Ca2+-Induced Increases in Steady-State Concentrations of Intracellular Calcium Are Not Required for Inhibition of Parathyroid Hormone Secretion

It has been well established that increases in extracellular calcium concentration ([Ca²⁺]) inhibit parathyroid hormone (PTH) secretion. The effects of [Ca²⁺] are mediated through a G-protein-coupled receptor that has been cloned and characterized. Additionally, it has been demonstrated in parathyroid cells that an increase in [Ca²⁺] results in an increase in steady-state levels of intracellular calcium ([Ca²⁺]_i). At present, it has not been fully resolved whether changes in [Ca²⁺]_i are related to changes in PTH secretion. In the current study, the effect of increased [Ca²⁺] on PTH secretion and the connection regarding changes in concentrations of intracellular calcium [Ca²⁺]_i have been examined in primary cultures of bovine parathyroid cells. PTH secretion was measured by radioimmunoassay and intracellular calcium was determined by single cell calcium imaging. Bovine parathyroid cells pre-incubated with either 0.5 or 1 mM calcium responded to rapid increases in [Ca²⁺] (≥0.5 mM) with an immediate and sustained increase in steady-state levels of [Ca²⁺]_i that persisted for time intervals greater than 15 minutes. Although the magnitude of the sustained increase in [Ca²⁺]_i varied among individual cells (∼40% to >300%), the overall pattern and course of time were similar in all cells examined (n = 142). In all trials, [Ca²⁺]_i immediately returned to baseline levels following the addition of the calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Additional control studies, however, suggest that sustained increases in [Ca²⁺]_i do not correlate with regulation of parathyroid hormone secretion. Sustained elevations of [Ca²⁺]_i were not observed when [Ca²⁺] was gradually increased by the addition of 0.1 mM increments at 1 minute intervals. Furthermore, the effect on inhibition of PTH secretion was the same regardless of whether [Ca²⁺] was increased by gradual or rapid addition.     

Functional dedifferentiation of parathyroid cells results both from a defective regulation and action of cytoplasmic Ca2+

Monolayer culture of bovine parathyroid cells for 24 hours resulted in a right-shift of the dose-effect relationships for Ca²⁺-inhibition of parathyroid hormone (PTH) release and the dependence of the cytoplasmic Ca²⁺ concentration (Ca²⁺) on extracellular Ca²⁺ as well as in a less suppressible hormone release. After 4 days of culture, hormone secretion was almost non-suppressible and Ca _i ²⁺ increased poorly in response to a rise in extracelluiar Ca²⁺. Ionomycin, a Ca²⁺ ionophore, raised Ca _i ²⁺ , but there was only a small inhibition of PTH release and the correlation between Ca _i ²⁺ and secretion was weak. A deteriorated Ca _i ²⁺ regulation and a decreased inhibitory action of cytoplasmic Ca²⁺ on PTH release were also found in ceils from human parathyroid adenomas. Functional dedifferentiation of the parathyroid cell thus results from both defective regulation and action of cytoplasmic Ca²⁺.     

Cytoplasmic Ca2+ concentration of single normal human and bovine parathyroid cells measured by dual wavelength microfluorometry

