The adult cerebral cortex contains nicotinic acetylcholine (ACh) receptors vital to cortical function. However, little is known about the assembly of embryonic nicotinic receptor subunits into functional receptors or whether they play an active role in cortical development. We now report evidence of functional nicotinic acetylcholine receptor channels in fetal mouse cerebral cortex as early as embryonic day 10 (E10), when the cortex consists of dividing stem and progenitor cells. Patch-clamp electrophysiological measurements indicate that nicotine and ACh evoke sizable inward currents characteristic of nicotinic receptors, that are strongly rectifying with a reversal potential near 0 mV. Three different nicotinic agonists, ACh, nicotine, and dimethylphenylpiperazinium, evoked cytosolic Ca(2+) signals. Agonist-evoked Ca(2+) signals and electrophysiological responses were found in greater than 70% of all E10-E11 cells tested and were blocked by nicotinic receptor antagonists. The Ca(2+) response to nicotinic agonists was markedly prolonged in cells from early embryonic stages relative to later stages of development. alpha3, alpha4, and alpha7 receptor subunit proteins were detected immunocytochemically in cortical cells from E10 to birth. The incidence of each subunit declined with embryonic age, suggesting a role in early development. We discuss the possible function of nicotinic receptors in early cortical development and their role as a target for nicotine in the developmental pathologies associated with the fetal tobacco syndrome.

Bovine adrenal chromaffin cells were isolated by removal of the cortex and sequential collagenase digestion of the medulla. The catecholamine secretory function of these cells was characterized with respect to acetylcholine stimulation, cation requirements, and cytoskeletal elements. The dose-response curve for stimulated release had its half-maximum value at 10-5 M acetylcholine, and maximum secretion was on the average 7 times that of control basal secretion. The differential release of epinephrine versus norepinephrine after stimulation with 0.1 mM acetylcholine occurred in proportion to their distribution in the cell suspension. The cholinergic receptors were found to be predominantly nicotinic. The kinetics of catecholamine release were rapid, with significant secretion occurring in less than 60 sec and 85% of maximum secretion within 5 min. A critical requirement for calcium in the extracellular medium was demonstrated, and 80% of maximum secretion was achieved at physiologic calcium concentrations. Stimulation by excess potassium (65 mM KCI) also induced catecholamine secretion which differed from acetylcholine stimulation in being less potent, in having a different dependence on calcium concentration, and in its response to the local anesthetic tetracaine. Tetracaine, which is thought to inhibit membrane cation permeability, was able to block acetylcholine-stimulated but not KCI-stimulated secretion. The microtubule disrupting agent vinblastine was able to block catecholamine release whereas the microfilament disrupter cytochalasin B had little effect. The results show the isolated bovine chromaffin cells to be viable, functioning, and available in large quantity. These cells now provide an excellent system for studying cell surface regulation of hormone and neurotransmitter release.The term "stimulus-secretion coupling" was originally coined by Douglas and Rubin (1, 2) more than a decade ago to describe the sequence of events initiated by acetylcholine (ACh) stimulation of adrenal chromaffin cells and leading to secretion of catecholamines by exocytosis. They had in mind the close similarity to the phenomenon of "excitation-contraction coupling" in muscle (namely, the key role of calcium in mediating both secretion and contraction and a parallel set of electrical and ionic events at the plasma membrane in response to ACh). Much progress has been made during the past 2 decades in elucidating the various steps in the mechanism of secretion, with a substantial portion of the data deriving from studies on the perfused adrenal gland (2-4). There remain, however, many important aspects of cell surface receptor regulation of secretion that are still poorly understood, such as (i) the molecular nature of the calcium permeability sites that are altered by activated cholinergic receptors and potassium depolarization, (ii) the mechanism by which calcium entry into the cell induces membrane fusion and exocytosis, (iii) the possible role of cyclic nucleotides coupled with ion translocation in mediating horm...

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.

Adsorption isotherms of water on human red cell membranes have been measured and the results used to determine the Brunauer-Emmett-Teller monolayer coverage, saturation hydration, and relevant heats and entropies of hydration. The tightly bound monolayer corresponds to a complete covering of only one membrane surface and the saturation hydration is greater than values for globular proteins and phospholipids. A hysteresis loop is observed in the adsorption-desorption cycle.Circular dichroism spectra of membrane films have been measured as a function of hydration in the ultraviolet region of the spectrum. The spectra resemble those of membrane suspensions with reduced scattering. The circular dichroism did not change upon dehydration of the membrane films from 92 to 0% relative humidities, implying a stable membrane protein structure independent of both water content and lipid phase.