Calcium Pool Research Articles

Newcomer insulin secretory granules as a highly calcium-sensitive pool

Insulin secretion is biphasic in response to a step in glucose stimulation. Recent experiments suggest that 2 different mechanisms operate during the 2 phases, with transient first-phase secretion due to exocytosis of docked granules but the second sustained phase due largely to newcomer granules. Another line of research has shown that there exist 2 pools of releasable granules with different Ca(2+) sensitivities. An immediately releasable pool (IRP) is located in the vicinity of Ca(2+) channels, whereas a highly Ca(2+)-sensitive pool (HCSP) resides mainly away from Ca(2+) channels. We extend a previous model of exocytosis and insulin release by adding an HCSP and show that the inclusion of this pool naturally leads to insulin secretion mainly from newcomer granules during the second phase of secretion. We show that the model is compatible with data from single cells on the HCSP and from stimulation of islets by glucose, including L- and R-type Ca(2+) channel knockouts, as well as from Syntaxin-1A-deficient cells. We also use the model to investigate the relative contribution of calcium signaling and pool depletion in controlling biphasic secretion.

A Thalassemic Adolescent Presented With Bone Pain, Tetanic Contractions and Growth delay: Combined vitamin D and Parathyroid defects

Parathyroid hormone (PTH) and l,25-(OH) 2 vitamin D play key roles in the regulation of calcium and phosphate balance. Circulating levels of PTH can change within seconds after an alteration in serum calcium. When the serum calcium falls PTH is rapidly released and acts quickly to promote calcium reabsorption in the distal tubule and the medullary thick ascending limb of the loop of Henley. PTH also stimulates the release of calcium from the rapidly exchangeable pool of bone calcium and a chronic elevation in serum PTH increases renal l,25-(OH) 2 vitamin D production.

Study on calcium net absorptivity by 41Ca labeling calcium pool of rats

Study on calcium net absorptivity by 41Ca labeling calcium pool of rats

CONTROL OF MUSCLE RYANODINE RECEPTOR CALCIUM RELEASE CHANNELS BY PROTEINS IN THE SARCOPLASMIC RETICULUM LUMEN

1. Many biological processes that are governed by intracellular calcium signals rely on intracellular stores, which provide a reliable, controlled release of calcium into the cytoplasm. Calcium release through the ryanodine receptor (RyR), the main ion channel in the sarcoplasmic reticulum (the calcium store in muscle) is the key determinant of muscle force. 2. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the RyR and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. Until recently, how calsequestrin communicated the store Ca(2+) load to the RyR remained unknown. 3. Calsequestrin 1 (skeletal calsequestrin) has been shown to both inhibit and activate the skeletal RyR1, dependent on whether it's bound to the RyR1 directly or indirectly via anchoring proteins. 4. The phosphorylation status of calsequestrin 1 is deemed important: it influences the Ca(2+) binding capacity of calsequestrin, the way in which calsequestrin 1 regulates the RyR1 and how calsequestrin 1 interacts with the key anchoring protein junctin. 5. In skeletal muscle, junctin plays a more critical role than triadin in the mechanism that controls Ca(2+) release from the sarcoplasmic reticulum. 6. The close relationship between altered expression and dysfunction of calsequestrin in several skeletal and cardiac disorders highlights the critical role that calsequestrin plays in maintaining Ca(2+) homeostasis and regulation of muscle contraction.

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca(2+) precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca(2+)](cyt), and in pollen and style maturation during the progamic phase.

Age‐related alterations in Ca2+ signals and mitochondrial membrane potential in exocrine cells are prevented by melatonin

Information regarding age-induced Ca(2+) signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca(2+) signals and mitochondrial potential, a parameter involved in both Ca(2+) signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca(2+) homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22-24 months old) mice by collagenase dispersion. Ca(2+) signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca(2+) oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca(2+) homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca(2+) responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca(2+) signals and mitochondrial potential of aged exocrine cells.

Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events ("sparks") that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30-40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 microM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article.

