RESUMO
The regulated Cl(-) secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca(2+), cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux (J(w) = -0.16 +/- 0.02 microl.min(-1).cm(-2)) in parallel with moderate short-circuit current values (I(sc) = 1.55 +/- 0.23 microA/cm(2)). The secretory J(w) reversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I(sc), but, unexpectedly, J(w) was not affected. Bumetanide, an inhibitor of basolateral Na(+)-K(+)-2Cl(-) cotransporter, completely blocked secretory J(w) with no change in I(sc). Conversely, serosal forskolin increased I(sc), but J(w) switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J(w) and I(sc). No difference between the absorptive and secretory unidirectional Cl(-) fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl(-) flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J(w) values cannot be shown by I(sc) and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.
Assuntos
Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Água/metabolismo , Aquaporinas/genética , Toxinas Bacterianas/farmacologia , Transporte Biológico/efeitos dos fármacos , Bumetanida/farmacologia , Calcimicina/farmacologia , Linhagem Celular , Cloretos/metabolismo , Colforsina/farmacologia , Cultura em Câmaras de Difusão/instrumentação , Enterotoxinas/farmacologia , Proteínas de Escherichia coli , Humanos , Mucosa Intestinal/citologia , Ionóforos/farmacologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
Monoclonal antibodies are one of the most important products of biotechnology and laboratories and companies all over the world are pursuing their large-scale production. Herein we report a protocol for hybridoma cell cultivation over small glass cylinders inside a 3 liter bioreactor vessel which leads to the production and purification--in order of grams--of one MAb intended for human therapeutic use. This protocol proved to be simple, reproducible and cost effective. Three trials are reported: the first two using conventionally serum-supplemented medium culture and producing 3.15 and 2.1 g of purified MAb in 30 and 21 days respectively, and the third one using serum-free medium culture and producing 6 g of purified MAb in 36 days. We have ascertained the stability of the hybridoma by its cloning directly in serum-free medium. The downstream processing of the serum-free trial was done in a single step, concentrating large volumes of supernatant while simultaneously purifying the antibody.