Cytosolic Ca Levels Research Articles

O-339 - Effects of cyclopiazonic acid, a novel Ca-ATPase inhibitor, on agonist-induced contraction and cytosolic Ca level in skinned and intact smooth muscles

O-339 - Effects of cyclopiazonic acid, a novel Ca-ATPase inhibitor, on agonist-induced contraction and cytosolic Ca level in skinned and intact smooth muscles

On the mechanism of action of dexamethasone in a rat mast cell line (RBL-2H3 cells). Evidence for altered coupling of receptors and G-proteins.

Prolonged exposure of rat basophilic leukemia (RBL-2H3) cells, a cultured analog of rat mast cells, to 0.1 microM dexamethasone resulted in global suppression of various stimulatory events in response to Ag and a global enhancement of the same stimulatory events to the adenosine analog, N-(ethylcarboxamide)adenosine (NECA). We had previously shown that Ag and NECA both activate phospholipase C but by different mechanisms; cells that had been treated with cholera or pertussis toxin, for example, responded to Ag but not to NECA with the release of inositol phosphates, increase in levels of cytosolic Ca2+, and secretion. Because the toxins still inhibited the responses to NECA in dexamethasone-treated cells, the effects of dexamethasone may have been exerted at the level of receptor/G-protein coupling rather than at the level of effector systems. Additional evidence for this was the following: 1) NECA-induced hydrolysis of the inositol phospholipids was still enhanced after permeabilizing (with streptolysin O or Staphylococcus alpha-toxin) and washing the cells; 2) the response to the G-protein stimulant, guanosine 5'-(3-O-thio)triphosphate was also enhanced in permeabilized, dexamethasone-treated cells and 3) binding and kinetic studies suggested that the enhanced responsiveness to NECA was attributable in part to an increase in receptor number. The suppressive action of dexamethasone on Ag-induced hydrolysis of inositol phospholipids, however, was readily lost by permeabilizing RBL-2H3 cells. The results indicate, therefore, that treatment with dexamethasone leads to changes in receptor-coupling mechanisms that are either resistant to (i.e., NECA-mediated responses) or reversed by (i.e., Ag-mediated responses) cell permeabilization.

Inhibitory effects of cadralazine and its metabolite, ISF-2405, on contractions and the level of cytosolic Ca 2+ in vascular smooth muscle

The inhibitory effects of a hypotensive agent, cadralazine and its metabolite, ISF-2405, on the level of cytosolic Ca 2+ ([Ca 2+] cyt) and on contractions were examined in isolated vascular smooth muscle. Cadralazine slightly inhibited the transient norepinephrine-induced contraction in rabbit aorta and canine femoral, renal and mesenteric arteries and saphenous vein, and prostaglandin F 2α-induced contractions in canine basilar and coronary arteries. In contrast, ISF-2405 inhibited the contractions induced by prostaglandin F 2α in canine basilar and coronary arteries and those induced by norepinephrine in canine renal and femoral arteries and rabbit aorta. In aorta, ISF-2405 inhibited the increase in [Ca 2+] cyt and muscle tension caused by norepinephrine. A Ca 2+ channel blocker, verapamil, inhibited the norepinephrine-stimulated increase in [Ca 2+] cyt more potently than it inhibited the increase in muscle tension, and ISF-2405 inhibited the verapamil-resistant part of the contraction. In Ca 2+-free solution, norepinephrine induced transient increases in [Ca 2+] cyt and muscle tension. ISF-2405 inhibited these changes. However, ISF-2405 did not inhibit the transient contraction induced by caffeine in the aorta. These results suggest that cadralazine is metabolized to ISF-2405 and inhibits vascular smooth muscle contraction by inhibiting receptor-mediated Ca 2+ influx, Ca 2+ release and Ca 2+ sensitivation of contractile elements.

Greater Contraction Is Induced by Pilocarpine than by Carbachol at the Same Levels of Cytosolic Ca2 + Concentration in Isolated Longitudinal Muscle of Guinea Pig Ileum

Pilocarpine, which activates propylbenzi1y1cho1ine mustard (PrBCM)-sensitive cho1inoceptors, and carbachol, which activates PrBCM-resistant ones, induced an increase in cytosolic Ca2+ concentration ([Ca2+]i) and tension development in a concentration dependent manner in isolated longitudinal muscle of guinea pig ileum. A positive correlation between [Ca2+]i and tension development due to both of the agonists was noted. The slope of regression line between two values for pilocarpine was steeper than that for carbachol, suggesting that pilocarpine induced greater tension for a given increase in [Ca2+]i than did carbachol. Thus an activation of PrBCM-sensitive cholinoceptors might enhance the Ca2+-sensitivity of the contractile elements.