The Calcium Sensing Receptor: From Understanding Parathyroid Calcium Homeostasis to Bone Metastases

The cloning of the calcium sensing receptor (CaR) confirmed that parathyroid cells monitor extracellular calcium concentration ([Ca2+]ext) via a receptor-type mechanism. This lead to the hypothesis that abnormalities in the expression and/or function of the CaR could explain the biochemical abnormalities in primary hyperparathyroidism (PHPT). Cultured cells from parathyroid adenomas of patients operated for PHPT were used to monitor real-time changes in intracellular calcium concentration ([Ca2+]i) as measured by fluorescent microscopy using the Fura-2/AM dye. We found that CaR agonists trigger release of intracellular calcium pools and such responses are amplified by increasing the affinity of IP3 receptors. Using confocal microscopy to monitor membrane trafficking in living parathyroid cells labelled with the fluorescent dye FM1-43, we found that a decrease in [Ca2+]i rather than an absolute change in [Ca2+]ext is the main stimulus for exocytosis from human parathyroid cells. These data suggest that, in PHPT, a defective signalling mechanism from the CaR allows cells from parathyroid adenomas to maintain low [Ca2+]i with uninhibited PTH secretion in the face of hypercalcaemia. Over longer periods of time, CaR controls parathyroid proliferation via changes in tyrosine phosphorylation. We found that multiple proteins of molecular weight 20-65 kDa are phosphorylated within 10-60 min in response to CaR agonists. Further work demonstrated that high [Ca2+]i stimulates the expression of bcl-2 oncoprotein in cultured human parathyroid cells and that, in parathyroid adenomas, predominant expression of bcl-2 rather than bax oncoprotein might prevent apoptosis and explain the slow growth rate of these tumours. More recently, it became apparent that CaR stimulates cell proliferation in several cell types not involved in calcium homeostasis. Using archived histological material from 65 patients who died with metastatic breast cancer, we identified CaR expression predominantly in tumours from patients who developed bone rather than visceral metastases (35 of 49 versus 7 of 16; P < 0.01, chi-squared test). These data suggest that CaR expression has the potential to become a new biological marker predicting the risk of bone metastases in patients with breast cancer. A prospective study should investigate if patients with CaR-positive tumours are more likely to develop bone metastases and whether they could benefit more from prophylactic treatment with bisphosphonates or the newly developed CaR antagonists.

Synthetic human calcitonin in Paget's disease of bone and osteoporosis (author's transl)

Synthetic human calcitonin was used in the treatment of 26 patients over a period of 1-14 months. 17 patients had Paget's disease of the bone, 6 postmenopausal osteoporosis and 3 Sudeck's syndrome. Subjective improvement (reduction of pain, improvement of mobility) was found in 15 patients with Paget's disease, in 4 females with postmenopausal osteoporosis and in all 3 patients with Sudeck's syndrome. Radiographic improvement of bone changes developed only very slowly. These results were confirmed by diminution of the exchangeable calcium pool indicating reduction of rates of osseous degradation. Calcitonin tolerance was acceptable. Transitory nausea and occasional vomiting occurred in 3 patients.

Phosphorylation of skeletal muscle calsequestrin enhances its Ca2+ binding capacity and promotes its association with junctin

Calcium signaling, intrinsic to skeletal and cardiac muscle function, is critically dependent on the amount of calcium stored within the sarcoplasmic reticulum. Calsequestrin, the main calcium buffer in the sarcoplasmic reticulum, provides a pool of calcium for release through the ryanodine receptor and acts as a luminal calcium sensor for the channel via its interactions with triadin and junctin. We examined the influence of phosphorylation of calsequestrin on its ability to store calcium, to polymerise and to regulate ryanodine receptors by binding to triadin and junctin. Our hypothesis was that these parameters might be altered by phosphorylation of threonine 353, which is located near the calcium and triadin/junctin binding sites. Although phosphorylation increased the calcium binding capacity of calsequestrin nearly 2-fold, it did not alter calsequestrin polymerisation, its binding to triadin or junctin or inhibition of ryanodine receptor activity at 1 mM luminal calcium. Phosphorylation was required for calsequestrin binding to junctin when calcium concentration was low (100 nM), and ryanodine receptors were activated by dephosphorylated calsequestrin when it bound to triadin alone. These novel data shows that phosphorylated calsequestrin is required for high capacity calcium buffering and suggest that ryanodine receptor inhibition by calsequestrin is mediated by junctin.

