Sensitivity of liver cells formed after partial hepatectomy to glucagon, vasopressin and angiotensin II

During the active proliferation which follows partial hepatectomy, the sensitivity of liver cells to glucagon is markedly diminished. In hepatocytes obtained from rats partially hepatectomized 3 days before experiments were performed, the dose-response curves to glucagon were shifted to the right by about two orders of magnitude as compared to those of the control cells. Later on (7 days after surgery) the dose-response to glucagon was still shifted to the right but by only one order of magnitude. These data are consistent with the diminution in the number of glucagon receptors in liver plasma membrane during liver regeneration reported by other authors. No stimulation of glycogenolysis, gluconeogenesis or ureogenesis was produced by vasopressin or angiotensin II in hepatocytes from rats partially hepatectomized 3 days before experimentation. However, phosphatidylinositol labeling was stimulated in these cells to a similar extent as in the controls. The ionophore A23187 was also ineffective in stimulating glycogenolysis in these cells. Later, 7 days after surgery, the hepatic responsiveness to vasopressin and angiotensin II was restored. The data suggest that, during the initial stages of liver regeneration, the enzymatic machinery of the hepatocyte is not sensitive to calcium-signalling.     

Rapid stimulation by vasopressin, oxytocin and angiotensin II of glycogen degradation in hepatocyte suspensions

1. The hormonal control of glycogen breakdown was studied in hepatocytes isolated from livers of fed rats. 2. Glucose release was stimulated by [8-arginine]vasopressin (10pm–10nm), oxytocin (1nm–1μm), and angiotensin II (1nm–0.1μm). These responses are all at least as sensitive to hormone as is glucose output in the perfused rat liver. 3. The effect of these three hormones on glucose release was critically dependent on extracellular Ca²⁺, unlike that of glucagon. Half-maximal restoration of the vasopressin response occurred if 0.3mm-Ca²⁺ was added back to the incubation medium. 4. Glycogen breakdown was more than sufficient to account for the glucose released into the medium, in the absence or presence of hormones. Lactate release by hepatocytes was not affected by vasopressin, but was inhibited by glucagon. 5. If Ca²⁺ was omitted from the extracellular medium, vasopressin stimulated glycogenolysis, but not glucose release. 6. The phosphorylase a content of hepatocytes was increased by vasopressin, oxytocin and angiotensin II; minimum effective concentrations were 0.1pm, 0.1nm and 10pm respectively. This response was also dependent on Ca²⁺. 7. These results demonstrate that hepatocytes can respond to low concentrations of vasopressin and angiotensin II, i.e. these effects are likely to be relevant in the intact animal. The role of extracellular Ca²⁺ in the effects of these hormones on hepatic glycogenolysis and glucose release is discussed.     

Effects of glucagon, Arg-vasopressin, and angiotensin II on rat hepatic zinc thionein levels

Rat hepatic zinc thionein levels can be modulated by a variety of external and internal stimuli. Metals, such as zinc or copper, induce levels 20 to 50 fold over controls. Catecholamines can increase levels 10 to 20 fold, while glucocorticoids, such as dexamethasone, can increase levels modestly by 2-6 fold. We have investigated the ability of additional hormones, which have receptors on hepatocytes, to modulate the levels of hepatic zinc thionein. Glucagon, angiotensin II, and Arg-vasopressin were administered intravenously and intraperitoneally, one time and three times, over an 11 hour period. Zinc thionein levels in rat liver were increased 1.7 to 5.6 fold by glucagon and 1.7 to 3.6 fold by angiotensin II, but not at all by Arg-vasopressin, as compared to appropriate controls. Glucagon and angiotensin II, when administered in vivo, can modulate zinc thionein levels in rat liver to an extent similar to glucocorticoids. Hepatic zinc thionein levels must now be recognized to be affected in vivo by metals, glucocorticoids, catecholamines, and polypeptide hormones.     

