Fluoride interactions with stimulus-secretion coupling of normal and pathological parathyroid cells.

Effects of the GTP binding protein (G-protein) activator NaF on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP content of bovine as well as normal and pathological human parathyroid cells were studied using precautions to avoid CaF2 precipitation. In 0.5 mM external Ca2+, NaF inhibited PTH release and lowered the cAMP content by 50-70% of the effects attained with 3.0 mM Ca2+. The NaF-induced increase of [Ca2+]i was considerably smaller than that obtained with rise of external Ca2+. It seems likely that NaF activates the inhibitory G1-protein involved in the regulation of cAMP generation. However, it is unclear whether the sluggish rise of [Ca2+]i induced by NaF is due to a direct effect of a G-protein on Ca2+ entry, or somehow related to the G-protein mediated formation of inositol 1,4,5-trisphosphate, which is part of the signal transduction pathway normally initiated by Ca2+ binding to its receptor on the parathyroid cell surface. Inhibition of PTH release by NaF probably results from the combined effects on [Ca2+]i and cAMP content. In hyperparathyroidism (HPT) the actions of NaF were not markedly affected despite severe impairments of Ca(2+)-inhibited PTH release and Ca2+ triggered increase of [Ca2+]i. Consistent with observations of down regulation of the parathyroid Ca2+ receptor in HPT, the present results indicate that the disease perturbs signal transduction at a level proximal to the site of action for NaF.     

Hormone-mediated Ca2+ transients in isolated renal cortical thick ascending limb cells

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca²⁺ ([Ca²⁺]_i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca²⁺]_i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca²⁺]_i in a dose-dependent manner. [Ca²⁺]_i maximally increased from 85±5 nmol/l to 608±99 nmol/l with PTH, 10^–6M, and to 766±162 nmol/l with AVP, 10^–7 M. The increment in [Ca²⁺]_i by both hormones was by intracellular Ca²⁺ release and entry through plasma membrane Ca²⁺ channels. 8-Bromoadenosine-3, 5-cyclic monophosphate (8-BrcAMP), 10^–4M, increased [Ca²⁺]_i(basal 83±3 to 427±121 nmol/l) but only from internal sources as nifedipine (10 mol), ([Ca²⁺]_i changes: 86±4 to 390±29 nmol/l) and removal of bath Ca _o ²⁺ , ([Ca²⁺]_ichanges: 84±6 to 517±142 nmol/l), were without effect on agonist-induced [Ca²⁺]_i. Thapsigargin, 1.5 mol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca²⁺ transients, and partially inhibited PTH-mediated Ca²⁺ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca²⁺ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca²⁺]_i transients. The responses partially returned following down-regulation of PKC with prolonged exposure to phorbol esters. These data suggest that PKC activation modulates agonist-induced Ca²⁺ release and entry, possibly through actions on intracellular release mechanisms. In summary, PTH and AVP stimulate Ca²⁺ signals by similar pathways involving cAMP and inositol 1,3,4-trisphosphate activity at similar sites on the endoplasmic reticulum and plasma membrane. These results suggest that peptide hormones may act through Ca²⁺ and be modulated by different pathways which may have diverse effects on cTAL function.     

Evidence for a function of calcium influx in the stimulation of hormone release from the parathyroid gland in the goat

The acute effects of various drugs on the release of parathyroid hormone (PTH) in goats were studied by local infusions in vivo. Infusions of Ca²+ or Sr²+ reduced the PTH secretion rate, whereas hypocalcemia induced by EDTA increased the PTH release. Blockers of voltage sensitive Ca²+ channels (verapamil, D-600 and nifedipine) lowered the PTH secretion rate, while infusion of 4-aminopyridine, which is a blocker of voltage sensitive K+ channels, increased the PTH release. These effects were not due to altered βadrenergic tonus, since the effects persisted when the drugs were administered during contineous infusion of the β-blocker propranolol. We suggest that the parathyroid cells possess voltage sensitive K+ and Ca²+ channels, and that exocytosis of stored PTH depends on the influx of extracellular Ca²+ as in other secretory cells. In order to explain the inverse relationship between the plasma Ca²+ level and the PTH release, we postulate a suppressive effect of the plasma Ca²+ on the membrane permeability to Ca²+ in parathyroid cells.     

