Structural and functional features of bile canaliculi in adult rat hepatocyte spheroids

Abstract: Spheroids of adult rat hepatocytes are spherical cell aggregates which retain three-dimensional architecture and hepatocyte specific functions. In this study, we investigated the detailed structure and function of bile canaliculi in spheroids. Hepatocytes were prepared from adult rat liver and cultured with epidermal growth factor (50 ng/ml). Hepatocytes formed floating spheroids 4 days after inoculation. The morphology of hepatocyte spheroids was investigated after fluorescent staining for actin using confocal laser scanning microscopy and electron microscopy. To study the function of bile canaliculi, the transcellular transport of fluorescein diacetate was observed. These experiments were performed in a control group and in a group treated with the actin inhibitor cytochalasin B. In a control group, spheroids contained bile canalicular structures which were surrounded by actin filaments. Added fluorescent dye was secreted and pooled in bile canaliculi. Cytochalasin B caused marked distention of bile canaliculi and prominent accumulation of secreted fluorescent dye in dilated bile canaliculi. This phenomenon was based on the impairment of contractile movement of bile canaliculi. These results demonstrate that hepatocyte spheroids maintain functional and morphological peculiarity, and therefore this model may be useful in investigation of the mechanism of bile formation and intrahepatic cholestasis.     

Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR- rats.

BACKGROUND/AIMS: Fluorescent bile acids have proved useful for characterizing bile salt transport mechanisms. The aim of this study was to further validate the use of lysyl-fluorescein conjugated bile acid analogues as surrogate bile acids. METHODS: We analyzed biliary excretion kinetics of cholyl lysyl fluorescein (CLF), lithocholyl lysyl fluorescein (LLF) and sulfo-lithocholyl lysyl fluorescein (sLLF), both in the isolated rat hepatocyte couplet model and in TR- rats with a selective canalicular transport defect of non-bile acid organic anions. RESULTS: CLF and LLF, which like their natural nonsulfated bile acid congeners are expected to be handled by the canalicular bile salt export pump, were transferred into the bile canaliculus much faster than sLLF, a putative substrate for the canalicular multispecific organic anion transporter in both the in vivo and the in vitro models employed. The contention that different transport systems are involved in sulfated and non-sulfated lysyl fluorescein conjugated bile acids biliary excretion was supported further by studies using TR- rats, in which the cumulative biliary excretion of sLLF was reduced to 6% as compared with that of normal Wistar rats, in good agreement with values for its naturally-occurring radiolabeled parent compound sulfoglycolithocholate. In contrast, CLF and LLF were reduced to 66% and 52%, similar values to these for their congeners, [14C] glycocholate and [14C] lithocholate. CONCLUSION: The close similarity in behavior of lysyl fluorescein conjugated bile acids to that of their naturally-occurring parent compounds in these different models gives support for both sulfated and nonsulfated lysyl fluorescein conjugated bile acids as substitute molecules for studies of bile acid transport.     

Effect of brefeldin a on transcytotic vesicular pathway and bile secretion: A study on the isolated perfused rat liver and isolated rat hepatocyte couplets

This study investigated the effect of Brefeldin A (BFA) on the transcytotic vesicular pathway labeled with horseradish peroxidase (HRP) in both isolated rat hepatocyte couplets (IRHC) and the isolated perfused rat liver (IPRL). To evaluate the role of the transcytotic vesicular pathway on bile secretion, the effect of BFA on bile secretion in the IPRL was then investigated. In the basolateral area of IRHC, BFA showed no effect on the density and percentage of area of HRP-labeled vesicles. However, HRP-labeled vesicles tended to accumulate in the juxtanuclear area of BFA-treated hepatocytes (P < .001 vs. controls). In the pericanalicular area, on the other hand, HRP-labeled vesicles were depleted compared with controls (P < .001). In keeping with these findings, although the early peak remained unchanged, BFA inhibited as much as 50% of the late peak of HRP excretion in bile, after a pulse load of HRP in the IPRL. Bile flow and the biliary secretion of bile salts (BS) and phospholipids were not modified by BFA in isolated livers perfused without BS in the perfusate or with 1 μmol min taurocholate (TCA). In BFA-treated livers, peak bile flow and BS output decreased by 20% (P < .05 vs. controls) only when a 5 μmol TCA bolus was administered. In conclusion, this study demonstrates that BFA inhibits the transcytotic vesicular pathway in the liver. However, BFA has no significant effect on bile secretion either in basal conditions or during perfusion with physiological amounts of BS. BFA slightly decreases bile flow and BS output only after an overload of BS, providing evidence against the physiological relevance of the transcystotic vesicular pathway in the process of bile formation.459.)     

