Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin.

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.     

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.     

Effect of hypothyroidism on the cytosolic free Ca2+ concentration in rat hepatocytes during rest and following stimulation by noradrenaline or vasopressin

The mean resting concentration of cytosolic free Ca2+ [( Ca2+]i) in parenchymal liver cells, as determined with the intracellular Ca2+ indicator quin2, was lowered by about 30% in hypothyroidism (0.17 microM vs. 0.27 microM in normal cells). The [Ca2+]i level in hypothyroid cells at 10 s following stimulation by noradrenaline (1 microM) was about 64% lower than in normal cells (0.33 microM vs. 1.0 microM). The response to noradrenaline in hypothyroid cells was slower in onset (significant at 5 s vs. 3 s in euthyroid cells), and the maximum of the initial [Ca2+]i increase was reached later (14 s vs. 8 s in normal cells). In hypothyroid hepatocytes the initial increase was followed by a slow but prolonged secondary increase in [Ca2+]i. With vasopressin similar results were found. Chelation of extracellular Ca2+ with EGTA immediately prior to stimulation had no effect on the initial [Ca2+]i increase. Treatment with T3 in vivo (0.5 micrograms/100 g body weight daily during 3 days) completely restored the basal and stimulated [Ca2+]i in hypothyroid cells. The half-maximally effective dose of noradrenaline was the same in euthyroid and hypothyroid liver cells (1.8 X 10(-7) M). Hypothyroidism had no significant effect on the number of alpha 1-receptors determined by [3H]prazosin labeling in crude homogenate fractions, while the Kd for [3H]prazosin was 21% lower than in the euthyroid group. These results show that thyroid hormone has a general stimulating effect on intracellular Ca2+ mobilization by Ca2+-mobilizing hormones, probably at a site distal to the binding of the agonist to its receptor. The results also support our idea that thyroid hormone may control metabolism during rest and activation, at least partially, by altering Ca2+ homeostasis.     

Alpha 1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: evidence for compartmentalization of quin-2 and fura-2

This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to alpha 1-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10(-5) M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180-300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells "scrape-loaded" with fura-2 revealed a four-fold increase in [Ca2+]i concentration following stimulation with phenylephrine.     

Characterization of responses of isolated rat hepatocytes to ATP and ADP

In isolated rat hepatocytes, ATP and ADP (10(-6) M) rapidly mobilize intracellular Ca2+ and increase the concentration of free cytosolic Ca2+ ([Ca2+]i) within 1-2 s. The increase in [Ca2+]i is maximal (2.5- to 3-fold) by about 10 s and is dose-dependent, with ATP and ADP being half-maximally effective at 8 X 10(-7) and 3 X 10(-7) M, respectively. At submaximal concentrations, the rise in [Ca2+]i is transient due to hydrolysis of the agonist. The increase in [Ca2+]i in response to ATP or ADP can be potentiated by low concentrations of glucagon (10(-9) M). In addition, the [Ca2+]i rise can be antagonized in a time- and dose-dependent manner by the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. Adenosine, at concentrations as high as 10(-4) M, does not alter [Ca2+]i. AMP is ineffective at 10(-5) M, but at 10(-4) M it increases [Ca2+]i approximately 1.5-fold after a 30-s lag and at a slow rate. Conversely, high concentrations (10(-4) M) of adenosine and AMP increases cell cAMP about 2- to 3-fold. ATP and ADP, at concentrations (10(-6) M) which near-maximally increase [Ca2+]i, do not affect hepatocyte cAMP. ATP and ADP increase the cellular level of myoinositol 1,4,5-trisphosphate (IP3), the putative second messenger for Ca2+ mobilization. The increase in IP3 is dose-dependent and precedes or is coincident with the [Ca2+]i rise. There is an approximate 20% increase in IP3 with concentrations of ATP or ADP which near-maximally induce other physiological responses. It is concluded that submicromolar concentrations of ATP and ADP mobilize intracellular Ca2+ and activate phosphorylase in hepatocytes due to generation of IP3. These effects may involve P2-purinergic receptors. In contrast adenosine and AMP interact with P1 (A2)-purinergic receptors to increase cAMP.     