Interactive α- and β-adrenergic actions of norepinephrine in rat cardiac myocytes

We investigated the effects of the physiological neurotransmitter norepinephrine on the contractile properties and Ca 2+ dynamics of isolated cardiac myocytes, with particular emphasis on possible interactions between α- and β-adrenergic effects. Individual rat ventricular myocytes were electrically stimulated at a frequency of 1 Hz. Norepinephrine (10 −9 to 10 −5 m) increased extent and velocity of shortening and decreased the contraction duration. β-Adrenergic activation gave a greater enhancement of extent and velocity of shortening than did norepinephrine alone (i.e. α plus β). Neither α 1 nor α 2 adrenergic activation individually produced a significant impact upon contraction. Using suspensions of myocytes loaded with Quin-2, we also studied resting levels of cytosolic Ca 2+ ([Ca 2+] c), the increase of [Ca 2+] c due to caffeine-addition (as an index of sarcoplasmic reticulum Ca 2+ content) and the subsequent increase in [Ca 2+] c due to depolarization with 30 m m K + (as an index of sarcolemmal voltage-dependent Ca 2+ channel activity). Norepinephrine decreased resting [Ca 2+] c, increased sarcoplasmic reticulum Ca 2+ content and increased Ca 2+ channel activity. β-Adrenergic activation produced the same effect on resting [Ca 2+] c and sarcoplasmic reticulum content, but gave significantly greater activation of sarcolemmal Ca 2+ channel activity, than did norepinephrine (α plus β). By contrast, α-adrenergic stimulation had no effect on resting [Ca 2+] c or sarcoplasmic reticulum Ca 2+ content. We conclude that β-mediated effects predominate in the action of the physiological agonist norepinephrine on cardiac myocytes. However, α (specifically α 1)-adrenergic effects are significant in diminishing the potentiation of the extent and velocity of shortening, and of depolarization-induced entry of Ca 2+ into the cell, which is seen on β-stimulation alone. Thus, there may be an intrinsic feedback effect in the actions of norepinephrine on the cardiac myocyte.

Characterization of the relation between sodium channels and electrical activity in cultured rat skeletal myotubes: regulatory aspects

The relation among sodium channel density, frequency of electrical activity and maximal rate of rise of the action potential was studied in developing and mature rat skeletal myotubes in culture. The number of tetrodotoxin (TTX)-sensitive Na-channels was determined by measurements of the amount of [ 3H]saxitoxin (STX) bound to the cultures, and electrical properties were recorded with intracellular microelectrodes. The EC 50 for TTX-induced decreases in maximal STX-binding, frequency and rate of rise of action potentials was in the range 8–20 nM. The 3 variables increased in parallel with age in culture to reach peak values at age 7–8 days, and then decreased in parallel until 10–12 days in culture. The age-related increase in Na-channel density was decreased, but not abolished, by prevention of myoblast fusion. Treatment with the Ca 2+ ionophore, A23187, down-regulated, and blockade of Ca-channels with verapamil up-regulated the number of Na-channels. Na-channel density was also increased by chronic treatment with TTX and elevated external [K +], which eleminated spontaneous electrical and contractile activity. Parallel effects were observed on frequency and rate of rise of action potentials. Up-regulation of Na-channels was prevented by simultaneous treatment of myotubes with inhibitors of protein synthesis. We conclude that electrical and mechanical activity of cultured myotubes regulate de novo synthesis of Na-channels through alterations in the level of cytosolic Ca 2+.

Greater tension is developed at the same level of cytosolic Ca2+ concentration in the presence of clonidine, an adrenergic partial agonist, than in the presence of norepinephrine.

Fura 2-loaded thoracic aorta strips from rabbits were used. Norepinephrine and clonidine induced an increase in cytosolic Ca2+ concentration ([Ca2+]i) and in muscle tension in a concentration-dependent manner. A positive correlation between [Ca2+]i and tension development due to norepinephrine and clonidine was noted in muscle strips both untreated and treated with phenoxybenzamine (PBZ). As the total receptor population was reduced by the PBZ treatment, norepinephrine acted as a partial agonist in the treated strips. The slope of a regression line for norepinephrine in the untreated muscle strips was significantly smaller than the slopes for the same drug in PBZ-treated strips and for clonidine, suggesting that norepinephrine in the PBZ-treated strips and clonidine induced a greater tension at same [Ca2+]i than did norepinephrine in the untreated strips.