Release of bile canalicular membrane antigen into blood in experimental extrahepatic cholestasis of the rat

The level of a bile canalicular membrane antigen in serum during extrahepatic cholestasis was serially analyzed using HAM.4, a monoclonal antibody against a bile canalicular membrane glycoprotein of normal rat hepatocytes. After bile duct ligation, the level of HAM.4 antigen in serum promptly increased within 1 hr, reached a maximum at 3 hr, and declined somewhat until 48 hr, where it plateaued. Elevated levels of HAM.4 antigen in serum preceded those of well-known biliary marker enzymes activities. Immunohistochemical studies showed that the expression of HAM.4 antigen in hepatocytes and bile duct cells was not altered appreciably after bile duct ligation even when HAM.4 antigen in serum reached a maximal level. The serum and hepatic HAM.4 antigen had a molecular weight of 110 kDa. These results suggest that HAM.4 antigen may be regarded as a potential marker of the early stage of cholestasis, with release occurring before apparent histological changes.     

Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat

BACKGROUND/AIMS: Alterations in hepatobiliary transporters may render fatty livers more vulnerable against various toxic insults. METHODS: We therefore studied expression and function of key organic anion transporters and their transactivators in 8-week-old obese Zucker rats, an established model for non-alcoholic fatty liver disease. RESULTS: Compared to their heterozygous littermates, obese animals showed a significant reduction in canalicular bile salt secretion, which was paralleled by significantly diminished Oatp2 mRNA and protein levels together with reduced nuclear HNF3beta, while expression of bile salt export pump, organic anion transporter (Oatp) 1 and multidrug resistance-associated protein (Mrp) 4 were unchanged. Impaired bile salt-independent bile flow in obese rats was associated with a 50% reduction of biliary secretion of the Mrp 2 model-substrates glutathione disulfide and S-(2,4-dinitrophenyl)glutathione. In line Mrp2 protein expression was reduced by 50% in obese rats. CONCLUSIONS: Oatp2 and Mrp2 expression is decreased in fatty liver and may impair metabolism and biliary secretion of numerous xenobiotics. Reduction of bile salt secretion and absence of biliary GSH excretion may contribute to impaired bile flow and posthepatic disorders associated with biliary GSH depletion.     

Effect of enkephalins and morphine on insulin secretion from isolated rat islets

Summary The direct effects of an enkephalin analogue, (D-Ala²/MePhe⁴/Met/(O)-ol) enkephalin (DAMME), on insulin release from isolated islets of Langerhans of the rat have been investigated. DAMME had a dose-dependent effect on insulin secretion: low concentrations (10^–10 to 10^–8 mol/l) were stimulatory while high concentrations (10^–5mol/l) were inhibitory in the presence of 8 mmol/l glucose. Similar effects were found with met-enkephalin, and with the longer acting alanine substituted metenkephalin. Morphine sulphate (5 sx 10^–7 mol/l) also stimulated insulin release. The effects of enkephalin and morphine were blocked by the specific opiate antagonist naloxone hydrochloride (1.2 × 10^–6 mol/l). The insulin secretory response of perifused islets to enkephalins and morphine was rapid, corresponding to the first phase of glucose induced insulin release. These observations suggest that there may be opiate receptors in islets, and that opioid peptides could modulate insulin release.     

Afterload- and preload-dependent interactions in the isolated biventricular working rat heart

OBJECTIVES:

The afterload- (AL) and preload- (PL) dependent interactions between the left and right ventricle (LV, RV, respectively) of an isolated biventricular ejecting rat heart were measured in terms of left (L) and right (R) intraventricular peak pressure (LP_max and RP_max, respectively) and aortic and pulmonary flow (AF, PF, respectively).

METHODS:

Starting with standardized loading conditions, LVPL was varied in six steps for each of five distinct LVALs (n=28) and then RVPL was varied in seven steps for each of five distinct RVALs (n=37). Thus, the entire range of loading conditions was covered.