Parathyroid Hormone and Vitamin D Levels are Independently Associated With Calcific Aortic Stenosis

In calcific aortic valve disease, the early lesion is similar to atherosclerotic plaque, but later calcification prevails. Parathyroid hormone (PTH) and vitamin D are the principal calcium pool regulators, so the present study was designed to assess their association with aortic stenosis (AS) in patients with significant coronary artery disease (CAD), and preserved renal function. The 122 consecutive patients with AS (mean gradient > or =30 mmHg) plus CAD, and 101 patients with nonobstructive aortic sclerosis (mean gradient < or =10 mmHg) plus CAD, as controls, were prospectively enrolled. The AS patients were older (71+/-7 vs 66+/-7 years; p<0.001), had higher serum intact (i)PTH (51.4 [39-70] vs 37.4 [27-50] pg/ml; p<0.001), and lower plasma vitamin D (32.0 [25-40] vs 35.8 [27-55] nmol/L; p=0.003) levels than those with aortic sclerosis. The groups did not differ significantly in creatinine level (93 [82-105] vs 96 [85-107] micromol/L, p=0.19), calcium - phosphate product, occurrence of hypertension, smoking, diabetes, dyslipidemia, or body mass index. The iPTH (odds ratio (OR) 1.04, 95% confidence interval (CI) 1.02-1.05; p<0.001) and vitamin D levels (OR 0.97, 95% CI 0.95-0.99; p=0.003) were independently associated with AS. Higher serum iPTH with lower vitamin D levels were independently associated with calcific AS in CAD patients.

Bartonella-Induced Endothelial Cell Proliferation is Mediated by Release of Calcium from Intracellular Stores

The facultative intracellular bacterium Bartonella henselae induces unique angiogenic lesions in immunocompromised hosts. To determine the role of intracellular calcium pools in B. henselae-induced endothelial cell proliferation, we generated B. henselae-conditioned medium (BCM) and tested the ability of these cell-free proteins to induce human umbilical vein endothelial cell (HUVEC) proliferation, CXCL8 production, and intracellular Ca2+ signals. HUVECs incubated with BCM for 3 days had higher cell numbers than controls. In addition, HUVECs produced increased amounts of CXCL8 in response to BCM when compared to medium controls. When BCM was added to HUVECs and the intracellular Ca2+ response measured with the calcium-sensitive dye fura-2/AM, a Ca2+ rise was demonstrated. It was determined that this Ca2+ rise originated from intracellular Ca2+ stores through the use of the Ca2+ ATPase inhibitor thapsigargin. Further, it was demonstrated that BCM enhanced CXCL8 production and HUVEC proliferation in a Ca2+-dependent manner. Conditioned medium from B. henselae causes an intracellular Ca2+ rise in HUVECs, which is involved in B. henselae-induced HUVEC proliferation and CXCL8 production. These results implicate intracellular Ca2+ pools in B. henselae-induced angiogenesis and may lead to increased understanding of the mechanisms of pathogen-induced angiogenesis.

The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cgamma1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation.

Parathyroid hormone-related protein and calcium regulation in vitamin D-deficient sea bream (Sparus auratus)

Gilthead sea bream (Sparus auratus L.) were fed a vitamin D-deficient diet for 22 weeks. Growth rate, whole body mineral pools and calcium balance were determined. Plasma parathyroid hormone-related protein (PTHrP) and calcitriol levels were assessed. Expression of mRNA for pthrp and pth1r was quantified in gills and hypophysis. Fish on vitamin D-deficient diet (D- fish) showed reduced growth and lower calcium turnover (calcium influx, efflux and accumulation rates decreased) and unaltered plasma calcium levels. Plasma calcitriol levels became undetectable, PTHrP levels decreased in the D- fish. In controls, a significant increase in plasma PTHrP level over time was seen, i.e. it increased with body mass. Relationships were found between plasma PTHrP and the whole body pools of calcium, phosphorus and magnesium, indicative of a role for PTHrP in bone development. Expression of pthrp and pth1r mRNA was down-regulated in the hypophysis of D-fish, whereas in gill tissue, pthrp and pth1r mRNA were up-regulated. We conclude that lower pthrp mRNA expression and plasma values in D- fish reflect lower turnover of PTHrP under conditions of hampered growth; up-regulation of pthrp mRNA in gills indicate compensatory paracrine activity of PTHrP during calcitriol deficiency to guarantee well-regulated branchial calcium uptake. This is the first report to document a relation between PTHrP and calcitriol in fish.