Inhibition by staurosporine of mitogen-induced calcium mobilisation in human T lymphoblasts

Addition of monoclonal antibodies (UCHT1) directed against the antigen receptor/CD3 complex or the addition of the mitogens concanavalin A or phytohemagglutinin to human T lymphoblasts induced an elevation of the cytosolic free calcium concentration ([Ca 2+] i) as monitored by the fluorescence of the intracellular fura-2. The rise in [Ca 2+] i induced by these agents was effectively inhibited by staurosporine, an agent which inhibits protein kinase (PK)C and PKA. Conversely the PKC activator 12- O-tetradecanoyl phorbol-13-acetate decreased the delay and accelerated the rate of elevation of [Ca 2+] i induced by the mitogens and UCHT1. Increasing the concentration of cAMP or cGMP in the T lymphoblasts had no effect on agonist-induced elevation of [Ca 2+] i. Our data indicate that PKC can exert a positive feedback on the level of cytosolic Ca 2+ in T lymphocytes, in contrast to what has been observed with other cells.

Mobilization of Ca2+ from intracellular stores of pancreatic beta-cells by the calcium store blocker TMB-8.

TMB-8 has been used experimentally in many cell types, including endocrine cells, because of its ability to block the efflux of Ca2+ from intracellular stores without affecting influx. Unexpectedly, TMB-8 potentiates stimulated insulin release from pancreatic islets, a process believed to be dependent on the level of cytosolic Ca2+. In the present study, while having no effect on basal insulin release (in the presence of 2.8 mM glucose), TMB-8 (10, 30, and 100 microM) caused a concentration-dependent increase in 45Ca2+ efflux from 45Ca2+-preloaded islets. TMB-8 (100 microM) stimulated 45Ca2+ efflux even in the absence of extracellular Ca2+. In the presence of 5.6 mM glucose, TMB-8 (30 and 100 microM) potentiated insulin release and again increased 45Ca2+ efflux in a concentration-dependent manner. Similarly, insulin release stimulated by isobutylmethylxanthine (IBMX) was potentiated significantly, and IBMX-stimulated 45Ca2+ efflux was increased by the simultaneous introduction of 30 microM TMB-8. Thus, in pancreatic islets, TMB-8 appears to mobilize Ca2+ from intracellular stores, rather than inhibit the efflux as has been commonly accepted. In further studies, using insulin-secreting beta-cells of the RINm5F cell line, TMB-8 was shown to increase the cytosolic Ca2+ concentration in the presence and absence of extracellular Ca2+. This confirmed that mobilization of intracellular Ca2+ was occurring in the pancreatic beta-cell in response to TMB-8. Furthermore, a rise in cytosolic Ca2+ of not more than 10 nM (as induced with KCl) was found to mimick the effect of TMB-8 in conjunction with IBMX. No additional effect of TMB-8 to alter Ca2+ handling at the plasma membrane was found when 45Ca2+ uptake experiments were performed. Therefore, the paradoxical mobilization of beta-cell Ca2+ by TMB-8 appears to be a sufficient explanation for its potentiating effect on the rate of insulin secretion.

Relationship between Cytosolic Free Calcium Concentration and 2-Deoxyglucose Uptake in Adipocytes Isolated from 2- and 12-Month-Old Rats*

We have examined the relationship between insulin-stimulated 2-deoxyglucose uptake and cytosolic free calcium concentrations, [( Ca2+]i), in adipocytes isolated from 2- and 12-month-old rats. The basal rates of glucose uptake and the levels of cytosolic Ca2+ were only minimally reduced in 12-month-old animals. In contrast, insulin-stimulated glucose up-take and [Ca2+]i were significantly decreased in older adipocytes at all insulin concentrations (P less than 0.01). When the rate of glucose uptake was plotted as a function of [Ca2+]i, insulin-stimulated glucose uptake was almost identical in older and younger animals at any given level of [Ca2+]i. Similar to insulin, glyburide and K+ increased [Ca2+]i in both younger and older adipocytes. However, glyburide- and K+-elicited responses were lower in older rats (P less than 0.01). The effects of insulin, glyburide, and K+ on [Ca2+]i are mediated via voltage-dependent Ca2+ channels. Thus, the present observations suggest an impairment in either function and/or availability of the voltage-dependent Ca2+ channels in older animals. This was supported by the finding of reduced [3H]nitrendipine binding in adipocytes isolated from older animals (6.5% vs. 3.3% in 2- and 12-month-old rats, respectively; P less than 0.01). The results of these experiments indicate that the postreceptor changes in adipocyte responsiveness to insulin in aging may involve inadequate increases in [Ca2+]i. The latter probably occurs as a result of decreased availability and/or function of the voltage-dependent calcium channels.