Dual wavelength microfluorometry was utilized to measure the cytoplasmic calcium concentration (Ca_i ²⁺) of single parathyroid cells loaded with the indicator fura-2. The method enabled the first registrations of Ca_i ²⁺ of normal human parathyroid cells, available only in minute numbers. At 0.5 mM extracellular Ca²⁺, the Ca_i ²⁺ levels were similar in normal human and bovine cells. Both cell types responded with an initial Ca_i ²⁺ transient followed by a sustained increase when raising extracellular Ca²⁺ to 3.0 mM. The sustained effect exhibited a sigmoidal relation to extracellular Ca²⁺ in the 0.5–3.0 mM range. Although the increase was somewhat greater in the human cells, the half maximal responses were obtained at almost identical extracellular Ca²⁺ concentrations. Whereas K⁺ depolarization decreased Ca_i ²⁺, the Ca_i ²⁺ channel blocker D-600 had dual actions, raising Ca_i ²⁺at 0.5 mM Ca²⁺ and decreasing it at 3.0 mM Ca_i ²⁺, and the effects were similar in the bovine and human cells. The present experimental approach verified the validity of utilizing bovine cells as controls in studies of human parathyroid tissue and it appears suitable for analysis of the role of different subpopulations of parathyroid cells in the abnormal parathyroid tissue of patients with hyperparathyroidism.     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca²⁺ concentration ([Ca²⁺]_o) and modulation of cellular processes associated with acute or sustained changes in [Ca²⁺]_o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca²⁺]_o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca²⁺]_o activated PKC-α and PKC-ε in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca²⁺]_o required influx of Ca²⁺through Ni²⁺-sensitive Ca²⁺channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -ε with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca²⁺]_o. Activation of ERK1/2 by high [Ca²⁺]_o was not necessary for the [Ca²⁺]_o-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca²⁺]_o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Modulation of the Ca2+-sensing function of parathyroid cells in vitro and in hyperparathyroidism

When raising the extracellular Ca²⁺ concentration stepwise from 0.5 to 3.0 mM, bovine parathyroid cells reacted with initial transient and sustained elevations of the cytoplasmic Ca²⁺ concentration (Ca²⁺_i), as well as more than 50% inhibition of parathyroid hormone (PTH) release. Human parathyroid adenoma cells and bovine cells cultured for 1 day or exposed to a low concentration of a monoclonal antiparathyroid antibody exhibited right-shifted dependencies of PTH release and Ca²⁺_i on extracellular Ca²⁺ and reduced Ca²⁺_i transients. The protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) further right-shifted the dose response relationship for Ca²⁺ regulated Ca²⁺_i of the adenoma cells, whereas the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) tended to normalize it, without affecting Ca²⁺_i of normal bovine cells. In cells from an oxyphil adenoma and a parathyroid carcinoma as well as in bovine cells cultured 4 days or exposed to a high concentration of the antiparathyroid antibody, there were no Ca²⁺_i transients, very small increases in steady-state Ca²⁺_i and nonsuppressible PTH release. The results suggest that reduced availability of a putative Ca²⁺-receptor and increased protein kinase C activity may be important factors in the decreased Ca²⁺ sensitivity of abnormal parathyroid cells.     

Effects of parathyroid hormone on cyclic AMP-formation and cytoplasmic free Ca2+ in the osteosarcoma cell line UMR 106-01

The effects of parathyroid hormone (PTH) on cytoplasmic free CA²⁺ (Ca _i ²⁺ ) and cAMP-formation were investigated in the rat osteosarcoma cell line UMR 106-01.In fura-2 loaded adherent single cells bPTH 1-34 (10 nM–1M) induced a rapid transient increase in Ca _i ²⁺ in 11% of the studied cells. In fura-2 tracings from UMR 106-01 cells in suspension, bPTH 1-34 (0.1 M) induced a transient increase in Ca _i ²⁺ in 20% of the experiments. The transient increase in Ca _i ²⁺ seen in suspensions of cells was not abolished by addition of EGTA (2.5 mM) prior to challenge with PTH, suggesting that the increase in Ca _i ²⁺ was derived from intracellular stores.A marked rapid increase in cAMP-formation was observed in all experiments with cells in suspension, also in the experiments where PTH did not affect Ca _i ²⁺ .These data show that PTH causes a release of Ca²⁺ from intracellular stores in a small percentage of osteosarcoma UMR 106-01 cells, and that PTH is capable of inducing an increase in cAMP-formation without affecting Ca _i ²⁺ in osteoblasts.     

