Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells

The effect of extracellular ATP on intracellular free Ca2+ was characterized in quin2-loaded parotid acinar cells. ATP specifically increased the intracellular Ca2+ concentration six-fold above a basal level of 180 nM. Of other purine nucleotides tested, only adenylylthiodiphosphate (ATP gamma S) had significant activity. ATP and the muscarinic agonist carbachol increased intracellular Ca2+ even in the absence of extracellular Ca2+. Both agonists stimulated K+ release, which was followed by reuptake of K+, even in the continued presence of agonist. In the absence of Mg2+, ATP was much more potent but no more efficacious in elevating intracellular Ca2+, suggesting that ATP4- is the active species. The effect of ATP was reversed by removal with hexokinase, arguing against a role for an active contaminant of ATP and against a non-specific permeabilizing effect of extracellular ATP. Lactate dehydrogenase release was unaffected by a maximally effective concentration of ATP. These observations are consistent with a possible neurotransmitter role for ATP in the rat parotid gland.     

Two modes of regulation of the phospholipase C-linked substance-P receptor in rat parotid acinar cells.

In rat parotid acinar cells prelabelled with [3H]inositol, substance P (100 nM) induced the formation of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 reached a maximum 7 s after substance P stimulation, and thereafter decreased and reached a stable value at 60 s. When the cells were exposed to substance P for 10, 30, 60, or 300 s, washed, and re-exposed to this peptide, the formation of [3H]inositol trisphosphate (InsP3) was attenuated in a time-dependent manner. In the cells pretreated as described above, the number of [3H]substance-P-binding sites (Bmax) was also decreased. Possible role(s) of Ca2+ and protein kinase (protein kinase C) control mechanisms in regulating substance P responses were investigated. Desensitization of substance P-induced InsP3 was not affected by the Ca2+ ionophore ionomycin, nor was it dependent on Ca2+ mobilization. On the other hand, in the presence of 4 beta-phorbol 12,13-dibutyrate (PDBu) and 12-O-tetradecanoyl-4 beta-phorbol 13-acetate, known activators of protein kinase C, substance P-induced InsP3 formation was inhibited. However, PDBu had no effect on [3H]substance P binding, whether present during the assay or when cells were pretreated. The persistent desensitization of InsP3 formation induced by substance P was not affected by PDBu. These results suggest that the persistent desensitization of InsP3 formation induced by substance P is a homologous process involving down-regulation of the substance P receptor; the mechanism does not appear to involve, or to be affected by, the Ca2+ or protein kinase C signalling systems. Protein kinase C activation can, however, inhibit substance P-induced InsP3 formation, which may indicate the presence of a negative-feedback control on the substance P pathway.     

Rapid increases in cytosolic free calcium in response to muscarinic stimulation of rat parotid acinar cells

Carbachol-evoked rises in [Ca2+]i were measured in fura-2-loaded, rat parotid acinar cells. In suspensions of dissociated cells examined by dual wavelength excitation fluorimetry, a maximally effective concentration of carbachol produced a measured peak [Ca2+]i of 780 +/- 60 nM followed by a maintained elevation in the presence of 1 mM external Ca2+, and a peak of 630 +/- 95 nM followed by a return to resting values in the absence of external Ca2+. Stopped-flow, single wavelength fluorimetry was used to resolve the rising phase of the response. There was a dose-dependent lag of 70-220 ms before [Ca2+]i started to increase, and [Ca2+]i was maximal by 800-900 ms. These times were similar in the presence or absence of external Ca2+, although the initial rate of rise was faster in the presence of external Ca2+. These kinetics are consistent with a biochemical event, possibly phosphatidylinositol bisphosphate hydrolysis, mediating both internal release and Ca2+ entry, with a component of the initial rise being due to Ca2+ entry.     

Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells

In order to analyze the factors regulating agonist-stimulated Ca2+ mobilization, cytosolic free [Ca2+] ([Ca2+]i) was measured directly in fura-2-loaded rat parotid acinar cells. Stimulation of muscarinic receptors by carbachol produced a dose-dependent rise in [Ca2+]i. In the presence of external Ca2+, the initial transient rise was followed by a maintained elevation. The maintained elevation is dependent on the presence of external Ca2+. Removal of Ca2+ by addition of EGTA caused a rapid decline in [Ca2+]i back to base line. In the absence of external Ca2+, only an initial transient peak in [Ca2+]i was seen which then declined to base line; the maintained elevation in [Ca2+]i could then be evoked by addition of Ca2+ in the continued presence of carbachol. Muscarinic receptor occupation by carbachol is required to maintain the elevated level of [Ca2+]i; addition of the muscarinic antagonist, atropine, caused [Ca2+]i to decline back to the basal level. The maintained elevation in [Ca2+]i, but not the initial transient peak, can also be blocked by Ni2+ but was unaffected by the organic Ca2+ antagonists. Total substitution of external Na+ with the impermeant cation, N-methyl-D-glucamine, had no effect on either the initial or the maintained response to carbachol; however, total substitution of Na+ with K+ attenuated the maintained response while not affecting the initial peak. Refilling of the intracellular Ca2+ store was also studied and found to take place in the absence of agonist and with no substantial elevation in [Ca2+]i. These experiments also showed that not all of the intracellular vesicular Ca2+ stores can be released by agonists. From these results, we propose a model for the regulation of [Ca2+]i.     

Changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and beta-adrenergic agonists

The alteration in the concentration of cytosolic free calcium ([Ca2+]i) in isolated rat parotid cells caused by autonomic agents was directly measured using the Ca-sensitive fluorescent probe, quin2. [Ca2+]i of unstimulated cells was estimated to be 162.7 +/- 3.2 nM in normal medium. Carbachol (CCh) and isoproterenol (ISP) caused a rapid rise in [Ca2+]i in a dose-dependent manner. Maximum increases in [Ca2+]i induced by CCh and ISP were approximately 100% and 25% of resting level, respectively. In Ca-free medium, CCh produced a small, rapid rise in [Ca2+]i, followed by a slow decay and a return to resting level within 3-4 min, while all doses of ISP tested failed to change [Ca2+]i. These results suggest that CCh mobilizes Ca2+ from both extracellular and intracellular pools and then results in a rise in [Ca2+]i, whereas ISP may slightly mobilize only the extracellular Ca pool.     

The relation of elevation of cytosolic free calcium to activation of membrane conductance in rat parotid acinar cells

The relation between elevation of cytosolic free calcium and activation of membrane conductance has been studied in single acinar cells of the rat parotid. Outward and inward currents are activated by calcium elevation and oscillate in phase with oscillations of cytosolic calcium. The outward current results from activation of a large unit-conductance Ca2+ and voltage-dependent K+ channel, whereas the inward current is most likely carried predominantly by Cl-. Both these conductances have been previously described in exocrine cells. Buffering calcium at resting levels eliminated current responses to muscarinic agonists, suggesting that calcium is the only significant second messenger involved in the short-term control of this conductance by acetylcholine.     

Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca²⁺]_i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10–200) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentrationdependent increase in [Ca²⁺]_i with an EC₅₀ (mean ± SEM) value of 42 ± 11 μM. Inclusion of vanadate (200 μM) led to a significant increase (p < 0.05) in the peak [Ca²⁺]_i level to 190 ± 23 nM from a basal level of 102 ± 2 nM. At concentrations > 200 μM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50 % (due to a quenching effect), followed by a transient rise. H₂O₂, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca²⁺]_i. These data suggest that vanadate promotes vascular tone by elevating [Ca²⁺]_i in ASMC. However, [Ca²⁺]_i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.     

