Hepatocytes from alpha1B-adrenoceptor knockout mice reveal compensatory adrenoceptor subtype substitution

Abstract

1 Alpha1-adrenoceptors (ARs) play an important functional role in the liver; yet little is known about their cellular location. We identified the subtypes present in wild-type (WT) and alpha1B-AR knockout (KO) mice livers at 3 and 4 months of age, and investigated their distribution in hepatocytes. 2 The fluorescent alpha1-AR antagonist quinazolinyl piperazine borate-dipyrromethene (QAPB) was used to visualise hepatic alpha1-ARs and radioligand binding with [3H]-prazosin was used to quantify the alpha1-AR population. 3 QAPB and [3H]-prazosin bound specifically to hepatic alpha1-ARs with nanomolar affinity. The cellular distribution of alpha1-ARs was similar in WT and alpha1B-AR KO hepatocytes; QAPB binding was distributed diffusely throughout the cell with no binding evident on the plasma membrane. Radioligand binding produced Bmax values as follows: 3-month WT - 76+/-3.3 fmol mg(-1); 4-month WT - 50+/-3.1 fmol mg(-1); 3-month alpha1B-AR KO - 7.4+/-0.73 fmol mg(-1); 4-month alpha1B-AR KO - 30+/-2.0 fmol mg(-1). 4 In 3- and 4-month WT liver, all antagonists acted competitively. RS100329 (alpha1A-selective) and BMY7378 (alpha1D-selective) bound with low affinities, indicating the presence of alpha1B-ARs. In 4-month alpha1B-AR KO liver prazosin produced a biphasic curve, whereas RS100329 and BMY7378 produced monophasic curves of high and low affinity, respectively, indicating the presence of alpha1A-ARs. 5 In conclusion, we have made the novel observation that alpha1-ARs can compensate for one another in the absence of the endogenously expressed receptor; yet there appears to be no subtype-specific subcellular location of alpha1-ARs; the WT livers express alpha1B-ARs, while alpha1B-AR KO livers express alpha1A-ARs. This study provides new insights into both hepatocyte and alpha1-AR biology.

Figure 1

Typical QAPB binding on hepatocytes freshly isolated from livers of 3- and 4-month-old WT (a) and α_1B-AR KO (b) mice. Nonspecific binding was determined in the presence of 1 μM prazosin. Cells were plated on coverslips and examined by confocal microscopy with timelapse photography. Increasing concentrations (0.4–20 nM) were added cumulatively and images were collected at 1-min intervals. Images are presented in pseudocolour, where black indicates no staining, white indicates complete saturation and blue, cyan, yellow and red indicate increasing levels of saturation of the fluorophore, as indicated by pseudocolour scale. (c) 20 nM QAPB binding on WT liver slices. The image is a typical example of the results obtained (n=3).

Figure 2

Bar chart representing 5 nM QAPB binding to 4-month-old WT and α_1B-AR KO hepatocytes pre-incubated with 1 nM BMY7378 or RS100329. Inhibition of QAPB binding was measured as the average fluorescence intensity of the cell compared to the average fluorescence intensity in the absence of inhibitor (n⩾3).

Figure 3

QAPB (5 nM) binding on a hepatocyte from freshly isolated WT mouse liver. Image was collected by confocal microscopy and the three-dimensional reconstruction performed by the computer software IMARIS. QAPB binding indicating the presence of α₁-ARs is shown in blue and propidium-iodide-staining-identifying nuclei are shown in pink.