Antimalarial drugs disrupt ion homeostasis in malarial parasites

Plasmodium chabaudi malaria parasite organelles are major elements for ion homeostasis and cellular signaling and also target for antimalarial drugs. By using confocal imaging of intraerythrocytic parasites we demonstrated that the dye acridine orange (AO) is accumulated into P. chabaudi subcellular compartments. The AO could be released from the parasite organelles by collapsing the pH gradient with the K+/H+ ionophore nigericin (20 microM), or by inhibiting the H+-pump with bafilomycin (4 microM). Similarly, in isolated parasites loaded with calcium indicator Fluo 3-AM, bafilomycin caused calcium mobilization of the acidic calcium pool that could also be release with nigericin. Interestingly after complete release of the acidic compartments, addition of thapsigargin at 10 microM was still effective in releasing parasite intracellular calcium stores in parasites at trophozoite stage. The addition of antimalarial drugs chloroquine and artemisinin resulted in AO release from acidic compartments and also affected maintenance of calcium in ER store by using different drug concentrations.

Calcium‐sensing receptor and recovery from hypocalcaemia in thyroparathyroidectomized rats

Plasma ionized calcium (p-Ca(2+)) is kept within a very narrow range and deviations are rapidly corrected by flux of Ca(2+) between extracellular fluid and the labile calcium pool at the quiescent bone surface. The calcium sensing at the bone surface represents a physiological interesting model for the rapid minute-to-minute regulation of p-Ca(2+). Our aim was to study whether the calcium-sensing receptor (CaR) has a role in the rapid recovery of p-Ca(2+) from acute induced hypocalcaemia. Male Wistar rats were thyroparathyroidectomized (TPTX). Acute hypocalcaemia in the animals was induced by infusion of EGTA (40-50 mM EGTA, 3.0 mL h(-1) for 30 min). Thereafter the recovery of p-Ca(2+) was followed. Vehicle or the CaR activators, R-568 (2 mg as a bolus twice) or gentamycin were administrated intravenously. EGTA infusion resulted in significantly lower nadir of hypocalcaemia in R-568- or gentamycin-treated rats compared to vehicle-treated rats (P < 0.01). During recovery phase p-Ca(2+) remained significantly lower in R-568 rats (P < 0.001). As such p-Ca(2+) levels recovered to basal levels in the vehicle group within 70 min after stopping EGTA, while R-568 or gentamycin rats remained significantly hypocalcaemic. The CaR activators R-568 and gentamycin, both significantly delayed the recovery of p-Ca(2+) from acute EGTA-induced hypocalcaemia in TPTX rats. This novel finding suggests the existence of calcium sensing by bone of importance for the rapid minute-to-minute regulation of p-Ca(2+).

Hydrogen peroxide increases GABAergic mIPSC through presynaptic release of calcium from IP3 receptor‐sensitive stores in spinal cord substantia gelatinosa neurons

GABAergic interneurons of the spinal cord substantia gelatinosa regulate the transmission of nociceptive information. Hydrogen peroxide (H2O2) is likely a diffusible messenger contributing to the development of long-lasting pathological pain states after nerve injury. In this study, we examined the presynaptic effects of H2O2 on the inhibitory interneurons of mouse substantia gelatinosa (SG) using whole-cell patch-clamp recordings from spinal cord slices. H2O2 increased the frequency of GABAergic miniature inhibitory postsynaptic current (mIPSC) in a concentration-dependent (10-1000 microM) manner. The profound increase in mIPSC frequency was diminished by thapsigargin or cyclopiazonic acid suggesting that the intracellular stored pool was the source of presynaptic calcium. Further examination revealed the 2-aminoethoxydiphenil borate blockable inositol-(1,4,5) trisphosphate receptor (IP3R) regulated pool of stored calcium as the likely source. The phospholipase C (PLC) blocker, 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U73122), did not block the frequency increase, which suggested that the site of action of H2O2 lies downstream in the IP3 signalling pathway, and nifedipine-sensitivity of the frequency increase indicated a possible role of calcium-induced calcium-release. However, a direct examination of L-type voltage-gated calcium channels (VGCC) demonstrated that H2O2 did not increase the calcium influx through these channels. The H2O2 effect on mIPSC frequency was markedly reduced in the opisthotonus (Opt) mutant mice with a known deletion in the IP3R1 gene. We demonstrated that H2O2 increased presynaptic activity in the GABAergic interneurons by the release of calcium from the IP3R-regulated intracellular pool. The presynaptic IP3R could emerge as a novel target for preventing H2O2-induced synaptic plasticity in substantia gelatinosa leading to pathological pain states.