Rise in cytoplasmic Ca induced by monensin in bovine medullary chromaffin cells

Monensin, a exchanger, induces catecholamine secretion from adrenal chromaffin cells by an unknown mechanism. We found and report here that in bovine chromaffin cells, monensin evokes profound changes in [Ca²⁺]_i which were measured by means of the fluorescent Ca²⁺ indicator Indo-1. Application of monensin (10 μM) generated a marked [Ca²⁺]_i rise. Removal of external Ca²⁺ did not prevent the elevation of [Ca²⁺]_i, though it was significantly decreased. In the presence of nifedipine (10 μM) or tetrodotoxin (3 μM) the monensin-induced [Ca²⁺]_i rise remained unchanged. In contrast, in the absence of extracellular Na⁺ the [Ca²⁺]_i rise was abolished. Addition of caffeine (40 mM) at the peak response generated by monensin produced a further increase in [Ca²⁺]_i, which was independent of external [Ca²⁺] or [Na⁺]. After depletion of the IP₃-sensitive compartment by thapsigargin (1 μM), caffeine still induced a rise in [Ca²⁺]_i while the monensin response was absent. We concluded that the origin of the Ca²⁺ for the [Ca²⁺]_i increase elicited by the exchanger in chromaffin cells is not the extracellular space. Clearly there seems to be at least two intracellular Ca²⁺ stores, one of which is affected by monensin. This Ca²⁺ pool, which is different than the pool stimulated by caffeine, is sensitive to the extracellular [Ca²⁺] and to thapsigargin. Our data are compatible with the idea that the monensin mediated Na⁺ entry could activate the production of inositol trisphosphate and this in turn could trigger Ca²⁺ release from the endoplasmic reticulum.     

SK&F 96365 inhibits intracellular Ca pumps and raises cytosolic Ca concentration without production of nitric oxide and von Willebrand factor

The effects of the imidazole compound SK&F 96365 on Ca²⁺ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca²⁺ gradient, 30 μM SK&F 96365 doubled [Ca²⁺]_i suggesting a Ca²⁺ release from intracellular stores. The SK&F 96365-induced [Ca²⁺]_i rise was not accompanied by detectable NO and vWF production, while 1 μM thapsigargin enhanced [Ca²⁺]_i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 ± 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca²⁺]_i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca²⁺, from internal pools, its effect and that of thapsigargin on the ATP-dependent ⁴⁵Ca²⁺, uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of ⁴⁵Ca²⁺ uptake. In conclusion, we demonstrate that, in the absence of Ca²⁺ entry from the extracellular space, the [Ca²⁺]_i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca²⁺ influx in endothelial secretion processes.     

Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

Calcium-dependent control of volume regulation in renal proximal tubule cells: II. Roles of dihydropyridine-sensitive and-insensitive Ca2+ entry pathways

Summary The Ca^2– entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca^2–] from 1 mM to 1 M caused intracellular free calcium concentration ([Ca²⁺]_i) to fall close to zero. Treatment with 10 M verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca²⁺ also induced fluctuations in cell volume. However, isotonic treatment with 10 M nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca²⁺]_i or cell volume, indicating that the endogenous Ca²⁺ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca²⁺, they swelled and underwent normal RVD while [Ca²⁺]_i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991.J. Membrane Biol. 123:149–160). When cells were swollen in the presence of verapamil or low bath [Ca²⁺], RVD was abolished and [Ca²⁺]_i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca²⁺]_i were abolished, but [Ca²⁺]_i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca²⁺ entry but that Ca²⁺ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca²⁺ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca²⁺ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive baseline Ca²⁺ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca²⁺ entry pathway, which is responsible for the supply of Ca ions to the Ca²⁺-dependent mechanism by which cell volume regulation is achieved.     

An Interaction Between ATP and High K+: Mutual Impairment of ATP- and High K+-Evoked [Ca2+]i Increase in NG 108–15 Cells

The interaction between ATP- and high K⁺-evoked increase in intracellular free calcium concentration ([Ca²⁺]_i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca²⁺]_i was measured in the neuronal model, neuroblastoma × glioma hybrid cells (NG 108–15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca²⁺, ATP induced a rapid, concentration-dependent increase in [Ca²⁺]_i. High K⁺ (50 mM) evoked a [Ca²⁺]_i rise from 109 ± 11 nM to 387 ± 81 nM (n = 16). The application of either of these two [Ca²⁺]_i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K⁺ strongly suggests that both agents rely at least in part on the same source of Ca²⁺ for elevation of [Ca²⁺]_i in NG 108–15 cells.     