Association of a protective paraoxonase 1 (PON1) polymorphism in Parkinson's disease

Intracellular second messengers play an important role in capsaicin- and analogous-induced sensitization and desensitization in pain. Fluorescence Ca²⁺ imaging, enzyme immunoassay and PKC assay kit were used to determine a novel mechanism of different Ca²⁺ dependency in the signal transduction of capsaicin-induced desensitization. On the average, capsaicin increased cAMP, cGMP concentration and SP release in bell-shaped concentration-dependent manner, with the maximal responses at concentrations around 1 μM, suggesting acute desensitization of TRPV1 receptor activation. Capsaicin-induced intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase depended on extracellular Ca²⁺ influx as an initial trigger. The Ca²⁺ influx by capsaicin increased PKC activation and SP release. These increases were completely abolished in Ca²⁺-free solution, suggesting that the modulation of capsaicin on PKC and SP are Ca²⁺-dependent. Interestingly, the maximal cAMP increase by TRPV1 activation was not blocked Ca²⁺ removal, suggesting at least in part a Ca²⁺-independent pathway is involved. Further study showed that cAMP increase was totally abolished by G-protein and adenylate cyclase (AC) antagonist, suggesting a G-protein-dependent pathway in cAMP increase. However, SP release was blocked by inhibiting PKC, but not G-protein or AC, suggesting a G-protein independent pathway in SP release. These results suggest that both Ca²⁺-dependent and independent mechanisms are involved in the regulation of capsaicin on second messengers systems, which could be a novel mechanism underlying distinct desensitization of capsaicin and might provide additional opportunities in the development of effective analgesics in pain treatment.     

Evidence for interference of vitamin D with PTH/PTHrP receptor expression in opossum kidney cells

 Vitamin D counters the phosphaturic action of parathyroid hormone (PTH) in rats in vivo. The present study was undertaken to examine this interaction using monolayers of Opossum kidney (OK) cells. ³²P uptake, cAMP generation, PTH/PTHrP receptor mRNA expression and intracellular Ca²⁺ [Ca²⁺]_i were measured in (1) control cells, (2) cells exposed to PTH, (3) cells pretreated with 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], and (4) 1,25(OH)₂D₃-pretreated cells exposed to PTH. ³²P uptakes were in (1) 5.00±0.20 (mean ±SE), in (2) 2.30±0.14 (P<0.001 versus group 1), in (3) 4.80±0.24 (P NS versus group 1) and in (4) 3.70±0.20 (P<0.001 versus group 2) nmol P_i/(mg·prot 10 mm). cAMP levels were in (1) 10±3, in (2) 210±8, in (3) 12±4, and in (4) 122±12 pmol cAMP/mg protein (P<0.001 versus group 2). PTH/PTHrP receptor mRNA expression was in relative units: (1) 100±0, (2) 99.5±6.2, (3) 68.7±2.6 (P<0.001 versus group 1), and (4) 34.8±3.3 (P<0.001 versus group 1). In groups 2 and 4 PTH induced equal transient increments in [Ca²⁺]_i. These experiments demonstrate that the effect of vitamin D on phosphate transport is associated with a commensurate diminution in PTH/PTHrP receptor gene expression and PTH-induced cAMP formation but not with Ca²⁺ transients. Vitamin D per se does not affect ³²P uptake or cAMP generation while it slightly decreased PTH/PTHrP receptor gene expression. These observations demonstrate that: (1) 1.25(OH)₂D₃ directly antagonizes the effects of PTH on ³²P uptake in OK cells, (2) this effect is mediated via inhibition of PTH-induced activation of AC/cAMP system, (3) the diminution in PTH-induced cAMP formation may stem at least in part from a decrease in the expression of PTH/PTHrP receptor mRNA. Received: 2 December 1997 / Received after revision: 19 January 1998 / Accepted: 28 January 1998     

Detection of dihydropyridine- and voltage-sensitive intracellular Ca2+ signals in normal human parathyroid cells

We recently showed dihydropyridine- and voltage-sensitive Ca²⁺ entry in cultured parathyroid cells from patients with secondary hyperparathyroidism. To determine whether normal parathyroid cells have a similar extracellular Ca²⁺ entry system, cells were isolated from normal (non-hyperplastic) human parathyroid glands. Fluorescence signals related to the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_I) were examined in these cells. Cells loaded with fluo-3/AM showed a transient increase in fluorescence (Ca²⁺ transient) following a 10-s exposure to a 150 mM K⁺ solution in the presence of millimolar concentrations of external Ca²⁺. The Ca²⁺ transient was reduced by dihydropyridine antagonists or 0.5 mM Cd²⁺, but enhanced by FPL-64176, an L-type Ca²⁺-channel agonist. Ca²⁺ transients induced by the 10-s exposure to 3.0 mM extracellular Ca²⁺ ([Ca²⁺]_o) were also inhibited by dihydropyridine antagonists or 0.5 mM Cd²⁺. These results provide the first evidence that normal human parathyroid cells express a dihydropyridine-sensitive Ca²⁺ entry system that may be involved in the [Ca²⁺]_o-induced change in [Ca²⁺]_I. This system might provide a compensatory pathway for negative feedback regulation of parathyroid hormone secretion under physiological conditions.     