Synergistic stimulation of the Ca2+ influx in rat hepatocytes by glucagon and the Ca2+-linked hormones vasopressin and angiotensin II

Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin II, and the effects on the initial 45Ca2+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly increased the initial 45Ca2+ uptake rate. When glucagon was added together with vasopressin or angiotensin II--both of which when added separately increase the initial 45Ca2+ uptake rate but did not affect the cellular content of cyclic AMP--the measured initial 45Ca2+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca2+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or forskolin, suggesting that cyclic AMP augments both the resting Ca2+ and the vasopressin- or angiotensin II-stimulated influx. Measurement of the initial 45Ca2+ uptake rate as a function of the extracellular Ca2+ concentration indicated that the increase in the Ca2+ influx resulting from single or combined glucagon and vasopressin administration occurred through a homogeneous population of Ca2+ gates. These hormones were found to raise both the apparent Km for external Ca2+ and the apparent Vmax of the Ca2+ influx. The maximal increase in these two parameters was observed when the two hormones were added together. This suggests that glucagon and vasopressin synergistically stimulate the same Ca2+ gating mechanism. The dose-response curves for the action of glucagon or vasopressin applied in the presence of increasing concentrations of vasopressin or glucagon, respectively, showed that each hormone increases the maximal response to the other without affecting its ED50. It is proposed that glucagon and the Ca2+-linked hormones control the cellular concentration of two intermediates which are both necessary to allow Ca2+ entry into the cells.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by glycogenolytic hormones. Effects of angiotensin, vasopressin, adrenaline, ionophore A23187 and calcium-ion deprivation

1. The effects on phosphatidylinositol metabolism of three Ca(2+)-mobilizing glycogenolytic hormones, namely angiotensin, vasopressin and adrenaline, have been investigated by using rat hepatocytes. 2. All three hormones stimulate both phosphatidylinositol breakdown and the labelling of this lipid with (32)P. 3. The response to angiotensin occurs quickly, requires a high concentration of the hormone and is prevented by [1-sarcosine, 8-isoleucine]angiotensin, a specific angiotensin antagonist that does not prevent the responses to vasopressin and to adrenaline. This response therefore seems to be mediated by angiotensin-specific receptors. 4. [1-Deaminocysteine,2-phenylalanine,7-(3,4-didehydroproline),8-arginine] vasopressin, a vasopressin analogue with enhanced antidiuretic potency, is relatively ineffective at stimulating phosphatidylinositol metabolism. This suggests that the hepatic vasopressin receptors that stimulate phosphatidylinositol breakdown are different in their ligand selectivity from the antidiuretic vasopressin receptors that activate renal adenylate cyclase. 5. Incubation of hepatocytes with ionophore A23187, a bivalent-cation ionophore, neither mimicked nor appreciably changed the effects of vasopressin on phosphatidylinositol metabolism, suggesting that phosphatidylinositol breakdown is not controlled by changes in the cytosol Ca(2+) concentration. This conclusion was supported by the observation that hormonal stimulation of phosphatidylinositol breakdown and resynthesis persists in cells incubated for a substantial period in EGTA, although this treatment somewhat decreased the phosphatidylinositol response of the hepatocyte. The phosphatidylinositol response of the hepatocyte therefore appears not to be controlled by changes in cytosol [Ca(2+)], despite the fact that this ion is thought to be the second messenger by which the same hormones control glycogenolysis. 6. These results may be an indication that phosphatidylinositol breakdown is an integral reaction in the stimulus-response coupling sequence(s) that link(s) activation of alpha-adrenergic, vasopressin and angiotensin receptors to mobilization of Ca(2+) in the rat hepatocyte.     

Role of angiotensin II and vasopressin in cisplatin-induced emesis

Cisplatin-containing chemotherapy regimens are known to produce intense nausea and vomiting. Angiotensin II (AII) and vasopressin (AVP) have been shown to have emetic properties. The role of these two peptides on cisplatin-induced vomiting was investigated in beagle dogs. Cisplatin (2 mg/kg, IV over 5 min) produced consistent emesis in all dogs after a mean latency time of 144 +/- 4 min. Serum Angiotensin Converting Enzyme (ACE) and plasma AII levels did not significantly change 3 hr after cisplatin administration (at the time of nausea and emesis) in control animals. AVP levels rose from 0.3 pg/ml to 7.5 pg/ml 3 hrs after cisplatin. Complete inhibition of ACE with enalapril (given at 3 mg/kg p.o., 3 hrs prior to cisplatin) reduced AII levels by 70%, but failed to significantly modify the increase in AVP levels (7.2 +/- 2.2 pg/ml), the latency time to emesis (149 +/- 2 min) and the number of emetic episodes induced by cisplatin. These results suggest that AII does not mediate cisplatin-induced emesis, nor does it mediate the increase in AVP observed at the time of emesis. We propose that AVP may be a good marker for nausea and emesis, and that increases in AVP may be neurally-mediated. The large increase in circulating AVP may represent a desirable water conservation response in anticipation of fluid losses induced by vomiting.     