Cyclic AMP Analogues Potentiate k-Opiate and Attenuate α2-Adrenoceptor Agonist Effects on Intrasynaptosomal Free Calcium

The intrasynaptosomal free calcium concentration ([Ca2+]i) was measured in quin2-loaded synaptosomes prepared from rat cerebral cortex. Membrane-permeant cyclic adenosine-3',5'-monophosphate (cAMP) analogues [8-bromo-cyclic adenosine-3',5'-monophosphate (8-Br-cAMP) and dibutyryl-cyclic adenosine-3',5'-monophosphate (db-cAMP)] increased [Ca2+]i in a dose-dependent manner; The maximal increases were approximately 50% for 8-Br-cAMP and 35% for db-cAMP and occurred at approximately 10 microM with both analogues. Clonidine (1 microM) alone reduced [Ca2+]i by 26.5%; db-cAMP and 8-Br-cAMP attenuated this reduction to 14.2 and 8.2%, respectively. In contrast, the reduction (19.9%) in [Ca2+]i induced by the preferential kappa-opiate agonist dynorphin A(1-13) was not attenuated by the cAMP analogues; in fact, db-cAMP and 8-Br-cAMP potentiated the effect of dynorphin A(1-13) (1 microM), producing decreases in [Ca2+]i of 33.6 and 29.6%, respectively. We conclude that although alpha 2-adrenergic and kappa-opiate receptors both reduce [Ca2+]i, the alpha 2-adrenoceptor-mediated response and the kappa-opiate receptor-mediated response involve different effector mechanisms. It appears that presynaptic alpha 2-adrenoceptor agonist effects are linked to reductions in adenylate cyclase activity and cAMP production and a resultant increase in Ca2+ sequestration, Ca2+-channel blockade, or both. On the other hand, the kappa-opiate-mediated effects possibly involve an increase in cAMP production and a blockade of Ca2+ entry.     

Elevation of cytosolic [Ca2+] due to intracellular Ca2+ release retards carbachol stimulation of divalent cation entry in rat parotid gland acinar cells

This study examines the activation of divalent cation entry into rat parotid gland acinar cells by using Mn2+ as a Ca2+ surrogate cation. Following muscarinic-cholinergic stimulation of dispersed parotid acini with carbachol (10 microM), the onset of internal Ca2+ release (cytosolic [Ca2+], [Ca2+]i, increase) and the stimulation of Mn2+ entry (increase in fura2 quenching) are not simultaneously detected. [Ca2+]i elevation, due to intracellular release, is detected almost immediately following carbachol addition and peak [Ca2+]i increase occurs at 6.0 +/- 0.8 sec. However, there is an interval (apparent lag) between carbachol addition and the detection of stimulated Mn2+ entry. This apparent lag is decreased from 26 +/- 3.1 sec to 9.2 +/- 1.5 sec when external Mn2+ ([Mn2+]0) is increased from 12.5 to 500 microM. It is not decreased further with increase in [Mn2+]0 from 500 microM to 1 mM (9.8 +/- 2.1 sec), although both intracellular free Mn2+ and [Mn2+-fura2]/[fura2] increase. Thus, at [Mn2+]0 < 500 microM, the observed lag time is partially due to a limitation in the magnitude of Mn2+ entry. Furthermore, neither peak [Ca2+]i nor the time required to reach peak [Ca2+]i is significantly altered by [Mn2+]0 (12.5 microM to 1 mM). At every [Mn2+]0 tested (i.e., 12.5 microM-1 mM), the apparent lag is significantly greater than the time required to reach peak [Ca2+]i. However, when carbachol stimulation of the [Ca2+]i increase is attenuated by loading the acini with the Ca2+ chelator, 2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), there is no detectable lag in carbachol stimulation of Mn2+ entry (with 1 mM [Mn2+]0). Importantly, in BAPTA-loaded acini, carbachol stimulates Mn2+ entry via depletion of the internal Ca2+ pool and not via direct activation of other divalent cation entry mechanisms. Based on these results, we suggest that the apparent lag in the detection of carbachol stimulation of Mn2+ entry into parotid acinar cells is due to a retardation of Mn2+ entry by the initial increase in [Ca2+]i, due to internal release, which most likely occurs proximate to the site of divalent cation entry.     

