Agonist-induced isotonic contraction of cultured mesangial cells after multiple passage

Contractile cells under conditions of prolonged culture lose their ability to contract in the usual manner (i.e., isotonically). One explanation for this may be that contraction is prevented by tight cell-to-substrate adhesion. Two models in which substrate adhesiveness was expected to be diminished were used to test this hypothesis. In one, cells were seeded onto collagen-coated dishes and used within 40 min of plating. In the other, cells were plated onto dishes coated with poly-2-hydroxyethyl methacrylate (poly-HEMA) and used, depending on thickness of the poly-HEMA substrate, up to periods of 1 wk. Cells plated onto such substrates contracted when challenged with either PGE₂ (2 x 10^-6 and 2 x 10^-9 M), arginine vasopressin (AVP, 10^-6-10^-9), or the calcium ionophore A23187 (5 μg/ml). Contraction took place within 5-15 min at 37°C. The contraction seen with AVP was due to its pressor action because 1-desamino-8-D-arginine vasopressin (dDAVP), the antidiuretic analogue, did not cause contraction and the antipressor analogue [1-(β-mercapto-ββ-cyclopentamethylene propionic acid)-4-valine 8-D-arginine]-vasopressin [d(CH₂)₅-VDAVP] blocked contraction by AVP. The contraction seen with AVP was dependent on extracellular calcium, whereas that observed with prostaglandin E₂ (PGE₂) was not. The contraction observed with PGE₂ appeared to depend upon release of stores of intracellular calcium because pretreatment of mesangial cells with the intracellular calcium antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) prevented contraction. Cells returned to their normal configuration between 15 min and 3 h after the washout of the agonist. With poly-HEMA, the length of time required to reach relaxation depended on the thickness of the substrate (between 30 min and 3 h). In contrast to these two models, cells similarly plated on plastic did not show isotonic contraction. Thus cell-substrate interaction, and possibly adhesiveness, may play a role in the expression of isotonic contraction by cultured mesangial cells in response to agonists. This may apply also to other contractile cells in culture such as smooth muscle. This technique should facilitate the study of contraction in cultured contractile cell types.