α-Adrenergic regulation of rat parotid acinar cell cytosolic ca levels and cl efflux

α-Adrenergic regulation of rat parotid acinar cell cytosolic ca levels and cl efflux

Atriopeptins decrease resting and hormone-elevated cytosolic Ca in cultured mesangial cells.

The purpose of this work was to investigate the effects of atrial peptides on the cytosolic Ca levels of cultured, adherent, mesangial cells. Resting Ca levels, measured by the use of fura-2, were dose-dependently decreased by up to 30%, by atriopeptin 23. The half-maximal effect was elicited by approximately 30 pM atriopeptin-23. Atriopeptin 21 also decreased resting Ca levels by up to 30%, but this peptide was approximately 30-fold less potent than atriopeptin 23. Atriopeptin 23 (100 nM) inhibited both the Ca transient and the peak Ca value elicited by 1 nM vasopressin, but the atrial peptide had no significant effect on the Ca transient elicited by higher vasopressin concentrations. At concentrations lower than 100 nM, atriopeptin 23 had no effect on the vasopressin-induced Ca transient. Atriopeptin 23 also decreased the Ca transient and the peak Ca value elicited by 100 nM angiotensin II. Similar to vasopressin, the angiotensin II-induced Ca transient was inhibited by 100 nM but not lower concentrations of atriopeptin 23. In contrast to the effect of atriopeptin 23, atriopeptin 21 (100 nM) had no effect on the Ca transient elicited by vasopressin. These results demonstrate that atriopeptins function as modulators of resting and, under certain conditions, of hormone-increased Ca levels in cultured mesangial cells.

Effect of diltiazem on altered cellular calcium regulation during endotoxic shock.

Cellular free and exchangeable Ca2+ were quantitated in livers of rats injected with saline (control), Salmonella enteritidis endotoxin (20 mg/kg), or diltiazem (0.8-1.2 mg/kg) plus the endotoxin (20 mg/kg). Injection of endotoxin alone resulted in signs of endotoxic shock in animals 5 h later. All rats were killed 5 h postinjection, and their livers were excised. Hepatocytes were prepared for the measurement of cytosolic [Ca2+] under basal and hormone-stimulated (1 and 10 microM epinephrine) conditions by means of quin 2 fluorescence technique. Liver slices were labeled with 45Ca, and the radioactivity was washed out to determine cellular exchangeable Ca2+. The apparent basal level of cytosolic Ca2+ in endotoxic rat hepatocytes [525 +/- 92 (SE) nM] was significantly (P less than 0.05) higher than in controls (146 +/- 23). A significant increase in the basal [Ca2+] occurred after stimulation with epinephrine in controls but not in endotoxic rat hepatocytes. In the endotoxic rats receiving diltiazem, basal [Ca2+] was significantly lower than in rats given endotoxin alone, and a significant elevation of cytosolic [Ca2+] with epinephrine also occurred. The cellular exchangeable Ca2+ in endotoxic rats (564 +/- 27 nmol/g) was significantly higher than in controls (427 +/- 23). Diltiazem treatment of endotoxic rats restored the exchangeable Ca2+ level to that found in controls. The endotoxic accumulation of cellular Ca2+ and its restoration toward control levels by diltiazem could be due to catecholamine-mediated Ca2+ influx and its blockade by diltiazem.

