles (1, 2). Although a sliding filament mechanism has been proposed to explain the role of microtubules in the movement of the above-mentioned processes, which is similar to that shown for actin in the movement involved in muscle contraction, any relationship of tubulin and/or microtubules to the mechanism of contraction of muscle remains unknown.There have been, however, three scattered reports (3-5), one of which comes from the laboratory of T.J.L. (5), suggesting that tubulin-binding agents can affect the rate of spontaneously beating cardiac cells growing in vitro (3-5).These previous reports confined their studies to one or two tubulin-binding drugs, colchicine and/or nocodazole, and thus it is not known whether other tubulin-binding agents have effects on cardiac contractility.In this study, we have addressed the hypothesis that the stimulation of cardiac muscle beating rate is a general property of tubulin-binding agents. In addition, by using taxol, a tubulin-binding compound that does not depolymerize microtubules, we have investigated whether cardiostimulation is associated with microtubule depolymerization. The physical association of tubulin with the contractile elements of cardiac muscle cells has also been studied by use of a monoclonal antibody (mAb) specific for a-tubulin. Furthermore, antiarrhythmic properties oftubulin-binding agents are presented, which opens the possibility of uncovering an additional class of antiarrhythmic drugs. MATERIALS AND METHODSEstablishment of Priary Cardiac Cultures. The procedure used here for isolating heart cells from newborn rats is a modification of that of Mark and Strasser (6) and has been described (7). Plastic plates (35-mm diameter) were seeded with 0.1-ml droplets of the primary cardiac cell suspension (2.3 x 106 cells per ml) and incubated at 3TC (5% C02/95% air) for 18 hr, at which time fresh medium (2 ml) was added to each plate. For fluorescence microscopy studies, cells were seeded on 12-mm glass coverslips. Medium changes were made every 48 hr thereafter. Drug treatments began 7 days after the cells were seeded, at which time cultures beated rhythmically and synchrono...