Inhibitory action of Ca2+ on spontaneous transmitter release at motor nerve terminals in a high K+ solution

The inhibitory effect of a high external Ca²⁺ ([Ca²⁺]_o) on spontaneous transmitter release in a high K⁺ solution (Gage and Quastel 1966; Birks et al. 1968) was studied at the frog neuromuscular junction, based on the hypothesis that an increased intracellular free Ca²⁺ ([Ca²⁺]_i) in the nerve terminal plays a key role in the depression. Three procedures were employed to increase [Ca²⁺]_i; increasing [Ca²⁺]_o, application of caffeine and tetanic nerve stimulation. All of these procedures increased m.e.p.p. frequency in normal Ringer. However, as the basic m.e.p.p. frequency was increased by raising the external K⁺ concentration (7–15 mM), their facilitatory effects on m.e.p.p. frequency decreased, disappeared and eventually reversed to depressant actions. Since a rise in the external K⁺ concentration would increase the steady state level of [Ca²⁺]_i, it is suggested that when the [Ca²⁺]_i is preset at a high level, manipulations so as to further increase [Ca²⁺]_i depress spontaneous release of transmitter. Possible mechanisms for this inhibition was discussed in relation to a question whether or not the rate of spontaneous transmitter release is a monotonic function of [Ca²⁺]_i.     

Influence of acid-base changes on the intracellular calcium concentration of neurons in primary culture

The influence of changes in intra- and extracellular pH (pH_i and pH_e, respectively) on the cytosolic, free calcium concentration ([Ca²⁺]_i) of neocortical neurons was studied by microspectrofluorometric techniques and the fluorophore fura-2. When, at constant pH_e, pH_i was lowered with the NH₄Cl prepulse technique, or by a transient increase in CO₂ tension, [Ca²⁺]_i invariably increased, the magnitude of the rise being proportional to pH_i. Since similar results were obtained in Ca²⁺-free solutions, the results suggest that the rise in [Ca²⁺]_i was due to calcium release from intracellular stores. The initial alkaline transient during NH₄Cl exposure was associated with a rise in [Ca²⁺]_i. However, this rise seemed to reflect influx of Ca²⁺ from the external solution. Thus, in Ca²⁺-free solution NH₄Cl exposure led to a decrease in [Ca²⁺]_i. This result and others suggest that, at constant pH_e, intracellular alkalosis reduces [Ca²⁺]_i, probably by enhancing sequestration of calcium. When cells were exposed to a CO₂ transient at reduced pH_e, Ca²⁺ rose initially but then fell, often below basal values. Similar results were obtained when extracellular HCO ₃ ^- concentration was reduced at constant CO₂ tension. Unexpectedly, such results were obtained only in Ca²⁺-containing solutions. In Ca²⁺-free solutions, acidosis always raised [Ca²⁺]_i. It is suggested that a lowering of pH_e stimulates extrusion of Ca²⁺ by ATP-driven Ca²⁺/2H⁺ antiport.     

Oxytocin mobilizes calcium from a unique heparin-sensitive and thapsigargin-sensitive store in single myometrial cells from pregnant rats

Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was monitored using the fluorescence from the dye Fura-2-AM in single myometrial cells from pregnant rats. Oxytocin and acetylcholine applied to the cell evoked an initial peak in [Ca²⁺]_i followed by a smaller sustained rise which was rapidly terminated upon removal of acetylcholine or persisted after oxytocin removal. A Ca²⁺ channel blocker (oxodipine) and external Ca²⁺ removal decreased both the transient and sustained rises in [Ca²⁺]_i suggesting that Ca²⁺ influx through L-type Ca²⁺ channels participated in the global Ca²⁺ response induced by oxytocin. However, the initial peak in [Ca²⁺]_i produced by oxytocin was mainly due to Ca²⁺ store release: it was abolished by inclusion of heparin [which blocks inositol 1,4,5-trisphosphate (InsP ₃) receptors] in the pipette (whole-cell recording mode of patch-clamp) and external application of thapsigargin (which blocks sarcoplasmic reticulum Ca²⁺-ATPases). In contrast, the transient Ca²⁺ response induced by oxytocin was unaffected by ryanodine. Moreover, caffeine failed to induce a rise in [Ca²⁺]_i but reduced the oxytocin-induced transient Ca²⁺ response. The later sustained rise in [Ca²⁺]_i produced by oxytocin was due to the entry of Ca²⁺ into the cell as it was suppressed in external Ca²⁺-free solution. The Ca²⁺ entry pathway is permeable to Mn²⁺ ions, in contrast to that described in various vascular and visceral smooth muscle cells. Oxytocin-induced Ca²⁺ release is blocked by the oxytocin antagonist d(CH₂)₅[Tyr(Me)²,Thr⁴,Tyr-NH ₂ ⁹ ]OVT. The prolonged increase in [Ca²⁺]_i after oxytocin removal is rapidly terminated by addition of the oxytocin antagonist suggesting that oxytocin dissociation from its receptor is very slow. The oxytocin stimulation of [Ca²⁺]_i was insensitive to incubation with pertussis toxin, and blocked by a pipette solution containing anti-q/₁₁ antibody. These data show that myometrial cells possess an unique heparin-sensitive and thapsigargin-sensitive store that can be mobilized by activation of oxytocin receptors which couples with a Gq/G₁₁-protein to activate phospholipase C.     

Intracellular citrate induces regenerative calcium release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum was studied in voltage-clamped guinea-pig atrial myocytes. Cells were dialysed with a pipette solution containing the Ca²⁺ indicator 1- [2-amino-5-(6-carboxyindol-2-yl) phenoxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N,N-tetraacetic acid](Indo-1, 100 M) and as main anion either chloride or the low-affinity Ca²⁺ buffer citrate. Intracellular Ca²⁺ transients (Ca_i transients) were elicited by depolarizations from a holding potential of –50 mV. In chloride-dialysed cells, Ca_i transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. In citratedialysed cells, membrane depolarizations were associated with a small rise in [Ca²⁺]_i. These small changes in [Ca²⁺]_i were either followed by a large Ca_i. transient or failed to induce large changes in [Ca²⁺]_i. The peak amplitude of the large Ca_i transient did not vary with the amplitude of the depolarizing pulse. These results demonstrate that in the presence of intracellular chloride, Ca²⁺ release in atrial cells is a graded process triggered by Ca²⁺ influx. Using citrate as the main intracellular anoin, Ca²⁺ release triggered by Ca²⁺ entry was no longer graded but occurred in a regenerative manner. The results are discussed in terms of two models in which citrate, affects the spatial distribution of [Ca²⁺]_i or the loading state of the sarcoplasmic reticulum.     

Different Reactions of Aortic and Venular Endothelial Cell Monolayers to Histamine on Macromolecular Permeability: Role of cAMP, Cytosolic Ca2+ and F-actin

Endothelial cells assume a central role in the one process that the permeation of microvessels is accelerated in case of inflammation. We studied the effect of histamine on endothelial permeability, [Ca²⁺]_i, cAMP and F-actin, using same origin aortic and venular cultured endothelial monolayers. When HUVEC were treated with histamine (10^–7–10^–5 M), permeability of FITC-dextran (molecular weight 70,000) and [Ca²⁺]_i were increased, while cAMP content was unchanged, and F-actin content was reduced. When bovine vein-derived endothelial cells were treated with histamine, [Ca²⁺]_i was increased via H1 receptors, but permeability and F-actin content were not altered. When human aorta-derived endothelial cells were, [Ca²⁺]_i was increased via H1 receptors and cAMP content was increased via H2 receptors, while permeability and F-actin content were not changed. When bovine aorta-derived endothelial cells were, cAMP and F-actin content were increased, while permeability was reduced. These findings suggest that endothelial cells derived from different tissues clearly showed the different reactions to histamine, the increase in [Ca²⁺]_i led to the increase in endothelial permeability, while the increase in cAMP levels led to the reduction in permeability, and finally, F-actin regulated endothelial macromolecular permeability.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

The effects of thapsigargin on [Ca2+]i in isolated rat mesenteric artery vascular smooth muscle cells