Ca2+ permeability and cytosolic Ca2+ concentration are not impaired in beta-thalassemic and hemoglobin C erythrocytes

Total calcium content, determined by atomic absorption spectroscopy, Ca2+ influx, and cytosolic free Ca2+ concentration ( [Ca]i), estimated by a method involving the incorporation of a Ca2+ chelator (Quin 2), were measured in erythrocytes from beta-thalassemic (beta-thal) and hemoglobin C (CC) patients. Elevation of the total calcium content was observed in the cells from all patients, particularly in CC and splenectomized beta-thal. However, [Ca]i was within the normal range (approximately 25 nmol/L) in all the pathologic cells. Ca2+ influx in CC cells and in cells from nonsplenectomized beta-thal patients was also within the same range as that observed in control erythrocytes. In cells from splenectomized beta-thal patients, the kinetic of 45Ca influx was biphasic, indicating the existence of two pools of exchangeable Ca2+. Density fractionation of the cells from one splenectomized beta-thal patient showed that the rapid pool corresponded to the lightest cell fraction, which was also found to have the highest calcium content. The dense cells exhibited a normal Ca2+ influx as well as a smaller increase in total calcium content. It is suggested that, as in sickle cell anemia, the excess of Ca2+ in beta- thal cells is not free in the cytoplasm but trapped within endocytic vacuoles, especially in a population of abnormal cells that are normally removed by the spleen. In CC patients, who have a functional spleen, a different mechanism could be responsible for the calcium retention. In conclusion, the present results demonstrate that in these two cases of hemolytic anemia associated with high calcium content, Ca2+ permeability and the the level of cytosolic Ca2+ are normal.

Ca2+ Permeability and Cytosolic Ca2+ Concentration Are Not Impaired in β-Thalassemic and Hemoglobin C Erythrocytes

Total calcium content, determined by atomic absorption spectroscopy, Ca2+ influx, and cytosolic free Ca2+ concentration ( [Ca]i), estimated by a method involving the incorporation of a Ca2+ chelator (Quin 2), were measured in erythrocytes from beta-thalassemic (beta-thal) and hemoglobin C (CC) patients. Elevation of the total calcium content was observed in the cells from all patients, particularly in CC and splenectomized beta-thal. However, [Ca]i was within the normal range (approximately 25 nmol/L) in all the pathologic cells. Ca2+ influx in CC cells and in cells from nonsplenectomized beta-thal patients was also within the same range as that observed in control erythrocytes. In cells from splenectomized beta-thal patients, the kinetic of 45Ca influx was biphasic, indicating the existence of two pools of exchangeable Ca2+. Density fractionation of the cells from one splenectomized beta-thal patient showed that the rapid pool corresponded to the lightest cell fraction, which was also found to have the highest calcium content. The dense cells exhibited a normal Ca2+ influx as well as a smaller increase in total calcium content. It is suggested that, as in sickle cell anemia, the excess of Ca2+ in beta-thal cells is not free in the cytoplasm but trapped within endocytic vacuoles, especially in a population of abnormal cells that are normally removed by the spleen. In CC patients, who have a functional spleen, a different mechanism could be responsible for the calcium retention. In conclusion, the present results demonstrate that in these two cases of hemolytic anemia associated with high calcium content, Ca2+ permeability and the the level of cytosolic Ca2+ are normal.

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca2+, Na+ and H+ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca2+ was about 10-7M. We did not detect any change in Ca2+ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na+ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na+ and pH.

Direct evidence for the modulation of human platelet cytosolic free Ca 2+ by intracellular cyclic AMP produced with a photoactivatable derivative

We have previously reported that intraplatelet “cyclic AMP jumps” produced with newly synthesized photoactivatable cyclic AMP analogue, inhibited washed rat platelet aggregation and serotonin release as induced by thrombin. Using the same approach on human platelets, thrombin-induced platelet aggregation was dose-dependently inhibited only when a flash was delivered. The mechanism of action of intraplatelet cyclic AMP as resulting from photolysis could be by controlling the level of cytosolic Ca 2+. In order to test this hypothesis, the same protocol was used on human platelets preloaded with the internal Ca 2+ fluorescent indicator, Quin 2, we found that the extent and the rate of the rise of the cytosolic Ca 2+ induced by thrombin were dramatically decreased, in the presence of the photoactivatable cyclic AMP, only following photoirradiation. In addition, the flashes were produced, in the presence of photoactivatable cyclic AMP, after the thrombin-induced rise of internal Ca 2+ had reached its peak. In these conditions, photoirradiation caused a rapid fall in fluorescence. These experiments provide the first direct evidence that intracellular cyclic AMP is involved in the control of platelet cytosolic Ca 2+ by inhibition of its mobilization and by stimulation of its sequestration.