Sphingosine stimulates calcium mobilization in rat parotid acinar cells

In fura-2-loaded parotid acinar cells, 50-200 microM sphingosine induced an increase in cytosolic Ca2+ ([Ca2+]i). When extracellular Ca2+ was chelated by EGTA, 50 microM sphingosine failed to increase [Ca2+]i, but 100 or 200 microM sphingosine induced a slight and transient increase in [Ca2+]i. The addition of LaCl3 to the medium resulted in the same effect as chelation of extracellular Ca2+. When cells were incubated in low Ca2+ medium containing sphingosine, and extracellular Ca2+ was subsequently added, a rapid increase in [Ca2+]i depending on the concentration of sphingosine was shown. In low Ca2+ medium, a slight increase in [Ca2+]i induced by high concentrations of sphingosine was not shown after the transient increase in [Ca2+]i elicited by methacholine. Inhibitors of protein kinase C, H-7 and K252a, did not mimic the effect of sphingosine on [Ca2+]i. These results suggest that sphingosine stimulates Ca(2+)-influx and further stimulates the release of Ca2+ from agonist-sensitive intracellular pools by a mechanism that is independent of protein kinase C.     

Capacitative calcium entry in parotid acinar cells.

The intracellular Ca2+ indicator, fura-2, was used to monitor changes in cytosolic [Ca2+] in parotid acinar cells. When parotid cells were incubated in a medium containing low [Ca2+], and [Ca2+] was restored to the physiological range, there was a small increase in cytosolic [Ca2+]. If, however, the cells were first activated by a muscarinic agonist, and receptor activation was terminated before the addition of Ca2+ by the addition of a pharmacological excess of the muscarinic-receptor antagonist atropine, the initial increase in cytosolic [Ca2+] was faster and transiently larger than in the control cells which had not been previously stimulated. This suggested that a stimulation of Ca2+ entry occurred owing to the prior emptying of the agonist-regulated intracellular Ca2+ pool. This extra Ca2+ influx seen in pool-depleted cells persisted even when the interval between the addition of atropine and Ca2+ was increased from 1 to 20 min. Also, when the pool was allowed to refill by adding atropine in the presence of extracellular Ca2+, and Ca2+ was then sequentially removed and restored, the rise in cytosolic [Ca2+] after the addition of extracellular Ca2+ was not rapid, and resembled the increase seen in unstimulated cells. These results indicate that, when the agonist-sensitive Ca2+ pool is emptied by an agonist, Ca2+ influx across the plasma membrane is increased. This influx of Ca2+ occurs independently of the concentrations of inositol phosphates and probably of any second messengers linked directly to receptor activation. It appears rather to be a consequence of the empty state of the Ca2+ pool. Further, we suggest that, whenever the agonist-sensitive Ca2+ pool is emptied by agonist activation, the plasma-membrane permeability to Ca2+ will be increased, and this may account, at least in part, for the phenomenon of receptor-activated Ca2+ entry.     

Regulation of calcium handling by rat parotid acinar cells

Summary Salivary gland fluid secretion following neurotransmitter stimulation is Ca²⁺-dependent. We have studied the control of cellular Ca²⁺ following secretory stimuli in rat parotid gland acinar cells. After muscarinic-cholinergic receptor activation, cytosolic Ca²⁺ is elevated 4–5 fold, due to both intracellular Ca²⁺ pool mobilization and extracellular Ca²⁺ entry. Fluid movement ensues due to the Ca²⁺-activated enhancement of membrane permeability to K⁺ and Cl^–. Basal cytosolic Ca²⁺ levels are tightly controlled at 150–200 nM through the action of high affinity and high capacity ATP-dependent Ca²⁺ transporters in the basolateral and endoplasmic reticulum membranes. Activity of these Ca²⁺ transporters can be modulated to facilitate rapid responsiveness and a sustained fluid secretory response necessary for alimentary function.     

Activin A increases cytosolic free calcium concentration in rat pituitary somatotropes.