Hormone-induced increase in free cytosolic calcium and glycogen phosphorylase activation in rat hepatocytes incubated in normal and low-calcium media.

The action of alpha 1-adrenergic agonists (noradrenaline in the presence of propranolol), vasopressin and angiotensin on the intracellular free Ca2+ concentration, [Ca2+]i, was determined by using the fluorescent dye quin2 in isolated rat liver cells. In the presence of external Ca2+ (1.8 mM), 1 microM-noradrenaline induced an increase in [Ca2+]i up to about 800 nM without apparent delay, whereas 10 nM-vasopressin and 1 nM-angiotensin increased [Ca2+]i to values higher than 1500 nM with a lag period of about 6s. The successive addition of the hormones and of their specific antagonists indicated that the actions of the three Ca2+-mobilizing hormones occurred without apparent desensitization (over 6 min) and via independent receptors. The relative contributions of internal and external Ca2+ pools to the cell response were determined by studying the hormone-mediated [Ca2+]i increase and glycogen phosphorylase activation in low-Ca2+ media (22 microM). In this medium: (1) [Ca2+]i was lowered and the hormones initiated a transient instead of a sustained increase in [Ca2+]i; subsequent addition (2 min) of a second hormone promoted a lesser increase in [Ca2+]i; in contrast, the subsequent addition (2 min) of Ca2+ (1.8 mM) caused [Ca2+]i to increase to a value close to that initiated by the hormone in control conditions, the amplitude of the latter response being dependent on the concentration of Ca2+ added to the medium; (2) returning to normal Ca2+ (1.8 mM) restored the resting [Ca2+]i and allowed the hormone added 2 min later to promote a large increase in [Ca2+]i whose final amplitude was also dependent on the concentration of Ca2+ added beforehand. Similar results were found when the same protocol was applied to the glycogen phosphorylase activation. It is concluded that Ca2+ influx is required for a maximal and sustained response and to reload the hormone-sensitive stores.     

Effect of (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55,940) on intracellular Ca2+ levels in human osteosarcoma cells

The study was undertaken to explore the effect of CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a drug commonly used as a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in MG63 human osteoblast-like epithelial cells. [Ca2+]i was measured in suspended cells by using the fluorescent dye fura-2 as an indicator. At concentrations between 2-20 microM, CP55,940 increased [Ca2+]i in a concentration-dependent manner with an EC50 of 8 microM. The [Ca2+] signal comprised an initial rise, a slow decay, and a sustained phase. CP55940 (10 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists (AM-251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole3-carboxamide; AM-281, 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide). Extracellular Ca2+ removal decreased the maximum value of the Ca2+ signals by 50%. CP55,940 induced quench of fura-2 fluorescence by Mn2+ (50 microM), suggesting the presence of Ca2+ influx across the plasma membrane. CP55,940 (10 microM)-induced [Ca2+]i increase in Ca(2+)-free medium was inhibited by 84% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 10 microM CP55,940 in Ca(2+)-free medium abolished thapsigargin-induced [Ca2+]i increase. At 1 microM, nifedipine, verapamil, and diltiazem did not alter CP55, 940 (10 microM)-induced [Ca2+]i increase. CP55,940 (20 microM)-induced Ca2+ release was not affected when phospholipase C was inhibited by 2 microM U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione). CP55,940 (20 microM) did not induce acute cell death after incubation for 30 min as assayed by trypan blue exclusion. Collectively, CP55,940 induced significant [Ca2+]i increases in osteoblasts by releasing store Ca2+ from thapsigargin-sensitive stores and by causing Ca2+ entry. The CP55,940's action appears to be independent of stimulation of CB1 cannabinoid receptors.     