RESULTS:

Identification of AL-dependent systolic interactions revealed an important ΔLP_max–ΔRP_max gain of 0.25 (r²=0.78) and a still more dominant ΔRP_max–ΔPF gain of 0.45 (r²=0.84). At least 26% of maximal PF were attributable to LV systolic function. In contrast, R-L systolic interaction impeded PF; there was no global crosstalk pressure gain and no ipsilateral pressure-flow gain. Reduction of RV activity augmented AF by at least 15%. PL-dependent L-R interactions were absent except for minimal LVAL. In contrast, the reverse interaction reflected an inverse correlation between RVPL and AF, which is coincidential with other studies (−11% AF for a doubling of the standard RVPL). For the minimal RVAL, there was a biphasic response of AF to RVPL. Unloading the maximally loaded RV revealed an overall inhibition of AF by 37% for the standardized LV. Unloading the standardized RV revealed a basal inhibition of AF by 6% for the standardized LV and a 4.5% augmentation for the highly loaded LV. Consequently, basal contribution of RV to LV performance depended on the conditions of LV loading.

CONCLUSIONS:

The authors suggest a unidirectional transseptal R-L mechanism for diastolic interactions, and transseptal L-R and paraseptal R-L mechanisms for systolic interactions.     

Enhanced biliary excretion of canalicular membrane enzymes in estrogen-induced and obstructive cholestasis, and effects of different bile acids in the isolated perfused rat liver

Backgrounds/Aims: Canalicular membrane enzymes are normally released into bile by partially known processes. This study was undertaken to investigate whether hepatocellular cholestatis induced in rats by ethynylestradiol or obstructive cholestasis produced by complete biliary obstruction for 24 h is associated with an increased release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile, and to clarify how this process is affected by different bile acids.Methods: The studies were performed in the isolated perfused liver during infusion of sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate at increasing rates.Results: Maximum sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate secretory rates were decreased in both cholestatic groups (complete biliary obstruction>ethynylestradiol) compared with controls. Maximum biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase were significantly increased in the ethynylestradiol group during infusion of sodium taurocholate and taurochenodeoxycholate, but not of tauroursodeoxycholate, and were increased in the complete biliary obstruction group during the infusion of sodium taurocholate and tauroursodeoxycholate but not of taurochenodeoxycholate. The biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase showed a significant and direct linear relationship with sodium taurocholate and taurochenodeoxycholate secretory rates in both cholestatic groups. However, only in the complete biliary obstruction group did alkaline phosphatase and gamma-glutamyl transpeptidase excretion show a significant correlation with tauroursodeoxycholate secretory rates. The slope of the line, which indicated the mU of enzyme activity secreted per nmol of sodium taurocholate or taurochenodeoxycholate, was greater for gamma-glutamyl transpeptidase and alkaline phosphatase in both cholestatic groups (ethynylestradiol>complete biliary obstruction) than in the control group. Alkaline phosphatase activity in purified isolated canalicular and sinusoidal membranes was significantly increased in both cholestatic groups (complete biliary obstruction>ethynylestradiol), while gamma-glutamyl transpeptidase activity was unchanged compared with controls.Conclusion: The marked increase in sodium taurocholate and taurochenodeoxycholate-mediated release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile in cholestatic rats suggests an increased lability of these intrinsic membrane proteins to the detergent effects of secreted bile acids. It remains to be elucidated whether this phenomenon, which was particularly intense in ethynylestradiol induced cholestasis, is important in the pathogenesis and perpetuation of bile secretory failure. In contrast, tauroursodeoxycholate administration did not result in enhanced biliary excretion of these membrane enzymes, in either the control group or the ethynylestradiol group, supporting the concept that this bile salt lacks the membrane toxicity of common bile acids.     

Effects of galanin on insulin and glucagon secretion in the rat

Galanin-like immunoreactivity has been visualized in nerve fibers in the islets of Langerhans, suggesting an involvement of galanin in the neural regulation of islet function. In this study, we investigated the effects of galanin on basal and stimulated insulin and glucagon secretion by infusing the peptide at three different dose rates in rats. We also studied the direct effect of galanin on insulin secretion from freshly isolated rat islets. At 320 pmol/kg/min, but not at 20 or 80 pmol/kg/min, galanin lowered basal plasma insulin levels. In contrast, basal plasma glucagon levels were lowered by galanin already at 20 and 80 pmol/kg/min. Furthermore, galanin inhibited both glucose- and arginine-induced insulin release at all three dose levels, whereas arginine-induced glucagon release was not affected by galanin. Glucose-stimulated insulin secretion from isolated rat islets was dose-dependently suppressed by galanin (10^-6-10^-8M). Therefore, it is concluded that galanin in rats inhibits insulin secretion, both in vivo and in vitro, and that at lower dose levels, the peptide also inhibits basal glucagon release.     