Summary The effects of thapsigargin were studied on single cells isolated from side branches of the rat mesenteric artery. Thapsigargin (150 nM) produced a transient increase of [Ca²⁺]_i. This transient rise of [Ca²⁺]_i was unaffected by removing external Ca²⁺ ions. This suggests that thapsigargin is releasing Ca²⁺ ions from an intracellular store. In the absence of thapsigargin both noradrenaline and caffeine also produced a transient increase of [Ca²⁺]_i. These increases were abolished by prior exposure to thapsigargin. Correspondingly, the effects of thapsigargin were abolished by prior exposure to caffeine. These results show that thapsigargin releases Ca²⁺ from the noradrenaline and caffeine-sensitive stores.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca²⁺ ([Ca²⁺]_i). The increases of [Ca²⁺]_i evoked by TG was associated with significant changes of plasma membrane Ca²⁺ permeability, with [Ca²⁺]_i values following changes in extracellular [Ca²⁺]. Plasma membrane Ca²⁺ extrusion is activated rapidly as a consequence of the rise in [Ca²⁺]_i evoked by TG and the rate of extrusion is linearly dependent on [Ca²⁺]_i up to 1 μM Ca²⁺. In contrast, the activation of the Ca²⁺ entry pathway is delayed and the apparent rate of Ca²⁺ entry is independent of [Ca²⁺]_i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP₃), the increase of [Ca²⁺]_i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca²⁺]_i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca²⁺]_i increase (from 12.3±1.0 to 6.1±0.6 nM Ca²⁺/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca²⁺]_i. The amplitude of the [Ca²⁺]_i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La³⁺, which is known to inhibit the plasma membrane Ca²⁺-activated adenosine triphosphatase, induced a much higher peak of [Ca²⁺]_i. This increase was associated with an augmentation of the apparent rate of [Ca²⁺]_i increase (from 12.3±1.2 to 16.1±1.9 nM Ca²⁺/s). The inhibitory effect of caffeine, as well as the increase in [Ca²⁺]_i observed on caffeine removal was not affected by the presence of 1 mM La³⁺. These data indicate that an important component of the TG-evoked [Ca²⁺]_i increase is due to InsP₃-sensitive Ca²⁺ release which is probably mediated by the resting levels of InsP₃.     

Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

A hypo-osmotically induced increase in intracellular Ca2+ in lactating mouse mammary epithelial cells involving Ca2+ influx

 Using a fluorescent Ca²⁺-sensitive dye, we studied the effect of hypo-osmotic stress on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in acini freshly isolated from lactating mouse mammary gland. The basal [Ca²⁺]_i of mammary acini was unaffected by a 50% (v/v) dilution of suspensions with isotonic or hypertonic buffer, or after ionic (iso-osmotic) dilution (external Ca²⁺ was 3 mM). Hypo-osmotic dilution (50%) elicited a rapid increase in [Ca²⁺]_i comprising a large, transient elevation, followed by a maintained plateau phase. No hypo-osmotically induced rise in [Ca²⁺]_i was observed in the absence of extracellular Ca²⁺. Neither microtubule disassembly using nocodazole nor actin disruption with cytochalasin D prevented hypo-osmotically evoked stimulation of [Ca²⁺]_i. Pre-incubation of acini with nifedipine did not prevent hypo-osmotically induced stimulation of [Ca²⁺]_i, whereas a non-specific cation channel blocker, gadolinium, partially inhibited the increases in [Ca²⁺]_i induced by hypo-osmotic stress. Furthermore, the transient component was still apparent, and not diminished in magnitude, after [Ca²⁺]_i had been elevated by mobilisation of Ca²⁺ from intracellular stores using thapsigargin. The results demonstrate that hypo-osmotic stress generates an increase in [Ca²⁺]_i in lactating mammary epithelial cells, the major, transient component of which appears to be due to influx of extracellular Ca²⁺. Received: 15 October 1996 / Received after revision and accepted: 1 November 1996     

Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential

We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca²⁺]_i) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I₂ in endothelial cells. ATP (3 mol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca²⁺]_i in cultured endothelial cells. [Ca²⁺]_i remained elevated over several minutes. When the cells were depolarized, either by K⁺ (70–90 mmol/l) or by preincubation with the blocker of K⁺ channels tetraethylammonium (3 mmol/l), the initial peak of [Ca²⁺]_i remained unaffected but [Ca²⁺]_i returned significantly faster to resting levels, indicating a reduction in Ca²⁺ influx. In native, freshly isolated endothelial cells, K⁺ abolished increases in [Ca²⁺]_i induced by acetylcholine (3 mol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K⁺ (by 70%), whereas release of prostaglandin I₂ was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 mol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca²⁺]_i (twofold) after washout of the drug. Depolarization with K⁺ decreased thimerosal-induced EDRF release and [Ca²⁺]_i in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 mol/l), an activator of K⁺ channels. BRL 34915 also elicited increases in [Ca²⁺]_i, particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K⁺. We conclude that the initial rise in [Ca²⁺]_i in response to receptor-binding agonists, caused by mobilization of Ca²⁺ from intracellular stores, activates K⁺ channels, thereby inducing hyperpolarization. This hyperpolarization provides the driving force for transmembrane Ca²⁺ influx into endothelial cells and is thus an important signal for synthesis and release of EDRF.     

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells

We have studied the pathways by which extra-cellular bradykinin and adenosine 5-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca²⁺]_i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca²⁺]_i as assessed by fura-2 based micro-fluorimetry. The bradykinin-induced response consisted of an initial Ca²⁺ mobilization from an internal pool followed by a sustained increase in [Ca²⁺]_i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca²⁺ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca²⁺]_i, due to an increase in the driving force for Ca²⁺ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca²⁺]_i. Inward current and [Ca²⁺]_i were enhanced during hyperpolarizing steps indicating a substantial Ca²⁺ influx through ATP-activated channels. No release of Ca²⁺ from internal stores, but a large Na⁺ inward current, was observed in Ca²⁺-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.     

Effect of α1‐adrenergic stimulation of Cl− secretion and signal transduction in exocrine glands (Rana esculenta)

In the present work, the effect of stimulation of α‐adrenergic receptors on Cl^? secretion via exocrine frog skin glands was investigated. The α‐adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the β‐adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl^? secretion was measured as the amiloride‐insensitive short circuit current (I_SC). Addition of noradrenaline induced a biphasic increase in the I_SC. The increase in I_SC coincided with an increase in the net ³⁶Cl^? secretion. The noradrenaline‐induced increase in I_SC was dose‐dependent with an EC₅₀ of 13 ± 0.3 μM . Epifluorescence microscopic measurements of isolated, fura‐2‐loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca²⁺]_i) response. Application of noradrenaline induced a biphasic [Ca²⁺]_i response, which was dose‐dependent with an EC₅₀ of 11 ± 6 μM . The Ca²⁺ plateau unlike the peak‐response was sensitive to removal of Ca²⁺ from the extracellular medium. The noradrenaline‐induced increase in the Cl^? secretion as well as in [Ca²⁺]_i was sensitive to the α₁‐adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca²⁺]_i indicating that the release was independent of ryanodine‐sensitive Ca²⁺ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5‐trisphosphate (IP₃) content suggesting that the signal transduction pathway leading to the noradrenaline‐induced increase in Ca²⁺ involved IP₃ and a release of Ca²⁺ from IP₃‐sensitive stores.     

Actions of growth-hormone-releasing hormone on rat pituitary cells: intracellular calcium and ionic currents

Actions of growth-hormone-releasing hormone (GHRH) on single rat anterior pituitary cells were studied using indo-1 fluorescence to monitor changes in intracellular calcium, [Ca²⁺]_i, and perforated-patch recording to measure changes in membrane potential and ionic currents. GHRH elevated [Ca²⁺]_i in non-voltage-clamped cells by a mechanism that was dependent upon extracellular Na⁺ and Ca²⁺ and was blocked by the dihydropyridine Ca²⁺-channel blocker, nitrendipine. Resting cells had a fluctuating membrane potential whose a mean value depolarized by 9 mV in response to GHRH. The membrane-permeant cAMP analogue, 8-(4-chlorophenylthio)cAMP, mimicked the action of GHRH on membrane potential. Under voltage clamping, GHRH activated a small inward current (1–5 pA). Two types of response could be distinguished. The type I response had an inward current that was largest at more negative potentials (–90 mV), and the type II response had inward current that was larger at more positive potentials (–40 to –70 mV). Both types of response were reversible and blocked by removal of extracellular Na⁺. These results suggest that the rise in [Ca²⁺]_i produced by GHRH in non-voltage-clamped cells results from the activation via cAMP of a Na⁺-dependent conductance, which depolarizes the cell and increases the Ca²⁺ influx through voltage-gated Ca²⁺ channels.Dedicated in memory of the late Alexander P. Naumov.