Different effects of vasopressin and angiotensin II on baroreflexes

Our data indicate that vasopressin facilitates baroreflex inhibition of lumbar sympathetic nerve activity by two mechanisms: it sensitizes baroreceptors locally and shifts the stimulus-response curve so that a lower carotid sinus pressure results in a certain level of reflex sympathetic inhibition; it also produces a corresponding shift when given i.v. and excluded from baroreceptors implicating a second, central mechanism for facilitation of baroreflexes. In contrast, angiotensin II attenuates baroreflex inhibition of peripheral sympathetic function and this is accounted for totally by a central action. Why these differences occur present challenging new questions for future investigation.     

Trauma induced increases in plasma vasopressin and angiotensin II

Previous investigators have shown that hypotension will cause an increase in plasma levels of both vasopressin and angiotensin II. Significant increases in peripheral resistance after thermal trauma suggested that a similar increase in plasma vasopressin and angiotensin II levels might occur under this condition. This possibility has been studied in the pentobarbital anesthetized dog. Peripheral resistance was calculated from measured cardiac output and mean arterial blood pressure. Vasopressin and angiotensin II levels were measured by radio-immunoassay. The results of this study showed that vasopressin plasma levels increase 4 to 6 fold 15 minutes after thermal trauma and remained elevated (3 to 4 fold) for at least 6 hours. Angiotensin II increased in a linear manner from 15 minutes to 6 hours post trauma. At 6 hours post trauma angiotensin II plasma levels were 4 times pretrauma levels. For the first 4 hours post trauma there was a positive correlation between the sum of vasopressin and angiotensin II plasma levels and the increase in peripheral resistance. These results suggest that the trauma induced increase in peripheral resistance is due to increases in plasma vasopressin and angiotensin II.     

Effects of adrenaline, angiotensin and calcium on spontaneously active and potassium-depolarized rat portal veins.

1) Adrenaline and angiotensin increased both frequency and developed tension of spontaneously active rat isolated portal veins. Adrenaline 10(-7) M and angiotensin 10(-9) M, produced a maximal increment of the amplitude of twitch contraction. Greater concentrations elicited a tetanic state, which in all cases had the same amplitude as the maximal effects obtained on single twitches. This suggests that there exists a dissociation between membrane and electrical phenomena, so that the highest intensity of the contractile system activity is attained at agonist concentrations lower than those which result in maximal decrease of membrane stability. (2) Increasing K to a 106 mM final concentration caused a persistent contracture. Adrenaline and angiotensin exhibited pharmaco-mechanical properties, as later addition of both substances produced an increase of the contracture tension. (3) Ca dose-response curves were performed on previously Ca-depleted veins, after the addition of K 106 mM, K-plus-adrenaline or K-plus-angiotensin. Both substances made the Ca responses rise steeply, as evidenced by the leftward shift of ED50. This provides support to the belief that pharmacomechanical coupling is mediated through an increase of the membrane permeability to Ca.     

Stimulation of inositol trisphosphate formation in hepatocytes by vasopressin, adrenaline and angiotensin II and its relationship to changes in cytosolic free Ca2+.