The influence of cyclandelate on Ca++ translocation in human platelets.

A major event in signal transduction in platelets is the mobilisation of Ca++ ions by 2 mechanisms: influx of Ca++ from the extracellular medium and release from intracellular storage sites. The calcium modulating drug cyclandelate is known to inhibit platelet aggregation induced by various agonists. Thus, by studying the effects of cyclandelate on cytosolic Ca++ concentrations in gel-filtered human platelets, an explanation was sought of the mechanism which couples receptor stimulation with the processes that execute aggregation and secretion. Cyclandelate in concentrations between 10 and 50 mumol/L induced a dose-dependent increase in the basal level of cytosolic Ca++ of unstimulated platelets. In platelets stimulated with thrombin (5 U/ml) or platelet activating factor (1 mumol/L), cyclandelate strongly inhibited the increase of cystolic Ca++ in the presence of extracellular Ca++, but in the absence of extracellular Ca++ only a weak inhibition was observed. This inhibition was dose dependent and optimal at about 50 mumol/L, the concentration at which Ca++ mobilisation was suppressed to about 10% of control values, and at which cyclandelate inhibits platelet aggregation. In contrast, the metabolites cyclandelate alcohol and cyclandelate acid were without effect at the same concentrations (50 mumol/L). Concentrations of cyclandelate greater than 50 mumol/L were found to further increase the concentration of Ca++ in the unstimulated platelet, an effect which interfered with the stimulus-induced Ca++ translocation. Thus, at concentrations which inhibit platelet aggregation, cyclandelate prevents the mobilisation of Ca++ ions induced by such agonists as thrombin and platelet activating factor. Further study is required to determine the mechanism by which cyclandelate inhibits these platelet responses.

Effects of vasoactive peptides on cytosolic calcium in cultured mesangial cells.

We have used the "second generation" Ca indicator, fura-2, to measure cytosolic free Ca concentrations in superfused cultures of adherent primary renal mesangial cells. The basal cytosolic free Ca concentration in these cells was found to be 93 +/- 5 nM (n = 35). The Ca ionophore ionomycin (0.1 microM) increased cytosolic Ca levels to a peak value of fourfold above basal, followed by a decline to a steadily maintained concentration of twofold above basal. Two vasoactive peptide hormones, arginine vasopressin and angiotensin II, at maximally effective concentrations, transiently increased cytosolic free Ca levels to peak values of three- and sixfold, respectively, above basal levels. The angiotensin II-evoked increase declined to near basal values before rising again to a value of 1.5- to 2-fold above basal. Cells treated with vasopressin did not have a significant secondary increase of Ca above a small, time-dependent, spontaneous increase. Mesangial cells demonstrated tachyphylaxis to both peptides. However, cross-tachyphylaxis was not observed. Treatment of cells with angiotensin II in ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid-supplemented Ca-deficient medium, or with the Ca channel blockers nifedipine or verapamil, did not eliminate the transient phase of cytosolic Ca metabolism. In contrast, the Ca channel blockers completely inhibited the second sustained Ca response to angiotensin II. These results indicate that angiotensin II and vasopressin mobilize intracellular Ca in cultured adherent mesangial cells. Angiotensin II, but not vasopressin, also appears to increase cytosolic Ca by influx of extracellular Ca through specific channels.

Atriopeptin II decreases cytosolic free Ca in cultured vascular smooth muscle cells

We have examined the hypothesis that atriopeptin II decreases cytosolic free Ca concentrations in cultured aortic smooth muscle cells. Ca levels were measured, using the Ca indicator fura-2, by dual wavelength fluorescence spectroscopy, in superfused primary cultures of smooth muscle cells. Basal Ca values were between 40 and 100 nM. Angiotensin II (10 nM) elicited a transient threefold increase in Ca, followed by return to a sustained Ca level that was 30% higher than the original basal values. Atriopeptin II dose-dependently decreased basal Ca concentrations by 10-40%. Atriopeptin II did not have a significant effect on the transient Ca response elicited by 10 nM angiotensin II, but the atrial peptide dose-dependently decreased the sustained increase that followed the transient response. Atriopeptin II also decreased cytosolic Ca levels that were elevated by 50 mM KCl. These results support the hypothesis that atriopeptins relax vascular smooth muscle by decreasing basal and vasoconstrictor-elevated cytosolic free Ca levels and that these peptides may function as endogenous antagonists of Ca-mediated processes.