Effects of oleic acid and oxygen restriction followed by re-oxygenation on rhythm and contractile activity of the isolated rat heart; protective action of glucose

Isolated rat hearts beating intrinsic rhythm were perfused with balanced salt solution containing 0.15 mm albumin and glucose (11 mm) or oleic acid (FFA/albumin molar ratio 2.65–4.65). After a control period (30 min) oxygen supply was restricted by lowering pO₂ from 700 to 140 mm Hg. After 20 min of oxygen restriction oxygenation was resumed. In all hearts coronary flow increased substantially during oxygen restriction and returned to approximately pre-restriction levels after reoxygenation. Contraction amplitude decreased during oxygen restriction and re-established itself only party after resuming oxygenation. Decrease was significantly greater and recovery significantly less in hearts perfused with oleic acid. Ventricular pre-mature beats (VPBs) were significantly more numerous in oleic acid hearts and occurred mainly shortly after starting reoxygenation. The incidence of other ventricular arrhythmias was also greater in these hearts. In hearts perfused with the combination oleic acid (FFA/albumin molar ratio 4.65) and glucose (11 mm) incidence of VPBs was significantly lower and CA was much less depressed than in hearts perfused with oleic acid only. Apparently glucose protects the heart from the deleterious effects of oleic acid and oxygen restriction. Pyruvate (11 mm) showed no protecting action in this respect. The difference in electrical and contractile behaviour between oxygen restricted hearts perfused with oleic acid and those perfused with glucose was even more striking when the hearts were paced (5/s) instead of beating intrinsic rhythm.     

Divergent effect of glucagon antibodies on arginine and glucose-stimulated insulin secretion in the rat

Summary The effects of glucose and arginine on insulin secretion in the presence of glucagon antibodies were investigated in rats in vivo. In contrast to controls, animals given glucagon antibodies showed an inhibition of arginine-stimulated (p < 0.001), but not glucose-stimulated, insulin secretion. That these effects were not due to incomplete neutralisation of endogenous glucagon is evidenced by the presence of large antibody excess throughout the duration of the experiments. Both the glucagonotropic effect of arginine (319 ± 60ng/l, p < 0.01) and the insulinotropic effect of exogenous glucagon (8.3 ± 0.8 g/l, p < 0.001) were demonstrable under our experimental conditions in the absence of exogenous glucagon antibodies. These observations suggest that different mechanisms are involved in the stimulation of insulin release by arginine and by glucose, and that glucagon may play an important physiological role in the mediation and regulation of insulin secretion by secretogogues, such as arginine.     

Relationship of phosphorylation potential and oxygen consumption in isolated perfused rat hearts

In isolated mitochondria (State 4 to 3) respiration depends linearly on the phosphorylation potential (log [ATP]/[ADP] [Pi] of the medium. Experiments were performed to look for similar relationship in the intact myocardium. The following corrections for metabolite binding or compartmentation were carried out to obtain sarcoplasmic concentrations of the respective metabolites: (1) Correction for constant and proportional values according to literature. (2) Correction for ATP and Pi as (1) calculation of ADP from CPK equilibrium assuming a constant pH. (3) Correction as (2) assuming respiration dependent changes of cytosolic pH according to literature. (4) Calculation of Pi distribution correlated to H⁺ distribution according to literature. For (1) to (4) increasing coefficients of correlation (0.43 to 0.95) were obtained indicating that in the intact heart mitochondrial respiration is regulated in the same way as in mitochondrial suspensions by the phosphorylation potential or indirectly because of CPK equilibrium by the ratio $\text{[}\text{CrP}\text{] · [}\text{H}{}^{\mathrm{+}}\text{] ·}\text{K}{}_{\mathrm{<mtext>CPK</mtext>}}\text{[}\text{Cr}\text{] · [}\text{Pi}\text{]}$.     