Alpha 1-adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx

The regulation of [Ca2+]i in rat pinealocytes was studied using the fluorescent indicator quin2. Pinealocyte resting [Ca2+]i was approximately 100 nM; this rapidly decreased in low Ca2+ medium (approximately 10 microM), indicating there was a high turnover of [Ca2+]i in these cells. Norepinephrine (NE, 10(-6) M) increased [Ca2+]i to approximately 350 nM within 1 min; [Ca2+]i then remained elevated for 30 min. The relative potency of adrenergic agonists was NE greater than phenylephrine much greater than isoproterenol. Phentolamine (10(-6) M) and prazosin (10(-8) M) blocked the effects of adrenergic agonists; in contrast, propranolol (10(-6) M) or yohimbine (10(-6) M) had little or no effect. These observations indicate NE acts via alpha 1-adrenoceptors to elevate [Ca2+]i. The [Ca2+]i response to NE did not occur when [Ca2+]e was reduced to approximately 10 microM by adding EGTA 5s before NE, indicating an increase in net Ca2+ influx is involved rather than mobilization of Ca2+ from intracellular stores. The effect of NE was not blocked by nifedipine (10(-6) M), which did block a K+-induced increase in [Ca2+]i, presumably involving voltage-sensitive channels. Ouabain (10(-5) M) caused a gradual increase in [Ca2+]i; this increase was not blocked by nifedipine. Together these data indicate that pinealocyte [Ca2+]i may be influenced by mechanisms regulated by alpha 1-adrenoceptors, voltage-dependent Ca2+ channels, and perhaps a Na+/Ca2+ exchange mechanism stimulated by ouabain. These studies indicate that the pinealocyte is an interesting model to use to study the adrenergic regulation of [Ca2+]i because of the rapid and prolonged changes in [Ca2+]i produced by alpha 1-adrenoceptor activation.     

Effect of adrenalectomy on Ca2+ signaling in rat hepatocytes

To pursue our studies of the effects of adrenalectomy on the adrenergic regulation of phosphorylase a, cAMP, cell calcium, and Ca2+ signaling in rat hepatocytes (Studer, R.K., and Borle, A.B. (1984) Biochim. Biophys. Acta 804, 377-385; Freudenrich, C.C., and Borle, A.B. (1988) J. Biol. Chem. 263, 8604-8610), we have further examined the alpha 1-adrenergic pathway in adrenalectomized and sham-operated male rats. We measured the number and affinity of alpha 1-adrenergic receptors, the cytosolic free Ca2+ concentration [(Ca2+]i) of hepatocytes with aequorin, inositol triphosphate (IP3) accumulation, and Ca2+ influx and efflux across the plasma membrane. We also compared the effects of vasopressin with those obtained with epinephrine. We found that the number of alpha 1-adrenergic receptors was slightly depressed (-23%), but that their affinity was unchanged. However, IP3 accumulation evoked by epinephrine was decreased 50%. This is probably the main cause for the depressed peak rise in [Ca2+]i we previously observed and reported. We also found that the basal resting Ca2+ influx was increased after adrenalectomy. Experiments with the beta-blocker propranolol, which abolished the epinephrine-evoked increase in Ca2+ influx, suggest that this effect may be mediated by cAMP, at least in adrenalectomized animals. The effects of vasopressin on IP3 [Ca2+]i and Ca2+ influx and efflux were also significantly decreased after adrenalectomy, indicating that alpha 1-adrenergic-mediated and other IP3-dependent Ca2+ signaling pathways are depressed after adrenalectomy.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Novel effect of CP55,940, a CB1/CB2 cannabinoid receptor agonist,on intracellular free Ca2+ levels in bladder cancer cells

