Possible Regulation of P-Glycoprotein Function by Adrenergic Agonists II: Study with Isolated Rat Jejunal Sheets and Caco-2 Cell monolayers.

To clarify the regulation of drug absorption by the enteric nervous system, we investigated how adrenergic agonists (adrenaline (ADR), clonidine (CLO), dobutamine (DOB)) and dibutyryl cAMP (DBcAMP) affected P-glycoprotein (P-gp) function by utilizing isolated rat jejunal sheets and Caco-2 cell monolayers. ADR and CLO significantly decreased the secretory transport (Papptotal) of rhodamine-123 and tended to decrease the transport via P-gp (PappP-gp) and passive transport (Papppassive). In contrast, DBcAMP significantly increased and DOB tended to increase Papptotal and both tended to increase PappP-gpand Papppassive. Changes in P-gp expression on brush border membrane by adrenergic agonists and DBcAMP were significantly correlated with PappP-gp, while P-gp expression was not changed in whole cell homogenates, suggesting that the trafficking of P-gp would be responsible for its functional changes. Papppassive was inversely correlated with transmucosal or transepithelial electrical resistance, indicating that adrenergic agonists affected the paracellular permeability. Adrenergic agonists also changed cAMP levels, which were significantly correlated with PappP-gp. Furthermore, protein kinase A (PKA) or PKC inhibitor significantly decreased PappP-gp in Caco-2 cell monolayers, suggesting that they would partly contribute to the changes in P-gp activity. In conclusion, adrenergic agonists regulated P-gp function and paracellular permeability, which would be caused via adrenoceptor stimulation.

Possible Regulation of P-glycoprotein Function by Adrenergic Agonists in a Vascular-luminal Perfused Preparation of Small Intestine.

Although the functions of small intestine are largely regulated by enteric nervous system (ENS), an independent intrinsic innervation, as well as central nervous system (CNS), the neural regulation of drug absorption from the small intestine still remains to be clarified. To obtain some information on it, the effect of adrenergic agonists on P-glycoprotein (P-gp) function was investigated by utilizing a vascular-luminal perfused rat small intestine. Adrenaline significantly decreased the secretion of rhodamine-123 (R-123) into the intestinal lumen, but dibutyryl cAMP (DBcAMP) significantly enhanced R-123 secretion. The inhibition study with quinidine clearly indicated that the decrease in secretory clearance of R-123 by adrenaline or the increase by DBcAMP would be attributed to the decrease or increase in P-gp activity, respectively. Expression levels of P-gp in whole mucosal homogenates were not changed at all by any chemicals examined, but those on brush border membrane (BBM) of intestinal epithelial cells were significantly decreased or increased by adrenaline or DBcAMP, respectively. Furthermore, changes in P-gp activity caused by adrenergic agonists and DBcAMP were significantly correlated with changes in expression level of P-gp in BBM, suggesting that the trafficking of P-gp from cytosolic pool to BBM would be regulated by adrenergic agonists and DBcAMP.

Effect of adrenergic stimulation on drug absorption via passive diffusion in Caco-2 cells.

It is well known that the enteric nervous system (ENS) regulates the movement and function of the small intestine, but the effects of ENS on drug absorption from the small intestine still remain to be clarified. Focusing on adrenergic effect, we tried to evaluate how adrenergic stimulation influences the drug absorption via passive diffusion using Caco-2 cells as model epithelial cells, a terminal effector of ENS. Adrenaline, an adrenergic agonist, did not affect the transport of small molecules such as antipyrine, phenacetin and mannitol, but decreased the transport of large molecules such as FITC-dextran (FD)-20 and FD-40 without transepithelial electrical resistance (TEER) change. These results suggested that the transport of large molecules via paracellular route would be attenuated by adrenergic stimulation. Only clonidine, an alpha(2)-agonist, among selective adrenoceptor agonists decreased FD-40 transport across Caco-2 cell monolayers and the agonist also decreased intracellular cAMP. Furthermore, H-89, a protein kinase A inhibitor, significantly decreased FD-40 transport and dibutyryl cAMP, a cAMP derivative, increased it. These results suggest that the decrease in FD-40 transport would be mainly attributed to the decrease in intracellular cAMP and subsequent decrease in PKA activity via alpha(2)-receptor stimulation.