Effect of somatomedin A and insulin on cyclic AMP generation in isolated rat hepatocytes

Insulin and somatomedin A were shown to have inhibitory action on glucagon stimulated but not basal cyclic AMP production in isolated rat hepatocytes. The inhibition was dose-dependent and the potency per mol was about 100 fold higher for insulin than for somatomedin A.     

The effect of ionophore A23187, verapamil, and dibutyryl cyclic AMP on bile acid synthesis in isolated rat hepatocytes

The effect of the divalent cation ionophore A23187 and the calcium channel-blocker verapamil on bile acid synthesis in isolated hepatocytes in the presence and absence of dibutyryl cyclic AMP was studied. Both A23187 (1 microM) and verapamil (0.04 mM) caused a small (approximately 15-20%) but consistent decrease in total bile acid synthesis in the cells. When hepatocytes were incubated with dibutyryl cyclic AMP (1 mM) production of total bile acid was increased by about 25%, and this effect was unchanged by A23187 but abolished by verapamil. The relative proportions of the individual bile acids produced were not affected by either A23187 or verapamil. Dibutyryl cyclic AMP (1 mM) lowered the ratio of the amount of conjugated cholic acid to conjugated chenodeoxycholic + beta-muricholic acid formed in the cells by about 50%. Neither A23187 nor verapamil was able to prevent this change. These results suggest that the stimulatory effect of dibutyryl cyclic AMP on total bile acid synthesis is dependent on mobilisation of calcium from intracellular stores, but its effect on the relative proportions of bile acid formed via the cholic acid versus the chenodeoxycholic acid pathway is independent of calcium movement.     

Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes

Separate or combined addition of cyclic AMP-dependent and Ca2+-linked hormones to isolated rat hepatocytes suspended in a low Ca2+ medium reduced the total cellular Ca. When the hormones were administered together, their effects were not additive. This suggests that both types of hormones mobilize Ca2+ from a common intracellular pool. In the presence of 1.8 mM extracellular Ca2+, the Ca2+ influx counterbalanced or even exceeded the hormone-induced Ca2+ loss, depending on the ability of the hormones to stimulate the Ca2+ influx.     

Glucose synthesis by isolated guinea-pig hepatocytes. Effect of cyclic AMP and dibutyryl cyclic AMP.

In isolated guinea-pig hepatocytes, dibutyryl cyclic AMP stimulated gluconeogenesis from 2 mM galactose by 25 and 40% respectively. In the presence of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose synthesis from lactate and galactose by 26 and 34% respectively.     

Effect of sodium fluoride on cyclic AMP production in rat hepatocytes.

The effect of NaF on cAMP production was studied in hepatocytes isolated from fed and fasted rats. A four-six fold increase in hepatocyte cAMP production was observed in the presence of 10-20 mM NaF in cells isolated from either fed or fasted rats. The maximal stimulation of cAMP production was observed after a 10 min incubation in the presence of 1 mM theophylline. However, as little as 0.05-0.15 mM NaF induced a significant increase in cAMP production. It was also found that NaF would alter the production of glucose in isolated rat hepatocytes. When hepatocytes from fed rats were incubated with 0.05-5 mM NaF there was an increase in amount of glucose released from endogenous sources. Also NaF resulted in a decrease in lactate and pyruvate production. Similarly NaF stimulated glucose production in hepatocytes from fasted rats. The maximal stimulation was observed with about 0.15-0.25 mM NaF. At NaF concentrations greater than 1.5 mM a decrease in glucose production was observed. It is concluded that NaF increases the level of cAMP and alters glucose metabolism in intact hepatocytes.     

Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Na+/H+ exchange in acid-loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl-carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride-sensitive Na(+)-dependent recovery from cytoplasmic-acid-loading was found to be increased in cells treated with epidermal growth factor (EGF), 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (ClPhScAMP) or phorbol 12-myristate 13-acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid-loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride-inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.     

The effect of carbon tetrachloride on isolated rat hepatocytes

Isolated rat hepatocytes were incubated with carbon tetrachloride (CCl4) at a concentration of 0.2 mol CCl4/ml of incubation medium. The ultrastructural alterations and release of lactate dehydrogenase (LDH) and glutamate-oxaloacetate transaminase (GOT), were recorded after different periods of incubation. After 5 min incubation with CCl4, morphological changes observed by electron microscopy, involved the plasma membrane. The endoplasmic reticulum and mitochondria were altered later. These morphological alterations were accompanied by an early release of LDH and GOT into the incubation medium. It is concluded that, in contrast with its in vivo effects, in vitro CCl4 can induced an early morphological alteration of the hepatocyte plasma membrane before damaging the endoplasmic reticulum.     