At maximally effective concentrations, vasopressin (10(-7) M) increased myo-inositol trisphosphate (IP3) in isolated rat hepatocytes by 100% at 3 s and 150% at 6 s, while adrenaline (epinephrine) (10(-5) M) produced a 17% increase at 3 s and a 30% increase at 6 s. These increases were maintained for at least 10 min. Both agents increased cytosolic free Ca2+ [( Ca2+]i) maximally by 5 s. Increases in IP3 were also observed with angiotensin II and ATP, but not with glucagon or platelet-activating factor. The dose-responses of vasopressin and adrenaline on phosphorylase and [Ca2+]i showed a close correspondence, whereas IP3 accumulation was 20-30-fold less sensitive. However, significant (20%) increases in IP3 could be observed with 10(-9) M-vasopressin and 10(-7) M-adrenaline, which induce near-maximal phosphorylase activation. Vasopressin-induced accumulation of IP3 was potentiated by 10mM-Li+, after a lag of approx. 1 min. However the rise in [Ca2+]i and phosphorylase activation were not potentiated at any time examined. Similar data were obtained with adrenaline as agonist. Lowering the extracellular Ca2+ to 30 microM or 250 microM did not affect the initial rise in [Ca2+]i with vasopressin but resulted in a rapid decline in [Ca2+]i. Brief chelation of extracellular Ca2+ for times up to 4 min also did not impair the rate or magnitude of the increase in [Ca2+]i or phosphorylase a induced by vasopressin. The following conclusions are drawn from these studies. IP3 is increased in rat hepatocytes by vasopressin, adrenaline, angiotensin II and ATP. The temporal relationships of its accumulation to the increases in [Ca2+]i and phosphorylase a are consistent with it playing a second message role. Influx of extracellular Ca2+ is not required for the initial rise in [Ca2+]i induced by these agonists, but is required for the maintenance of the elevated [Ca2+]i.     

Release of purine and pyrimidine nucleosides and their catabolites from the perfused rat hindlimb in response to noradrenaline, vasopressin, angiotensin II and sciatic-nerve stimulation.

Uric acid and uracil were released at constant rates (0.95 and 0.4 nmol/min per g respectively) by the perfused rat hindlimb. Noradrenaline, vasopressin or angiotensin II further increased the release of these substances 2-5-fold, coinciding with increases in both perfusion pressure (vasoconstriction) and O2 uptake. The hindlimb also released, but in lesser amounts, uridine, hypoxanthine, xanthine, inosine and guanosine, and all but hypoxanthine and guanosine were increased during intense vasoconstriction. Uric acid and uracil releases were increased by noradrenaline in a dose-dependent manner. However, the release of these substances did not fully correspond with the dose-dependent increase in O2 uptake and perfusion pressure, where changes in the latter occurred at lower doses of noradrenaline. Sciatic-nerve stimulation (skeletal-muscle contraction) did not increase the release of uracil, uric acid or uridine, but instead increased the release of inosine (7-fold) and hypoxanthine (2-fold). Since the UTP content as well as the UTP/ATP ratio are higher in smooth muscle than in skeletal muscle, it is proposed that release of uric acid and uracil arises from increased metabolism of the respective adenosine and uridine nucleotides during intense constriction of smooth muscle.     

Effects of angiotensin II on autonomic components of nasopharyngeal stimulation in male conscious rabbits.

Angiotensin II (ANG II) is known to activate central sympathetic neurons. In this study we determined the effects of ANG II on the autonomic components of the cardiovascular responses to stimulation of nasopharyngeal receptors with cigarette smoke. Experiments were carried out in conscious New Zealand White rabbits instrumented to record arterial pressure and heart rate. Rabbits were exposed to 50 ml of cigarette smoke before and after subcutaneous osmotic minipump delivery of ANG II at a dose of 50 ng.kg(-1).min(-1) for 1 wk in one group and intracerebroventricular (icv) infusion at a dose of 100 pmol/min for 1 h in a second group. The responses were compared before and after heart rate was controlled by pacing. Autonomic components were evaluated by intravenous administration of atropine methyl bromide (0.2 mg/kg) and prazosin (0.5 mg/kg). ANG II given either systemically or icv significantly blunted the pressor response to smoke (P < 0.05) when the bradycardic response was prevented. This blunted response was not due to an absolute increase in baseline blood pressure after ANG II infusion (71.64 +/- 11.6 vs. 92.1 +/- 19.8 mmHg; P < 0.05) because normalization of blood pressure with sodium nitroprusside to pre-ANG II levels also resulted in a significantly blunted pressor response to smoke. The effect of smoke was alpha(1)-adrenergic receptor-mediated because it was essentially abolished by prazosin in both the pre- and the post-ANG II states (P < 0.05). These results suggest that elevations in central ANG II reduce the sympathetic response to smoke in conscious rabbits. This effect may be due to an augmentation of baseline sympathetic outflow and a reduction in reflex sensitivity similar to the effect of ANG II on baroreflex function.     

Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II

1,2-Diacylglycerol (DAG) was measured in neutral lipid extracts from isolated hepatocytes using high pressure liquid chromatography followed by refractive index detection. Maximally effective doses of epinephrine, angiotensin II, and vasopressin increased DAG by approximately 65, 80, and 180-250%, respectively, with maximal increases being observed at 8-10 min. Depletion of cellular Ca2+ resulted in a 50% decrease in DAG accumulation elicited by vasopressin. Other agents which increased DAG levels were the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (120% increase at 10(-6) M), the Ca2+ ionophore A23187 (385% increase at 10(-5) M), and ATP (180% increase at 1 mM). The concentration dependence of DAG accumulation in response to epinephrine, angiotensin II, and vasopressin was similar to that found for myoinositol triphosphate accumulation (Charest, R., Prpic, V., Exton, J. H., and Blackmore, P.F. (1985) Biochem. J. 227, 79-90), which was approximately 5-10 times less sensitive to hormone than was phosphorylase activation. Fatty acid analysis revealed that hormonally induced DAG was partially derived from sources other than inositol phospholipids. It is proposed from these studies that Ca2+-mobilizing hormones elicit a prolonged increase in the levels of hepatocyte DAG, which may activate protein kinase C.     

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.

The effects of glucagon and insulin administration $\text{in vivo}$ on hepatic mitochondrial Ca²⁺ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both $\text{in vivo}$ and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca²⁺ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca²⁺ by mitochondria, subsequently isolated. The possible implications of the results are discussed.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

Ca²⁺ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca²⁺-dependent enzymes. However, excessive mitochondrial Ca²⁺ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca²⁺ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca²⁺ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca²⁺ levels ([Ca²⁺]_c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca²⁺ content (Ca^2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca²⁺]_c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca²⁺ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca²⁺m that was not further increased during prolonged incubations, despite a sustained [Ca²⁺]_c elevation. By contrast, thapsigargin induced a net Ca²⁺ accumulation in mitochondria that continued for up to 30 min and reached Ca^2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca²⁺ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca²⁺ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca²⁺ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca²⁺ through artificial means, enabling mitochondria to function as a Ca²⁺ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)     

Inhibition of lipogenesis by vasopressin and angiotensin II in glycogen-depleted hepatocytes

Vasopressin and angiotensin II inhibited lipogenesis (measured with 3H2O) in hepatocytes from fed rats. Inhibition was also observed with hepatocytes from fed rats which had been depleted of glycogen in vitro and incubated with lactate + pyruvate (5 mM + 0.5 mM) as substrates. The inhibitory actions of the hormones are therefore independent of hormone-mediated changes in glycogenolytic or glycolytic flux from glycogen, and thus the site(s) of hormone action must be subsequent to the formation of lactate. (-)Hydroxycitrate, a specific inhibitor of ATP-citrate lyase, decreased lipogenesis in hepatocytes from fed rats incubated with lactate + pyruvate by approx. 51% but had little effect on lipogenesis in glycogen-depleted hepatocytes similarly incubated. There was parallel inhibition of incorporation of 14C from [U-14C]lactate into fatty acid and lipogenesis as measured with 3H2O in each case. Thus depletion of glycogen, or conceivably the process of glycogen-depletion (incubation with dibutyryl cyclic AMP) causes a change in the rate-determining step(s) for lipogenesis from lactate. Vasopressin and angiotensin II also decreased lipogenesis and incorporation of 14C into fatty acids in glycogen-depleted hepatocytes provided with [U-14C]proline as opposed to [U-14C]-lactate. However, proline-stimulated lipogenesis was inhibited by (-)hydroxycitrate, and proline-stimulated lipogenesis and incorporation of 14C from [U-14C]-proline were not decreased in parallel by this inhibitor (inhibition of 52% and 85% respectively). It is inferred that lactate and proline stimulate lipogenesis by different mechanisms and incorporation of 14C from [U-14C]proline and [U-14C]lactate into fatty acid occurs via different routes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of mitochondrial function. Description of a system capable of mimicking several effects of glucagon