Effect of short- and long-term alcohol feeding in rats on pancreatic enzyme content and enzyme secretion in isolated pancreatic lobules in vitro

Summary The effect of short-and long-term ethanol intake on digestive enzyme secretion was determined in isolated pancreatic lobules of rats. Groups of male Wistar rats were fed a modified Lieber-DeCarli diet containing either 5% (w/v) óf ethanol, isocaloric amounts of a liquid diet in which ethanol was substituted by starch, or solid rat chow; for 3 days, 1, 2, 4, 8 and 12 weeks. Basal and caerulein-stimulated secretion of lipase, amylase, chymotrypsin and trypsin and the enzyme content in the tissue were studied. Feeding the liquid control diet decreased the tissue content of the four enzymes as compared with the values obtained in the group receiving solid rat chow. While basal and stimulated amylase secretion was markedly reduced in the former group, the secretion pattern of the other enzymes exhibited only transient changes. Caerulein-stimulated secretion of lipase and the proteases was increased by ethanol, the effect being more pronounced during the initial phase of the experiment. Alcohol feeding stimulated the basal secretion of these enzymes only in weeks 1–4. In contrast to the other enzymes, basal and stimulated amylase secretion was not enhanced by ethanol feeding. The results suggest that the enzyme secretion of the rat pancreas is distinctly altered by chronic ethanol feeding. However, the response of the pancreatic enzymes is non-parallel, and changes with the duration of alcohol intake.     

Investigations on isolated islets in vitro. IX. Influence of food deprivation and glucose refeeding in vitro on insulin secretion

Summary Starvation resulted in a progressive decrease of insulin secretion in response to 15 mM glucose, but it neither inhibited insulin release completely nor changed the shape of the glucose response curve. Long-term fasting caused the glucose threshold to rise to 30 mM glucose during an incubation time of 15 min, whereas fed animals were characterized by a sigmoidal insulin response and a threshold level of 5 mM glucose during short-term incubation, too. Prolongation of incubation time (60 or 120 min) or pretreatment of isletsin vitro with 20 mM glucose for 60 min caused progressive lowering of the glucose threshold, indicating that glucoseper se influenced the effective stimulating concentration. The effect of starvation is not specific for glucose, since hormone secretion in response to glibenclamide, nicotinic acid and theophylline was also reduced, whereas theophylline can partly counteract the diminished release. Although the content of insulin as well as glucagon in the islets was reduced by food deprivation it is assumed that the islet hormone content is not responsible for the rise of the glucose threshold caused by starvation.     

Effects of hemorrhagic shock on alkaline secretion and mucosal tolerance to acid in rat duodenum

The effects of hemorrhagic shock (HE) on duodenal HCO₃ ^– secretion and mucosal tolerance to acid were investigated in anesthetized rats and compared with those of indomethacin. HE was performed by bleeding from the carotid artery to reduce arterial blood pressure to about 50 mm Hg (3 ml bleeding per 200 g of body weight) with a significant decrease in arterial pH and [HCO₃ ^–], and indomethacin was given subcutaneously in a dose of 5 mg/kg. The proximal duodenum (1.7 cm) secreted HCO₃ ^– at the rate of 1.5–1.8 eq/15 min (3.5–4.2 eq/cm/hr), and responded to luminal acid (10 mM HCl for 10 min) by a significant rise in HCO₃ ^– output. Indomethacin had no effect on basal HCO₃ ^– output but significantly inhibited the acid-induced HCO₃ ^– secretion, while under HE conditions duodenal HCO₃ ^– output significantly declined and failed to increase in response to luminal acidification. Subcutaneously administered 16, 16-dmPGE₂ (30 g/kg) significantly increased HCO₃ ^– secretion in the presence of indomethacin but had less effect on the impaired HCO₃ ^– output caused by HE. In contrast, intravenous infusion of NaHCO₃ (3 mmol/kg/hr) ameliorated the acid-base imbalance caused by HE, and significantly restored the impaired HCO₃ ^– responses induced by HE but not by indomethacin. Both HE and indomethacin induced extensive damage in the mucosa when the duodenal loop was perfused with 50 mM HCl for 1.5 hr, and these lesions were significantly reduced by NaHCO₃ infusion and 16, 16-dmPGE₂, respectively. These results suggest that HE impaired duodenal HCO₃ ^– secretion and reduced the tolerance of the mucosa to acid. This effect may be mainly a result of a decrease of HCO₃ ^– availability, but it is not accounted for by a deficiency of endogenous prostaglandins.     