The effect of activin A on the cytosolic free calcium concentration ([Ca2+]i) in normal rat pituitary cells was examined using a calcium sensitive fluorescent dye, indo 1 AM, and a digital imaging fluorescent microscope system. The cells showing an increase in [Ca2+]i in response to activin A were then characterized by comparison with cells responding to growth hormone releasing hormone (GRH), thyrotropin releasing hormone (TRH), corticotropin releasing hormone (CRH), and gonadotropin releasing hormone (GnRH) in monolayer cultures of normal rat pituitary cells. Activin A increased [Ca2+]i in some cells in a mixed population of normal rat pituitary cells. The cells that responded to activin A also responded to GRH. Most of these cells were not affected by other tropic hormones (CRH, TRH, and GnRH), but a few cells responded to both GRH and TRH. None of the activin A-responding cells responded to CRH or GnRH, and none of the CRH- or GnRH-responding cells responded to activin A. In a preparation of somatotropes purified 80-90% by fluorescence-activated cell sorting, activin A increased [Ca2+]i in 30% of the cells that shows a [Ca2+]i-response to GRH. These findings suggest direct involvement of somatotropes in activin A-induced biological events in the rat pituitary gland.     

Involvement of phospholipase D in the cAMP-regulated exocytosis of rat parotid acinar cells

The activation of beta-adrenergic receptors in rat parotid acinar cells causes intracellular cAMP elevation and appreciably stimulates the exocytotic release of amylase into saliva. The activation of Ca(2+)-mobilizing receptors also induces some exocytosis. We investigated the role of phospholipase D (PLD) in regulated exocytosis in rat parotid acinar cells. A transphosphatidylation assay detected GTPgammaS (a nonhydrolyzable analogue of GTP)-dependent PLD activity in lysates of rat parotid acinar cells, suggesting that PLD is activated by small molecular mass GTP-binding proteins. The PLD inhibitor, neomycin, suppressed cAMP-dependent exocytosis in saponin-permeabilized cells. Signaling downstream of PLD was disrupted by 1-butanol due to conversion of the PLD reaction product (phosphatidic acid) to phosphatidylbutanol. The stimulation of exocytosis by isoproterenol as well as by a Ca(2+)-mobilizing agonist (methacholine) was inhibited by 1-butanol. These results suggest that PLD is important for regulated exocytosis in rat parotid acinar cells.     

Cyclic AMP mediates beta-adrenergic-induced increases in N-linked protein glycosylation in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid cells by isoprenaline (isoproterenol) results in 2-3-fold increases in [3H]mannose incorporation into N-linked oligosaccharides. This occurs without perceptible lag and is linear with time for 60 min after agonist addition. Concomitantly, isoprenaline markedly increases cellular cyclic AMP. Examination of individual proteins by sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis reveals that glycosylation changes are primarily associated with four secretory proteins, of approx. Mr 17000, 32000, 38000 and 220000. Beta-Adrenoreceptor activation additionally elicits a slight increase in parotid protein synthesis. The greatest increase in [14C]leucine incorporation is that into another secretory protein (Mr approx. 24000). Exposure of cells to dibutyryl cyclic AMP yields results comparable with those after isoprenaline treatment. Forskolin, which increases parotid-cell cyclic AMP, also causes similar effects. Conversely, dibutyryl cyclic GMP shows no such response. The data are consistent with the notion that beta-adrenergic stimulation of N-linked protein glycosylation in rat parotid cells is mediated by cyclic AMP.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

Extracellular ATP increases cytosolic free calcium in thymocytes and initiates the blastogenesis of the phorbol 12-myristate 13-acetate-treated medullary population