The study was undertaken to explore the effect of CP55,940 ((-)-cis-3-[2-Hydroxy4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a drug commonly used as a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in several cell types [Ca2+]i was measured in suspended cells by using the fluorescent dye fura-2 as an indicator. At concentrations between 1-50 microM, CP55,940 increased [Ca2+]i in a concentration-dependent manner with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise, a slow decay, and a sustained phase. CP55940 (10 microM)-induced (Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists (AM-251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM-281, 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-m3thyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide). Extracellular Ca2+ removal decreased the maximum value of the Ca2+ signals by 50%. CPS5,940 (10 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 80% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 10 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increase. Nifedipine (10 microM) and verapamil (10 microM) did not alter CP55,940 (10 microM)-induced [Ca2+]i increase. CP55, 940 (10 microM)-induced Ca2+ release was not affected when phospholipase C was inhibited by 2 microM U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione). CP55,940 (5 microM) also increased [Ca22+] in Madin-Darby canine kidney cells, MG63 human osteosarcoma cells, and IMR-32 neuroblastoma cells. Collectively, CP,55940 induced significant [Ca2+]i increases in several cell types by releasing store Ca2+ from thapsigargin-sensitive pools and by causing Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors     

Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells

It is generally thought that receptor-operated Ca2+ entry is related to store-operated or capacitative Ca2+ entry mechanism. Recent evidence suggests that non-capacitative Ca2+ entry pathways are also involved in receptor activated Ca2+ influx in many different kinds of cells. In this study, we studied whether alpha1-adrenoreceptor (alpha1-AR)-activated Ca2+ entry is coupled to both capacitative and non-capacitative pathways in A10 vascular smooth muscle cells by fura-2 fluorescence probe and conventional whole-cell patch clamp techniques. We found that both thapsigargin (TG) and phenylephrine (Phe) induced transient increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in Ca2+-free medium, and subsequent addition of Ca2+ evoked a sustained [Ca2+]i rise. When the membrane potential was held at -60 mV, both TG and Phe activated inward currents, which were inhibited by GdCl3(Gd3+), 0Na+/0Ca2+ solution and 1-{beta[3-(4-mehtoxyphenyl)propoxy]-4-methoxypheneth-yl}-1H- imidazole hydro-chloride (SK&F96365), but not by nifedipine. When Ca2+ store was depleted by TG in Ca2+-free solution, Phe failed to further evoke [Ca2+]i rise. However, when capacitative Ca2+ entry was activated by TG in the medium containing Ca2+, 10 microM Phe further increased [Ca2+]i. At the same concentration, TG activated an inward cation current, subsequent addition of Phe also further induced an inward cation current. Furthermore, the amplitudes of [Ca2+]i increase and current density induced by Phe in the presence of TG were less than that induced by Phe alone. Our results suggest that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca2+ influx induced by activation of alpha1-AR in A10 vascular smooth muscle cells.     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.     

Characterization of benextramine as an irreversible alpha-adrenergic blocker and as a blocker of potassium-activated calcium channels

An irreversible alpha-adrenergic blocker, benextramine [N,N'-bis(o-methoxybenzylamine-n-hexyl)-cysteamine] was used as a probe to study the possible interrelationship between alpha-adrenoceptors and the K+-activated Ca2+-channels. Benextramine, a tetraamine disulfide, acts irreversibly both on the alpha 1-adrenoceptor (t 1/2 = 3 min) and the alpha 2-adrenoceptors. These studies were carried out on rat brain synaptosomes, [3H]prazosin and [3H]clonidine binding. Benextramine blocked Ca2+ influx in rat brain synaptosomes under both depolarizing (75 mM KCl) and normal conditions (5 mM KCl). Its action at the channel is reversible with IC50 = 10 +/- 5 microM of the net Ca2+ influx. This makes benextramine a most potent Ca2+ blocker compared to verapamil or nicardipine (IC50 = 200 microM and 170 microM, respectively). Pretreatment of rat brain slices with benextramine gave a synaptosomal preparation which was devoid of either alpha 1-adrenergic or alpha 2-adrenergic binding capacity due to the irreversible binding of benextramine, but with an undisturbed Ca2+ influx. Thus, these results suggest that the alpha-adrenoceptors and the Ca2+-channels are independent of each other, and that full occupancy of the alpha-receptors does not affect the net calcium flux.     

Mobilization of extracellular Ca2+ by prostaglandin F2 alpha can be modulated by fluoride in 3T3-L1 fibroblasts.

