Michael J. Sanderson

Mechanisms and function of intercellular calcium signaling

Intercellular Ca2+ waves initiated by mechanical or chemical stimuli propagate between cells via gap junctions. The ability of a wide diversity of cells to display intercellular Ca2+ waves suggests that these Ca2+ waves may represent a general mechanism by which cells communicate. Although Ca2+ may permeate gap junctions, the intercellular movement of Ca2+ is not essential for the propagation of Ca2+ waves. The messenger that moves from one cell to the next through gap junctions appears to be IP3 and a regenerative mechanism for IP3 may be required to effect multicellular communication. Extracellularly mediated Ca2+ signaling also exists and this could be employed to supplement or replace gap junctional communication. The function of intercellular Ca2+ waves may be the coordination of cooperative cellular responses to local stimuli.

GABA Has Excitatory Actions on GnRH-Secreting Immortalized Hypothalamic (GT1-7) Neurons

The effects of gamma-aminobutyric acid (GABA) on clonal gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons were investigated using patch-clamp and fura-2 imaging techniques. Local application of GABA (100 microM) to GT1-7 cells voltage-clamped in the whole-cell configuration immediately increased membrane conductance and noise consistent with activation of the GABAA receptor-Cl- channel complex. Depolarization activated transient Na+ currents which were abolished by tetrodotoxin (TTX; 0.5 microM), and more sustained Ca2+ currents. Under constant current conditions, GT1-7 cells fired spontaneous action potentials, and depending on the Cl- equilibrium potential, GABA either depolarized cells, causing a rapid activation of action potentials, or hyperpolarized cells. In order to determine the effect of GABA on intact cells, the cell-attached patch configuration was used to record extracellularly. Under these conditions, application of GABA (100 microM), but not the GABAB receptor agonist baclofen (10 microM), immediately evoked multiple action potentials. Measurement of [Ca2+]i using fluorescence video microscopy and fura-2 revealed spontaneous, transient, repetitive increases in [Ca2+]i which had a periodicity ranging from 1 to 60 s. These Ca2+ oscillations were abolished by TTX (1 microM) and by the removal of extracellular Ca2+. Application of GABA (1 and 10 microM) induced an immediate increase in [Ca2+]i in all cells and increased the frequency of Ca2+ oscillations in a dose-dependent manner. The GABA-induced increase in [Ca2+]i was abolished by bicuculline and by the removal of extracellular Ca2+, and was inhibited by TTX. Baclofen (1 microM) had no effect on [Ca2+]i. These results suggest that activation of GABAA receptors has an excitatory action on GnRH-secreting immortalized hypothalamic neurons caused by a Cl(-)-dependent depolarization. GABA has been reported to increase GnRH secretion; a direct stimulatory action of the neurotransmitter on GABAA receptors of GnRH-secreting hypothalamic neurons may be responsible for this effect.

Substance P stimulates IL-1 production by astrocytes via intracellular calcium

There is increasing evidence that local substance P (SP) exacerbates peripheral inflammations, partly by stimulating production of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). SP may play similar roles in certain central nervous system inflammations. Multiple sclerosis plaques, for example, form around veins which are innervated by unmyelinated SP-containing fibers, and astrocytes in multiple sclerosis plaques stain for SP. We tested whether SP could stimulate IL-1 and TNF alpha production by cultured astrocytes and whether calcium was the second messenger in this process. We found that both SP and the calcium ionophore A23187 raised intracellular calcium ([Ca2+]i) and stimulated IL-1 production in astrocytes. SP also nonsignificantly increased TNF alpha production by astrocytes. Treatment with dibromo BAPTA/AM, an intracellular calcium buffer, blocked SP-induced IL-1 production. These findings indicate that SP induces IL-1 production by astrocytes and uses calcium as a second messenger. Our results indicate local SP may play a role in multiple sclerosis and certain other central nervous system inflammations.

PREPARATION OF EXPLANT AND ORGAN CULTURES AND SINGLE CELLS FROM AIRWAY EPITHELIUM

Publisher Summary In this chapter, the methods for preparing explant and organ cultures as well as the enzymatic isolation of cells are described. The inaccessibility of the ciliated epithelium of the mammalian respiratory tract has hampered in vivo studies of its structure and function, including those regarding the regulatory mechanisms of mucociliary transport. Consequently, several in vitro approaches have been developed to maintain and study airway tissues in culture. These include explant cultures, where pieces of tissue are seeded on a substrate to encourage the spreading and outgrowth of the epithelial cells; organ cultures, in which the tissue is maintained to preserve its original structural and functional form; monolayer cell cultures, where enzymatically dissociated cells are plated onto a noncellular substrate; and feeder layer cultures, where isolated epithelial cells are grown on a cellular substrate such as treated fibroblasts. Explant cultures have been utilized for the measurement of ciliary beat frequency, including the timings of the phases of the beat cycle, of single and multiple cells, as well as for the analysis of intercellular Ca2+ signaling. Isolated epithelial cells have been used in patch–clamp studies and in assays to measure inositol trisphosphate levels. There is description of preparation of rat tail collagen, of collagen-coated coverslips, and tracheal epithelial explant culture—the materials to be used and the procedure. The technique is sterile. There is description of dissection of rabbit trachea, technique is aseptic. Details of preparation of organ culture and preparation of enzymatically isolated cells have been covered. In organ culture, the tracheal mucosa is supported at a liquid-air interface to mimic in vivo conditions of nutrient and gas exchange. Isolated cell preparations are obtained either from freshly dissected tissues or from tissues that had previously been placed in organ culture.

Regulation of ciliary activity in the mammalian respiratory tract

A computer-assisted transillumination, photoelectronic technique has been used to measure the beat frequency of cilia of rabbit tracheal cells grown in culture. When ciliated cells are mechanically stimulated with a microprobe the cells respond rapidly by increasing the beat frequency of their cilia. This mechanosensitive response is not limited to the stimulated cell, but is communicated in all directions to neighboring cells. To characterize the progression of this communicated response we used an automated computer-assisted imaging system to examine high-speed films of responding cells. The time it takes for the response to be transmitted between cells is slow (1-3 sec) with each cell responding after a lag-time that is proportional to the distance of the cell from the stimulated cell. We have confirmed that gap junctions are present between cells and that adjacent or non-adjacent ciliated, as well as non-ciliated, cells are electrically coupled. To correlate the mechanosensitive response with intracellular calcium fluxes we have used fura-2, a calcium-specific fluorescent dye, and digital video microscopy. Mechanical stimulation of the cultured ciliated cells, in the presence of extracellular calcium, resulted in an initial increase in intracellular calcium, which was communicated to neighboring cells. Without extracellular calcium, mechanosensitivity of cultured cells was lost and a small decrease in intracellular calcium was observed in the stimulated cell. However, neighboring cells still displayed an increase in intracellular calcium. The time course and general pattern of calcium increase in adjacent cells was similar to the responses in ciliary activity produced by mechanical stimulation. Ciliary beat frequency is also elevated by beta-adrenergic drugs independently of mechanosensitivity. These responses are important because they could provide a dual regulatory mechanism for the control of mucus transport. Adrenergic agonists could provide non-specific control by increasing ciliary activity throughout the airways while mechanosensitivity could provide local control by increasing activity in those regions of heavy mucus load.