Exogeneous nucleotides or nucleosides may influence lymphocyte functions such as proliferation and cytotoxicity. We report that ATP, and to a lesser extent ADP, at concentrations as low as 0.3 mM, are highly mitogenic for medullary mature thymocytes, when added in combination with phorbol myristate acetate (PMA), which is only weakly mitogenic by itself. Under the same conditions, the other nucleotides (AMP; GTP, ITP, 2'd-deoxyATP), the non-hydrolysable ATP analogs (p[NH]ppA, pp[CH2]pA) and adenosine are unable to trigger thymocyte blastogenesis. p[NH]ppA, a potent inhibitor of ATP hydrolysis, potentiates the ATP mitogenic effect. In contrast, T-cell-enriched splenocytes do not proliferate in response to ATP + PMA. These data and measurements of interleukin 2 synthesis suggest that ATP may efficiently deliver in thymocytes the calcium signal necessary for the initiation of blastogenesis (in medullary cells). Indeed, among all nucleotides tested, only ATP or ADP were able to increase the intracellular free calcium level in thymocytes, but not in splenocytes. Our results led us to suggest that thymocytes express on their surface receptors specific for ATP, which might be P2 type nucleotide receptors and could be involved in the lymphocyte response through the regulation of intracellular free calcium levels.     

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

The calcium requirement for stimulation of rubidium efflux from isolated rat parotid acinar cells by carbachol.

Stimulation of ⁸⁶Rb⁺ efflux from isolated parotid acinar cells by carbchol was biphasic. The phases of stimulated ⁸⁶Rb⁺ efflux were separated on the basis of their relative requirements for extracellular Ca²⁺. If the isolated cells were incubated in Ca²⁺ free buffer containing 1.0 mM ethylene glycol bis (β-aminoethyl ether) N, N¹ - tetra acetic acid (EGTA) for 30 min. before adding carbachol an initial phase of ⁸⁶Rb⁺ efflux was observed. A second phase of ⁸⁶Rb⁺ efflux was obtained upon addition of 2.0 mM Ca²⁺. However when cells were incubated for 60 min. in Ca²⁺ free buffer containing 1.0 mM EGTA the initial phase of release caused by carbachol was inhibited by 95 percent. If the EGTA was titrated with Ca²⁺ to give 1.0 mM Ca²⁺, following the 60 min. depletion regimen, the second phase was observed. Although 60 min. of Ca²⁺-depletion in EGTA buffer was required for complete inhibition of the effect of carbachol on the initial phase of ⁸⁶Rb⁺ efflux, the response was fully restored within 4 min. after the readdition of Ca²⁺.     

Activation of calcium oscillations by thapsigargin in parotid acinar cells.

The tumor promoter thapsigargin releases Ca2+ from intracellular stores by specific inhibition of microsomal Ca-ATPase activity without inositol phosphate formation. Recent studies of the actions of thapsigargin support the concept that the level of Ca2+ within the inositol (1,4,5)-trisphosphate (IP3)-sensitive intracellular pool regulates the Ca2+ permeability of the plasma membrane. We examined the effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) in single rat parotid cells using digital fluorescence microscopy. In the absence of extracellular Ca2+ (Ca2+o), thapsigargin transiently increased [Ca2+]i. Following the thapsigargin-induced [Ca2+]i transient, carbachol in the continued absence of Ca2+o was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes Ca2+ from the IP3-sensitive store. In the converse experiment, carbachol prevented a rise of [Ca2+]i by thapsigargin, suggesting that the IP3- and thapsigargin-sensitive Ca2+ pools are the same. Depletion of Ca2+ from the IP3-sensitive pool by thapsigargin enhanced plasma membrane Ca2+ permeability. Thapsigargin triggered sustained Ca2+ oscillations in Ca2(+)-containing medium which are highly reminiscent of agonist-induced oscillations in these cells. Carbachol addition rapidly raised IP3 levels during oscillations triggered by thapsigargin but did not elevate [Ca2+]i, indicating that the IP3-sensitive pool remains continuously depleted during [Ca2+]i fluctuations. The results from this study rule out the involvement of the IP3-sensitive pool in the mechanisms involved in thapsigargin-induced (and by analogy, agonist-induced) oscillations in parotid cells.