Isolated rat liver mitochondria were incubated at 0 degrees C in a medium consisting of 225 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM KH2PO4, 5 mM MgCl2 and 10 mM Tris-HCl, pH 7.4 (buffer 1) for 10 min, centrifuged and resuspended in 0.3 M sucrose. This treatment resulted in a stimulation of mitochondrial functions, mimicking several of the effects that follow glucagon treatment of the intact rat or isolated hepatocytes. Both phosphate and potassium are required for this effect; the addition of magnesium serves to enhance it. Mitochondrial respiration is essential for the development of the activated state as the stimulation is blocked by increasing concentrations of rotenone in the incubation. The intramitochondrial ATP/ADP ratio is increased, but when this increase was prevented by including low levels of rotenone or oligomycin in buffer 1, the stimulation of mitochondrial function was not diminished, thus demonstrating that an increased ATP/ADP ratio is not essential for activation. The rate of citrulline formation was unaffected by buffer 1 treatment unless glutamate was also included in the medium, indicating that control of this mitochondrial function differs from other functions studied.     

Cytosolic free calcium levels in monolayers of cultured rat aortic smooth muscle cells. Effects of angiotensin II and vasopressin

A rise in cytosolic free calcium ([Ca2+]i) is thought to be the principal mediator in vascular smooth muscle contraction. Quantitative changes of [Ca2+]i in response to two vasoconstrictor peptide hormones, angiotensin II and vasopressin, were directly measured in monolayers of adherent cultured rat aortic smooth muscle cells loaded with the fluorescent calcium indicator Quin 2. Angiotensin II induced rapid, concentration-dependent rises in [Ca2+]i from 1.53 +/- 0.27 X 10(-7) (n = 16) up to 1.2 X 10(-6) M, with ED50 of 0.45 X 10(-9) M, an effect which was blocked by the antagonist analogue [Sar1, Ala8]angiotensin II. Vasopressin also elicited transient rises in [Ca2+]i to peak levels of about 8 X 10(-7) M, with ED50 of 1.05 X 10(-9) M, and this response was completely abolished by a vasopressor antagonist. In calcium-free medium, basal [Ca2+]i levels fell to 0.92 +/- 0.24 X 10(-7) M (n = 4), and both hormones were still able to raise [Ca2+]i, although to a lesser extent. Readdition of extracellular calcium following the [Ca2+]i transient induced a second, slower [Ca2+]i rise. In calcium-containing medium, lanthanum ion (2 X 10(-5) M) reduced peptide-evoked [Ca2+]i rises to the values observed in calcium-free medium. Stimulation with each peptide completely desensitized the smooth muscle cells to a subsequent identical challenge, with little crosstachyphylaxis. Potassium ion (50 mM) only minimally affected [Ca2+]i levels. The calcium channel blocker nifedipine (10(-6) M) did not prevent the [Ca2+]i rises induced by angiotensin II, vasopressin, or potassium. These findings indicate that the two physiologically important vasoconstrictor hormones angiotensin II and vasopressin rapidly raise [Ca2+]i in cultured vascular smooth muscle cells, in part by mobilizing calcium from intracellular pools and in part through activation of receptor-operated calcium channels.     

Glycogenolytic responsiveness to glucagon, epinephrine, vasopressin and angiotensin II in the liver of developing hypothyroid rats. A comparative study of in vitro hormonal binding and in vivo biological response

Both dose-response curves and time-courses of plasma glucose levels after single maximal doses showed that in vivo glycogenolytic responsiveness to glucagon and epinephrine was significantly higher in developing hypothyroid rats, whereas it remained unchanged after vasopressin and angiotensin II injections. In contrast with the decreased basal activity of phosphorylase(a), the glucagon-stimulated activity increased in hypothyroid rats, whereas it was only slightly modified under vasopressin stimulation. Daily thyroxine treatment abolished these abnormalities. Thus, there is a close correlation between glucose output and enzyme activation. The maximal binding capacity of [3H]vasopressin and [125I]glucagon was significantly decreased in hypothyroid rats, without changes in the apparent dissociation constant of hormone from its specific receptor. Daily thyroxine treatment also abolished this deficit, which moreover appeared to be independent of possible changes in plasma hormone levels. With respect to glucagon action, neither basal nor Gpp(NH)p-stimulated adenylate cyclase activities were affected in hypothyroid rats. Glucagon-sensitive adenylate cyclase activity and the apparent activation constant appeared to be unaffected. The apparent discrepancy between the results obtained from in vivo and in vitro experiments is discussed on the basis of different membrane transducing phenomena and related intracellular mechanisms underlying the biological response to hormonal stimulation.