Epithelial strips: an alternative technique for examining arachidonate metabolism in equine tracheal epithelium.

The research explores a new method for studying the metabolism of arachidonic acid in the tracheal epithelium of horses. This method involves using strips of pseudostratified columnar epithelium attached to elastic tissue. The research showed these strips react differently to stimuli compared to the usual cell suspension method, and may therefore offer a more accurate way of studying this biological process.

Research Methods

• The researchers developed a new technique for studying arachidonic acid (AA) metabolism in equine tracheal epithelium. This technique involved using strips of pseudostratified columnar epithelium attached to a layer of elastic tissue approximately 80 to 130 microns thick.
• This new methodology was compared against the commonly used approach of enzyme-dispersed suspension of tracheal epithelium from the same horse.

Findings

• In comparison to the cell suspension method, the strips exhibited a different response when incubated with arachidonic acid and stimulated with A23187, a calcium ionophore. The strips incorporated around 40.8% of the added radioactivity and released about 2.55% of the incorporated radioactivity, while the cell suspensions incorporated 59.6% and released 1.9% respectively.
• When stimulated with other substances such as histamine or bradykinin, only the strips displayed significant release of free arachidonic acid.

Analysis of Released Eicosanoids

• High-performance liquid chromatography (HPLC) technique was used to analyze the molecular composition of the released eicosanoids. The profiles demonstrated that peaks coincided with free AA, prostaglandin (PG) E2, and PGF2 alpha for the strips, and free AA, leukotriene B4, and 5-HETE for the cell suspensions.
• The absence of PGE2 production by cell suspensions was confirmed by assessing the presence of this molecule in the test samples.

Implications

• Despite the lack of prostaglandin (PG) production by cell suspensions, the presence of PGH synthase – an enzyme involved in the synthesis of prostaglandin – was demonstrated in the epithelial cells of both sample types.
• These findings suggest that whereas the cell suspensions failed to synthesize cyclooxygenase metabolites (important components of AA metabolism), the epithelial strips did produce these compounds and responded appropriately to peptide agonists.