The role of cytoplasmic pH in the inhibitory action of high osmolarity on secretion from bovine adrenal chromaffin cells

Elevated osmolarity is known to inhibit secretion from a wide range of cells including bovine adrenal chromaffin cells. The mechanism of this inhibition is unclear but the elevated osmolarity has been proposed to oppose an osmotic driving force involved in exocytotic fusion. Using the fluorescent indicators quenel and fura2, we monitored the effect of elevated osmolarity on cytoplasmic pH (pH_i) and cytoplasmic free Ca²⁺ ([Ca²⁺]_i). Elevated osmolarity increased both pH_i and [Ca²⁺]_i, but had no effect on the [Ca²⁺]_i rise elicited by either K⁺ or nicotine. Elevating pH_i with NH₄Cl was shown to inhibit secretion from chromaffin cells. The elevation of pH_i by hyperosmolar solutions is proposed as one of the mechanisms by which elevated osmolarity inhibits secretion.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Mapping of a carboxyl-terminal active site of parathyroid hormone by calciummimaging

We recently showed that the C-terminal fragment PTH (52–84) effectively increases intracellular free calcium ([Ca²⁺]_i in a subset of growth plate chondrocytes not activated by the N-terminal PTH fragment (1–34). Here we characterize the active site on C-terminal PTH (52–84) with respect to calcium (Ca²⁺)-signaling and the mechanism involved by using synthetic PTH-subfragments in digital CCD ratio-imaging experiments. Our results show amino acids 73–76 to be the core region for increasing [Ca²⁺]_i. Ryanodine (1 μM), caffeine (10 mM), lithium (2 mM), or cyclopiazonic acid (2–5 μMI), agents that interfere with intracellular Ca²⁺ release, all failed to block PTH (52–84) induced [Ca²⁺]_i increases. Depletion of extracellular calcium ([Ca²⁺]_o) blocked PTH (52–84) induced [Ca²⁺]; increases, indicating a transmembrane Ca²⁺ influx. In contrast to voltage-gated and Ca²⁺ release activated Ca²⁺ influx, PTH (52–84) evoked Ca²⁺ influx was not blocked by nickel (1 mM). We conclude that PTH amino acids 73–76 are essential for activation of a nickel-insensitive Ca²⁺ influx pathway in growth plate chondrocytes that is likely to be of relevance for matrix calcification, a key step in endochondral bone formation.     

Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion

Cyclic AMP (cAMP) and Ca²⁺ are key regulators of exocytosis in many cells, including insulin-secreting β cells. Glucose-stimulated insulin secretion from β cells is pulsatile and involves oscillations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse β cells. These oscillations were preceded and enhanced by elevations of [Ca²⁺]_i. However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca²⁺]_i rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.     

Characterization of voltage operated R-type Ca2+ channels in modulating somatostatin receptor subtype 2- and 3-dependent inhibition of insulin secretion from INS-1 cells

