Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

Abstract

To elucidate mechanisms of glucagon-induced bicarbonaterich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pH1) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pH1, significantly stimulates the Cl-/HCO3 exchange activity.The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3-exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3 exchange was individually blocked by two specific and selective inhibitors of protein kinase A, uM) and H-89 (30 ,uM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl -channel blocker, NPPB (10 pmM), showed no effect on either the basal or the glucagon-stimulated Cl-/ HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 jpM), completely blocked the glucagon stimulation of the C1-/HCO3 exchange; however, this effect was achieved through a significant inhibition of the glucagonstimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3 exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pH1 or at acid pH, values. In contrast, glucagon, at basal pH1, stimulated the Na+-HCO-symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3 exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis. (J. Clin. Invest. 1995.

Gastrin inhibits cholangiocyte growth in bile duct–ligated rats by interaction with cholecystokinin-B/gastrin receptors viaD -myo-inositol 1,4,5-triphosphate–, Ca2+-, and protein kinase Cα–dependent mechanisms

“…A counter-regulatory effect of PKC on adenyl cyclase activity has been described in different cell types. 40,41 In rat hepatocytes glucagon-induced cAMP synthesis was counteracted by phorbol esters. 41 The effect of PKC could be mediated at the level of specific adenyl cyclase isoforms or at a receptor level by modification of the guanine nucleotide regulatory protein system.…”

“…40,41 In rat hepatocytes glucagon-induced cAMP synthesis was counteracted by phorbol esters. 41 The effect of PKC could be mediated at the level of specific adenyl cyclase isoforms or at a receptor level by modification of the guanine nucleotide regulatory protein system. 40,41 The inhibitory effect of gastrin on cholangiocyte DNA synthesis was observed in the absence of gastrin effects on basal intracellular cAMP levels, which plays an important stimulatory role in cholangiocyte growth in culture.…”

Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.

“…The composition of solutions used in the study have been described previously in detail (4,19,25,26). Secretin and ACh were made up as a concentrated solution in the appropriate perifusion buffer containing 1% (wt/vol) BSA.…”

“…pH i was measured using a microfluorimetric single-cell method (SPEX-AR-CM-micro system; Spex Industries, Edison, NJ) and BCECF-AM as a fluorescent pH i indicator (4,19,25,26). IBDU on glass coverslips were loaded with BCECF-AM (12 M) for 40 min, washed for 10 min in a BCECF-free medium and transferred into a thermostated perfusion chamber placed on the stage of a Nikon Diaphot inverted microscope.…”

“…The intrinsic buffering power ( ␤ i ) was determined at different pH i as described (4,19,25,26), by exposing the cells to 30-mM NH 4 Cl in Hepes, Na ϩ -free buffered solutions, and then decreasing the NH 4 Cl concentration by 5 or 10 mM for each step to 0 mM (n ϭ 9) from three IBDU preparations. The ␤ i was then calculated from the midpoint change in pH i at each step.…”