The effect of dibutyryl cyclic AMP on the excretion of taurocholic acid from isolated rat liver cells

Dibutyryl cyclic AMP (50-1000 microM) was found to increase the initial rate of efflux of taurocholic acid from isolated rat hepatocytes. Efflux of the bile acid was inhibited by sodium, and in the absence of sodium dibutyryl cyclic AMP failed to stimulate the rate. Increasing the concentration of calcium from 0 to 1.2 mM had no effect on the initial rate of taurocholic acid efflux from the cells, but the absence of calcium markedly reduced the effect of dibutyryl cyclic AMP. The results suggest that changes in the fluxes of sodium and calcium are involved in the effect of the cyclic nucleotide on taurocholic acid efflux from the cells.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Differential rates of glycoprotein secretion by isolated rat hepatocytes studied in terms of concanavalin A binding.

Using a concanavalin-A-based method which respects cell function, we have shown that the kinetics of glycoprotein secretion appear to depend on the nature of the oligosaccharide moiety. In 37 degrees C pulse/chase experiments using freshly isolated normal rat hepatocytes, we found that except for transferrin, whose rate of secretion was independent of its concanavalin A reactivity, the secretion of the concanavalin-A-retained forms of alpha 1 acid glycoprotein, T-kininogen, alpha 1 protease inhibitor and alpha 1 inhibitor III was slower than that of the concanavalin-A-non-retained forms. When hepatocytes were incubated at 20 degrees C, secretion was blocked with the accumulation of mainly endoglycosidase-H-sensitive forms. The secretion kinetics of the concanavalin-A-differentiated forms were still different when the temperature was shifted back to 37 degrees C. The divergence between the secretion rates of the concanavalin-A-differentiated forms would appear to be due to a late event in intracellular protein trafficking, which may depend on the sugar content and/or the number of carbohydrate chains of the glycoproteins.     

Formation of cyclic AMP from exogenous ATP by isolated hepatocytes and adipocytes

Suspensions of isolated rat hepatocytes and adipocytes converted exogenous ATP to cyclic AMP at a rate which was about 30--50% of that observed with homogenates of isolated cells. Formation of cyclic AMP was stimulated by hormones (isoprenaline in the case of adipose tissue and glucagon in the case of liver) and sodium fluoride. Experiments with [alpha-32P]ATP indicated that the conversion of exogenous ATP to cyclic AMP did not occur within the cells. It is proposed that in isolated hepatocytes ad adipocytes some catalytic units of adenylate cyclase are exposed on the outer surface of the cell membrane.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Effect of endogenous LH secretion on ovarian cyclic AMP and cyclic GMP levels in the rat

Injection of LH (2 and 10 μg) into proestrus rats increased ovarian cyclic AMP levels and concomitantly decreased the levels of cyclic GMP. When injected into diestrus rats, cyclic AMP increases were even greater, whereas cyclic GMP levels were not significantly different from controls receiving saline injections. Ovarian cyclic nucleotide levels were also examined on different days of the cycle. On the afternoon of proestrus (1700 h), the time when circulating levels of LH are at their maximum, the concentration of cyclic AMP showed a moderate but insignificant increase. At the same time, cyclic GMP levels were significantly decreased. An inverse relation between cyclic AMP and cyclic GMP levels was seen on each day of the cycle. When rats were injected with pentobarbital (35 mg/kg) on the afternoon of proestrus (1300 h) to block the LH surge, the expected increases in ovarian cyclic AMP and decreases in cyclic GMP were effectively blocked. These results indicate that ovarian cyclic AMP and cyclic GMP levels are regulated by circulating LH. The apparent differences in direction of nucleotide response to LH, suggest divergent roles for the nucleotides in ovarian function.     

An effect of glucagon on 3', 5'-cyclic AMP phosphodiesterase activity in isolated rat hepatocytes.

Glucagon was found to activate the low Km form of 3′,5′-cyclic AMP phosphodiesterase in intact isolated rat hepatocytes while the high Km phosphodiesterase was unaltered. Activation was concentration dependent and occurred at the same concentration required to observe an increase in 3′,5′-cyclic AMP levels in the cell. The maximal increase in activity occurred within 5 minutes of incubation with glucagon and was sustained for the 35 minutes assayed.     

Synthesis and secretion of ceruloplasmin by isolated rat hepatocytes

The synthesis and secretion of ceruloplasmin (Cp) by isolated rat hepatocytes were investigated. Cp released by liver cells appeared to have properties similar to those of the blood-circulating protein, i. e. Mr, oxidase activity, immunological specificity and the peptide set of tryptic fingerprints. The polypeptides with Mr of 130,000, 65,000, 48,000 and 18,000 were revealed in Cp isolated from the incubation medium. These results suggest the susceptibility of the single-chain protein molecule (Mr 130,000) to limited proteolysis which is accomplished by the proteases released from the cells. When fresh serum was added to the incubation medium, the proteolytic degradation of Cp proceeded at a much slower rate, which led to an increase in the content of excreted polypeptides with Mr 130,000. The secretion was strongly diminished by the addition of colchicine to the medium. The time of Cp molecule synthesis on membrane-bound polyribosomes (3.5 min) was determined.