Somatostatin (SST) inhibits Ca²⁺ entry into pancreatic B-cells via voltage-operated Ca²⁺ channels (VOCCs) of L-type, leading to the suppression of insulin secretion. Activation of R-type channels increases insulin secretion. However, the role of R-type Ca²⁺ channels (Ca_V2.3) in mediating the effects of SST on insulin secretion has not been so far investigated. Here, we identify the SST-receptor subtypes (SSTR) expressed on insulin-producing INS-1 cells by RT-PCR and by functional assays. The role of R-type channels in regulating [Ca²⁺]_i in response to SST-treatment was detected by cell fluorescence imaging and patch-clamp technique. INS-1 expressed SSTR2 and SSTR3 and agonists (ag.) selective for these receptors reduced 10 nM exendin-4/20 mM glucose-stimulated insulin secretion. Surprisingly, SST and SST2-ag. transiently increased [Ca²⁺]_i. Subsequently, these agonists led to a decrease in [Ca²⁺]_i below the basal levels. In contrast, SST3-ag. failed to induce a transient peak of [Ca²⁺]_i. Instead, a persistent minor suppression of [Ca²⁺]_i was detected from 25 min. R-type channel blocker SNX-482 altered [Ca²⁺]_i in SST- and SST2-ag.-treated cells. Notably, the inhibition of insulin secretion by SST and SST2-ag., but not SST3-ag. was attenuated by SNX-482. Taken together, SST and SSTR2 regulate [Ca²⁺]_i and insulin secretion in INS-1 cells via R-type channels. In contrast, the R-type calcium channel does not mediate the effects of SST3-ag. on insulin secretion. We conclude that R-type channels play a major role in the inhibition of insulin secretion by somatostatin in INS-1 cells.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Imaging of intracellular Ca waves induced by muscarinic receptor stimulation in rat parotid acinar cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca²⁺, the increase in [Ca²⁺]_i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca²⁺ wave toward the basolateral region. The [Ca²⁺]_i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca²⁺ was eliminated from the extracellular medium, the Ca²⁺ wave was a result of Ca²⁺ release from intracellular stores. The magnitude and velocity of the Ca²⁺ wave decreased with decreasing concentration of CCh, and the increase in [Ca²⁺]_i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca²⁺ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca²⁺ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca²⁺ pump, caused a slow and homogeneous increase in [Ca²⁺]_i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca²⁺]_i suggesting that the inositol-1,4,5-trisphosphate (InsP₃) and ThG-sensitive Ca²⁺ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP₃ is essential to trigger the Ca²⁺ wave and that the development of the Ca²⁺ wave may be attributed to regional differences in InsP₃ sensitivity of Ca²⁺ stores. The agonist-induced Ca²⁺ wave is probably a general phenomenon in exocrine acinar cells.     

The characterization and quantification of antigen-induced Caoscillations in a rat basophilic leukaemia cell line (RBL–2H3)

Using1999 Harcourt Publishers Ltdthe ratiometric Ca²⁺indicator, indo-1, the antigen-induced increase in intracellular Ca²⁺concentration ([Ca²⁺]_i) was measured in individual RBL–2H3 cells which had been passively sensitized with monoclonal antibody to the dintrophenyl (DNP) haptenic group. Antigenic stimulation using DNP-human serum albumin conjugate (DNP-HSA) induced concentration-dependent asynchronous Ca²⁺oscillations, or irregular spikes. To achieve a quantitative comparison of the effects of different concentrations of antigen on changes in [Ca²⁺]_i, the area under the curve (AUC) of Ca²⁺oscillations in each cell was calculated.The dose–response curve of the calculated AUC is consistent with the bell-shaped dose–response curve for antigen-induced mediator release, depolarization and⁸⁶Rb⁺-efflux. Ca²⁺oscillations induced by antigenic stimulation were abolished by removal of external Ca²⁺and the subsequent reintroduction of external Ca²⁺caused their resumption.To investigate the role of Ca²⁺oscillations in the secretory response, changes in [Ca²⁺]_iinduced by concanavalin A (Con-A), A23187, thapsigargin and NECA were also monitored. Con-A mimicked the response induced by antigen, whilst A23187 and thapsigargin induced a large transient non-oscillatory response. NECA, an adenosine receptor agonist, induced only a small transient rise in [Ca²⁺]_iwithout oscillatory behaviour. Since all these stimuli accept NECA-induced degranulation in these cells, it is suggested that, although Ca²⁺oscillations are not essential for the initiation of secretion, they probably underlie the in-vivo physiological response of mast cells and basophils to an antigenic challenge. They also seem to enhance the efficacy of the Ca²⁺signal.