Changes in the intracellular concentration of calcium [( Ca2+]i) have been shown to mediate the physiological effects of certain agonists. Ca2+ mobilization occurs through multiple mechanisms which involve both influx and internal release of Ca2+. Prostaglandin F2 alpha (PGF2 alpha) caused a transient mobilization of intracellular Ca2+ in 3T3-L1 fibroblasts. This effect was characterized by fluorescence measurements of trypsin-treated cells loaded with fura-2/AM. In the absence of extracellular Ca2+, the peak amount of Ca2+ mobilized by PGF2 alpha was decreased by 70%, a lag time before the onset of [Ca2+]i increase was observed, and the rate of rise of [Ca2+]i was slowed. Addition of NaF (10 mM) to fura-2-loaded 3T3-L1 cells caused a dose-dependent increase in [Ca2+]i after a brief (approximately 10 s) lag. Maximal effects (approximately 300 nM) were observed at 5-10 mM-NaF. This effect was dependent on the presence of extracellular Ca2+ and appeared to be independent of inositol phosphate production. After reaching a peak at around 40 s after fluoride addition, [Ca2+]i returned to near-baseline within 120 s. This return of [Ca2+]i to near-baseline after fluoride stimulation and the inability of the cells to respond to a subsequent addition of fluoride indicated that the response to fluoride underwent desensitization. Similarly, the pathway used by PGF2 alpha to mobilize Ca2+ underwent desensitization. Exposure of the cells to a maximally effective concentration of fluoride and subsequent addition of PGF2 alpha produced a [Ca2+]i response to PGF2 alpha which was similar in magnitude and kinetics to that seen for PGF2 alpha in the absence of extracellular Ca2+. Conversely, prior exposure of cells to PGF2 alpha diminished the ability of fluoride to mobilize Ca2+. PGF2 alpha also increased inositol phosphate formation, with a time course and dose-response consistent with its ability to increase [Ca2+]i. Prior exposure of cells to fluoride did not change the time course or dose-response characteristics of PGF2 alpha-induced generation of inositol phosphates. These data suggest that PGF2 alpha and fluoride share a common mechanism of activating Ca2+ influx in 3T3-L1 cells.     

Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes

The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.     

Glucagon and vasopressin interactions on Ca2+ movements in isolated hepatocytes.

The effects of glucagon and vasopressin, singly or together, on cytosolic free Ca2+ concentration [( Ca2+]i) and on the 45Ca2+ efflux were studied in isolated rat liver cells. In the presence of 1 mM external Ca2+, glucagon and vasopressin added singly induced sustained increases in [Ca2+]i. The rate of the initial fast phase of the [Ca2+]i increase and the magnitude of the final plateau were dependent on the concentrations (50 pm-0.1 microM) of glucagon and vasopressin. Preincubating the cells with a low concentration of glucagon (0.1 nM) for 2 min markedly accelerated the fast phase and elevated the plateau of the [Ca2+]i increase caused by vasopressin. In the absence of external free Ca2+, glucagon and vasopressin transiently increased [Ca2+]i and stimulated the 45Ca2+ efflux from the cells, indicating mobilization of Ca2+ from internal store(s). Preincubating the cells with 0.1 nM-glucagon accelerated the rate of the fast phase of the [Ca2+]i rise caused by the subsequent addition of vasopressin. However, unlike what was observed in the presence of 1 mM-Ca2+, glucagon no longer enhanced the maximal [Ca2+]i response to vasopressin. In the absence of external free Ca2+, higher concentrations (1 nM-0.1 microM) of glucagon, which initiated larger increases in [Ca2+]i, drastically decreased the subsequent Ca2+ response to vasopressin (10 nM). At these concentrations, glucagon also decreased the vasopressin-stimulated 45Ca2+ efflux from the cells. It is suggested that, in the liver, glucagon accelerates the fast phase and elevates the plateau of the vasopressin-mediated [Ca2+]i increase respectively by releasing Ca2+ from the same internal store as that permeabilized by vasopressin, probably the endoplasmic reticulum, and potentiating the influx of extracellular Ca2+ caused by this hormone.