Heme oxygenase (HO) catalyzes the conversion of heme into biliverdin, iron, and carbon monoxide (CO). Two isoforms of HO have been identified: the inducible HO-1 and the constitutive HO-2. CO, like nitric oxide, is an endogenous vasodilator that could contribute to modulation of systemic and local vascular tone. The aim of the present study was to determine the expression of HO isoforms in liver cells and splanchnic organs from portal hypertensive (PH) and sham-operated (SO) rats. Liver cells (hepatocytes, Kupffer and stellate cells), and splanchnic organs (liver, mesentery, intestine, colon, and spleen) were isolated from PH and SO rats. Expression of HO mRNA and protein was assessed by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. In SO rats, HO-1 mRNA expression was only detected in spleen. In contrast, in PH rats, HO-1 mRNA was expressed in hepatocytes, Kupffer cells, and in all the splanchnic organs studied. Moreover, levels of HO-1 protein in splanchnic organs were significantly higher in PH rats than in SO animals. In addition, HO-2 expression was observed in all liver cell types and splanchnic organs studied from both PH and SO rats. These results indicate that HO-2 is expressed in parenchymal and nonparenchymal liver cells, as well as splanchnic organs, of both PH and SO rats. In addition, HO-1 is up-regulated in hepatocytes and splanchnic organs of PH rats, compared with SO animals, suggesting a possible pathophysiological role of HO-1 in chronic portal hypertension. (HEPATOLOGY 1999;29: 1672-1679.)
Microsomal heme oxygenase (HO) is the rate-controlling enzyme for heme degradation in mammals. 1 HO metabolizes heme into biliverdin and releases free iron and carbon monoxide (CO). In most mammals, biliverdin is subsequently converted to bilirubin by biliverdin reductase. 1 Two isoforms of HO, transcribed from separate genes, have been characterized 2 : the inducible HO-1 and the constitutive HO-2. The expression of HO-1 is up-regulated, not only by its substrate heme, 3 but also by a variety of stress-inducing factors including transition metals, 3,4 heat shock, 3 hydrogen peroxide, 4,5 ultraviolet light, 5 sodium arsenite, 3-6 cytokines, 7 endotoxin, 8,9 hypoxia, 10 shear stress, 11 nitric oxide, 12,13 and glucagon. 14,15 More recently, a protein with sequence homology to HO-1 and HO-2 has been identified and referred to as HO-3, 16 although its physiological role more likely involves heme-binding or heme-sensing than heme catabolism, because its specific enzymatic activity is very low.
All products of HO activity are biologically active. Iron is a gene regulator. 1 Bilirubin and biliverdin are potent antioxidants. 17 CO is, like nitric oxide, an endogenous monoxide that activates soluble guanylate cyclase, 18 leading to the generation of cyclic guanosine monophosphate, which in turn mediates various physiological functions, such as smooth muscle relaxation 19 and inhibition of platelet aggregation. 20 Other mechanisms of action of CO, independent of cyclic guanosine monophosphate, are also possible. 19 Recent studies suggested that CO, generated by HO-2, may contribute to the regulation of local and systemic vascular tone, under basal conditions. [21][22][23][24][25] In addition, excessive production of CO, a consequence of HO-1 overexpression, could play an important role in modulating vascular tone under different pathological situations. 9,[26][27][28][29][30][31] Chronic portal hypertension (PH) is associated with hyperdynamic circulation, characterized by increased blood flow and reduced vascular resistance in the splanchnic and systemic circulation. 32,33 The mechanisms underlying the splanchnic hyperemia in PH are not fully understood, but it has been suggested that an overproduction of endogenous vasodilators, such as glucagon, 34 prostacyclin, 35,36 and nitric oxide, 37-40 could be involved.
The aim of the present study was to determine the expression of HO isoforms in parenchymal and nonparenchymal liver cells, as well as in splanchnic organs from PH rats and from sham-operated (SO) control animals.
Methods
Materials. Sprague-Dawley rats were obtained from Charles River Laboratories (Cambridge, MA). Ketamine hydrochloride was purchased from Fort Dodge Laboratories (Fort Dodge, IA). Polyethylene tubing was from ClayAdams Intramedic (Parsippany, NJ). Reverse-transcription and polymerase chain reaction buffers, deoxynucleotide triphosphate (dNTP) mix, Taq DNA polymerase (Thermus aquaticus strain YT1), avian myeloblastosis virus reverse transcriptase, and DNA markers were from Promega (Madison, WI).