Isolated hepatocyte suspensions were exposed to hypotonic and hypertonic stresses and serial cell volume measurements were made with an electronic particle size analyzer. With the exposure to hypotonic (160 mosM) buffer, hepatocytes swelled within 30-60 s as osomometers [relative volume (RV) = 1.44 +/- 0.08] and subsequently underwent regulatory volume decrease (RVD) back toward the resting (isotonic) level (1.16 +/- 0.05). This volume recovery was blocked by 65 mM extracellular K+ concentration and inhibited by barium (1 mM) and quinine (0.5 mM) but not by bumetanide (0.1 mM). Chloride depletion inhibited RVD by approximately 40% while 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) blocked the recovery by almost 90%. Calcium deprivation had no effect on RVD, nor did ouabain, amiloride, or sodium replacement. When exposed to buffer made hypertonic by addition of 200 mM sucrose, cells shrunk as osmometers (RV = 0.74 +/- 0.02) but did not exhibit regulatory volume increase (RVI). However, when cells that had first undergone RVD were reexposed to isotonic medium (relative hypertonic stress) RVI could be demonstrated from RV 0.77 +/- 0.17 to 0.91 +/- 0.20. This response was dependent on sodium, partially dependent on bicarbonate and chloride, and inhibited by the Na(+)-H+ exchange inhibitor amiloride (1 mM) but not by DIDS. Our findings suggest that RVD in rat hepatocytes is mediated by quinine- and barium-sensitive K+ conductance and DIDS-sensitive anion conductance, which is partly accounted for by Cl-; RVI is mediated by activation of Na(+)-H+ exchange coupled with a bicarbonate- and chloride-dependent but DIDS-insensitive process.

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In rat hepatocytes, basolateral Na(+)-H+ exchange and Na(+)-HCO3- cotransport function as acid extruders. To assess mechanisms of acid loading, intracellular pH (pHi) recovery from an alkaline load was analyzed in short-term cultured rat hepatocyte monolayers using the pH-sensitive dye BCECF. Electrophysiological techniques were also used to assess the role of the membrane potential (Vm). Cells were alkaline loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHo. After this maneuver, pHi rapidly rose by 0.13 +/- 0.03 pH units (pHu) and recovered to baseline at an initial rate of 0.026 +/- 0.009 pHu/min. Intracellular buffering power was estimated from the dependence of pHi on [NH4+]o and varied between 70 and 10.5 mM/pHu in a pHi range of 6.5-7.6. Initial pHi recovery corresponded to a rate of OH- efflux (JOH) of 1.76 +/- 0.71 mM/min and was blocked by 0.5 mM DIDS (0.003 +/- 0.002; JOH = 0.18 +/- 0.06) or by 1 mM H2DIDS (0.001 +/- 0.002; JOH = 0.26 +/- 0.08) and by removal of [Cl-]o (0.003 +/- 0.007; JOH = 0.28 +/- 0.07). The dependence of JOH on [Cl-]o exhibited saturation kinetics with an apparent Km for [Cl-]o of 5.1 mM. pHi recovery was Na+ independent and was not inhibited by substitution of Na+ with NMDG (0.045 +/- 0.09; JOH = 2.94 +/- 0.59). During an alkaline load, cell Vm hyperpolarized from -33.4 +/- 1.8 to -43.4 +/- 2.8 mV, mainly due to an increase in K+ conductance by a factor of 2.8 +/- 0.3.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of hormones in the regulation of bile secretion is not known; however vasoactive agents, which act via the phosphoinositide signal transduction pathway, may mediate changes in bile flow by altering the hepatic microvasculature. We therefore examined the effects of phorbol esters and diacylglycerol, agonists of the protein kinase C branch of the phosphoinositide cascade, on perfusion pressure and bile flow in a single-pass, hemoglobin-free, isolated perfused rat liver system with constant perfusate flow. The active phorbol ester, 12,13-phorbol dibutyrate, produced a dose-dependent (maximal effect at 10(-6) M), sustained and reversible decrease in bile flow from 1.09 +/- 0.18 to 0.61 +/- 0.09 microliters per min per gm liver (37.2 +/- 5.9%) while simultaneously increasing perfusion pressure from 12.3 +/- 0.7 to 21.5 +/- 2.5 cm H2O (74.0 +/- 4.3%). Both effects were inhibited by the synthetic protein kinase C antagonist H-7. 1,2-Dioctanoyl-sn-glycerol, a diacylglycerol, produced changes in bile flow and perfusion pressure that were similar to, but more marked than, those caused by 12,13-phorbol dibutyrate, whereas the inactive phorbol ester 4 alpha-phorbol didecanoate and the vehicle dimethyl sulfoxide had no effects on either parameter. 12,13-Phorbol dibutyrate infusion resulted in reversible decreases in oxygen consumption (23.3%) and a reversible vascular redistribution of trypan blue dye but did not alter hepatic venous effluent concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)

To assess the roles of acid-base transport systems in cell volume regulation in rat hepatocytes, intracellular pH (pHi) was measured in subconfluent monolayers loaded with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) after exposure to hypotonic and relative hypertonic media, interventions that stimulate regulatory volume decrease (RVD) and increase (RVI), respectively. During RVD, pHi decreased from 6.98 +/- 0.11 to 6.85 +/- 0.08 in the absence of HCO3- and from 7.26 +/- 0.10 to 7.19 +/- 0.06 in its presence. Omission of Na+ or addition of 1 mM amiloride prevented the decline in pHi. Acute withdrawal or replacement of Na+ in hypotonic medium resulted in a slower rate of fall or recovery in pHi, respectively, than when the same maneuvers were carried out in isotonic medium. In contrast, during RVI, pHi increased from 6.86 +/- 0.11 to 7.15 +/- 0.15 in the absence of HCO3-, a rise in pHi that was also completely abolished by Na+ removal or by 1 mM amiloride. In the presence of HCO3-, the rise in pHi was less marked than in its absence, although net acid efflux was greater because of a greater intracellular buffering capacity. Cl- removal in the presence of HCO3- had no effect on the change in pHi during either RVD or RVI. Perfusion with 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) during RVD lowered pHi further and accentuated the subsequent pHi rise seen after the return to isotonic medium. These data suggest that Na(+)-H+ exchange in rat hepatocytes is downregulated during RVD and activated during RVI. Cl(-)-HCO3- exchange does not appear to be involved in hepatocyte volume regulation.