Effect of extracellular pH on insulin secretion and glucose metabolism in neonatal and adult rat pancreatic islets

Changes in extracellular pH are known to affect glucose-stimulated insulin secretion. In the present study, glucose metabolism in pancreatic islets cultured at different pHs was investigated. Also, for islet transplantation purposes, insulin secretion and glucose metabolism were compared in neonatal and adult islets at different pHs to determine which islet preparation is more tolerant to acidity and alkalinity. The results revealed a dependency of insulin secretion on the external pH in both neonatal and adult islets. Reduction of insulin secretion was observed at both the acidic and alkaline sides of pH 7.3. Glucose stimulated increases of insulin secretion in all cases. Similar results were obtained for ATP and pyruvate contents. Intracellular insulin increased with the increase of pH value. In contrast, calcium content decreased with the increase of pH. The results demonstrate that neonatal islets are more acid tolerant than adult islets. Both basal and glucose-stimulated insulin secretions, as well as other parameters of neonatal islets were significantly higher than those of adult islets in response to low pH. The differences under alkaline conditions were not significant but give an indication that neonatal islets are more tolerant to alkalinity than are adult islets. Received: 10 February 2001 / Accepted in revised form: 29 June 2001     

The effects of cyclooxygenase and nitric oxide synthase inhibition on cardiodynamic parameters and coronary flow in isolated rat hearts

BACKGROUND:

Despite the widespread clinical use of cyclooxygenase (COX) inhibitors, dilemmas regarding the potential impact of these drugs on the cardiovascular system persist.

OBJECTIVE:

To estimate the effects of different COX inhibitors (meloxicam, acetylsalicylic acid [ASA] and SC-560) on cardiac function and coronary flow in isolated rat hearts, with special focus on the L-arginine/nitric oxide system.

METHODS:

The hearts of eight-week-old male Wistar albino rats (n=72; 12 rats per group; body mass 180 g to 200 g) were retrogradely perfused according to the Langendorff technique at gradually increased perfusion pressure (40 cmH₂O to 120 cmH₂O). After control experiments, the hearts were perfused with the following drugs: 100 μM ASA, alone or in combination with 30 μM N(ω)-nitro-L-arginine monomethyl ester (L-NAME), 0.3 μM meloxicam with or without 30 μM L-NAME, 3 μM meloxicam with or without 30 μM L-NAME, 30 μM L-NAME and 0.25 μM SC-560. In the control and experimental groups, the following parameters of heart function were continuously recorded: maximum rate of left ventricular pressure development, minimum rate of left ventricular pressure development, systolic left ventricular pressure, diastolic left ventricular pressure, heart rate and mean blood pressure. Coronary flow was measured flowmetrically. The amount of released NO₂⁻ was determined spectrophotometrically in coronary venous effluent.

RESULTS:

While meloxicam and SC-560 were found to have an adverse influence on cardiac function and coronary perfusion, ASA did not negatively affect the intact model of the heart.

CONCLUSION:

It appeared that interaction between COX and the L-arginine/nitric oxide system truly exists in coronary circulation and may explain the causes of the observed effects.     

Histamine-induced vasodilations mediated by H1- and H2-receptors in isolated rat common carotid arteries

Summary Using the cannula inserting method, the vasodilatory effects of histamine were analysed employing selective histamine H₁- and H₂-receptor agonists and antagonists in isolated, perfused rat common carotid arterial preparations which were preconstricted by a continuous infusion of phenylephrine with propranolol. Histamine, 2-pyridylethylamine (2-PEA) (a selective H₁-agonist) and dimaprit (a selective H₂-agonist) produced a vasodilation in a dose-related manner. The order of potency was histamine > dimaprit > 2-PEA. Histamine-induced dilations were significantly inhibited by either diphenhydramine (a selective H₁-antagonist) or cimetidine (a selective H₂-antagonist). 2-PEA-induced dilations were significantly inhibited by diphenhydramine but not by cimetidine. Dimaprit-induced dilations were significantly blocked by cimetidine but not by diphenhydramine. ACh-, histamine-, 2-PEA- and dimaprit-induced dilations were significantly suppressed by removal of the endothelium. From these results, it is concluded that (1) isolated rat common carotid arteries have both H₁-and H₂-receptors, (2) there are few vasoconstrictory H₁-receptors, (3) both H₁- and H₂-receptors mediate only vasodilation but not vasoconstriction, and (4) EDRF from the endothelium might participate in histamine-induced vasodilation via not only H₁- but also H₂-receptors.