An Investigative study on the mechanisms by which nicotine affects the secretory responses in rat primary pancreatic acinar cells

The current study examined the underlying mechanisms behind the nicotine –induced maximal secretory response to cholecystokinin ( CCK) in isolated pancreatic acinar cells. Methods: Acinar cells were isolated, purified and maintained in Hepes-Ringer buffer (HR) at pH 7.4. The cells were incubated with nicotine (100 uM) for 6 min and then with CCK (100pM) for 30 min in the presence or absence of a nicotinic receptor antagonist, calcium receptor antagonists and mitogen activated protein kinase inhibitors. Functional response, measured as an index of percent amylase release in response to CCK, in the incubation media was determined. Results: Exposure of cells to (100 uM)of nicotine significantly enhanced CCK-stimulated cell function. These effects remained unaltered in the presence of MAPK inhibitors but were suppressed by calcium channel receptor antagonists and inositol trisphosphate (IP3) receptor blockers, p <0.05. Mecamylamine, a nicotinic receptor antagonist, also prevented calcium mobilization resulting in decreased release of amylase in the presence of nicotine. Conclusions: These data suggest that downstream events regulating the exocytotic secretion of amylase in response to CCK in acinar cells are influenced primarily by intracellular calcium release. The results further implicate the role of nicotine in the mobilization of intracellular and extracellular calcium pools in isolated acinar cells. We conclude that nicotine plays an important role in calcium mobilization in acinar cells.

Prolactin stimulates prostate cell proliferation by increasing endoplasmic reticulum content due to SERCA 2b over-expression

Prolactin (PRL) has been shown to be involved in the differentiation and proliferation of numerous tissues, including the prostate gland. Moreover, variations in [Ca2+]ER (calcium concentration within the endoplasmic reticulum) may play a role in cell growth. However, few studies have focused on the regulation of calcium homoeostasis by prolactin. The present study evaluates the regulation of calcium pools as well as the possible role of [Ca2+]ER variations as a signal for growth modulation by PRL. We show that PRL stimulates the proliferation of normal SV40 immortalized epithelial prostate (PNT1A) cells with a maximum effect at a dose of 100 ng/ml. We also show that 100 ng/ml PRL increases the [Ca2+]ER when measured either by indirect quantification with Fura-2AM after application of 1 mM thapsigargin or by direct quantification with Mag-Fura-2AM within the endoplas-mic reticulum. Western blot analysis shows that the SERCA 2b (sarcoendoplasmic calcium ATPase 2b) is over-expressed in PNT1A cells treated with 100 ng/ml PRL for 24 h. A small inter-fering RNA SERCA 2a/b, used to down-regulate endogenous SERCA 2b expression, reduced both PNT1A cell proliferation and [Ca2+]ER. We thus identify [Ca2+]ER and SERCA 2b as protagonists in PRL-induced proliferation.

Signaling pathways involved in pilocarpine-induced mucin secretion in rat submandibular glands

We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE 2 levels and a positive ( r = 0.8870) and significant ( p = 0.0077) correlation between PGE 2 production and mucin released was determined. The participation of PGE 2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M 1 and M 4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M 1 and M 4 mAChR subtypes induces an increase of intracellular Ca 2+ concentration ([Ca 2+] I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE 2 released induces cAMP accumulation which, together with PKC are involved in the PGE 2 increased Ca 2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE 2 production.