Extracellular Calcium Ion Concentration Research Articles

How is plasma calcium held constant? Milieu interieur of calcium

Extracellular or plasma calcium ion concentration is held constant at 5 mg/dl through the actions of parathyroid hormone (PTH), vitamin D, and calcitonin, on their target organs, kidney and bone. The thresholds of renal tubular calcium reabsorption and bone resorption and formation are both set at 5 mg/dl. The set point of PTH secretion is also set at 5 mg/dl plasma calcium ion. Therefore, the sensing system (parathyroid cell) and the effectors, kidney and bone, are all set to maintain plasma calcium at 5 mg/dl, perhaps through membrane-bound calcium sensor proteins. The effectiveness of this system depends upon the presence of bone remodeling, which allows a swift shift of plasma calcium from and to bone in response to PTH and calcitonin, respectively. In this regard, directing hematopoiesis to bone marrow that provides bone resorbing osteoclasts is critical. It is likely that this shift of hematopoiesis occurs through evolution at the transition from the aquatic to the terrestrial life, and this event is directed by expression of "homing molecule" in bone marrow stromal cells. This brief review provides a factual and conceptual framework of the current understanding of the milieu interieur of the calcium ion.

Disorders of calcium ion sensing.

T HE PARATHYROID glands secrete parathyroid hormone (PTH) at a rate that is dependent upon and responsive to the extracellular calcium ion concentration (Ca*‘,) (1). Recent advances in molecular techniques have enabled the characterization of an extracellular calcium ionsensing receptor (Can) that initiates the afferent limb of the cellular pathways necessary for regulating this Ca2’0-responsive PTH secretion (2). Renal tubular calcium reabsorption, secretion of calcitonin by the thyroidal C-cells, and probably many other Ca2t0responsive cellular functions are dependent upon the CaR to permit the regulation of extracellular calcium homeostasis and other diverse cellular functions unrelated to calcium homeostasis, such as found in the brain. We will review the biochemical and molecular characteristics of the CaR as well as three disorders: familial benign hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia, which are caused by abnormalities of CaR function (3, 4).

A cloned extracellular Ca2+-sensing receptor: Molecular mediator of the actions of extracellular Ca2+ on parathyroid and kidney cells?

Calcium ions serve multiple biological roles; therefore, maintaining a nearly constant extracellular ionized calcium concentration (Ca2,) is a critical function of free-living terrestrial organisms. Tetrapods have evolved a homeostatic system for accomplishing this goal that has two key elements [1] (Fig. 1): The first are cells that sense (that is, recognize and respond to) small physiologically-relevant changes in Ca2, including parathyroid cells and thyroidal C-cells, which secrete, respectively, less parathyroid hormone (PTH) and more calcitonin (CT) in the presence of elevated levels of Ca20 [1, 21. The second element are the effector tissues that translocate Ca20 ions in response to calciotropic hormones (that is, kidney, bone and intestine) [1, 2]. The receptor-mediated mechanisms through which classical calciotropie hormones, such as PTH, calcitonin and 1,25-dihydroxyvitamin D regulate their target tissues has been the focus of intense studies. In contrast, much less has been known until recently about the mechanisms underlying Ca20-sensing by parathyroid and C-cells. Recent application of molecular techniques, however, has shown that Ca2)-sensing, like the sensing of many other extracellular first messengers, involves a cell surface, Ca20sensing receptor (CaR) that is a member of the superfanuly of G-protein-coupled receptors (GPCR) [3]. Moreover, the receptor is present not only in classic Ca20-sensing cells, like parathyroid cells, but also in effector elements of the mineral ion homeostatic system, such as the kidney [3, 4]. Finally, studies on the role of the CaR in inherited diseases of Ca20-sensing have yielded novel insights into the basis for previously poorly understood direct actions of Ca20 on renal function.

Calcium modulates circadian variation in cAMP-stimulated melatonin in chick pineal cells

In chick pineal cells, melatonin synthesis is regulated by both calcium and cAMP. Calcium-dependent regulation of melatonin is suggested by the monotonic decrease in melatonin observed with decreasing extracellular calcium ion concentrations ([Ca 2+] o), the stimulation of melatonin with Bay K8644, and the inhibition of nocturnal melatonin by several calmodulin antagonists. At submicromolar [Ca 2+] o, a stimulation of melatonin was observed in the presence of 8-Br cAMP, but not with Bay K8644, suggesting that this amount of stimulation of melatonin by 8-Br cAMP is independent of Ca 2+ influx through dihydropyridine-sensitive Ca 2+ channels. At micromolar [Ca 2+] o, there was a further increase in the stimulation of melatonin by 8-Br cAMP that was not blocked by nifedipine, a dihydropyridine-sensitive Ca 2+ channel antagonist. Micromolar [Ca 2+] o is required for the greater stimulation of melatonin by 8-Br cAMP during the night than during the day. Melatonin was stimulated by 8-Br cAMP to higher levels during the night than during the subjective day under normal [Ca 2+] o (1.3 mM). This difference in the amount of melatonin stimulated by 8-Br cAMP during the subjective night versus the subjective day was blocked by lowering [Ca 2+] o to a submicromolar concentration (0.2 μM). Both nifedipine and calmidazolium partially blocked nocturnal increases in melatonin, but were ineffective during the day. These results suggest that Ca 2+ plays an important role in the differential ability of cAMP to stimulate melatonin during the night versus the day.

Cloning and functional characterization of extracellular Ca2+-sensing receptors from parathyroid and kidney

Parathyroid cells recognize and respond to (i.e., "sense") minute perturbations in the extracellular ionized calcium concentration (Ca2+o), but the mechanisms underlying this process have remained obscure. Recently, we employed expression cloning in Xenopus laevis oocytes to isolate a cDNA coding for a Ca2+o-sensing receptor from bovine parathyroid. Like the native receptor, the cloned Ca2+o-sensing receptor stimulates phospholipase C (PLC) in a G-protein-dependent manner with a nearly identical pharmacological profile. Its deduced amino acid sequence confirms that it is a member of the superfamily of G-protein-coupled receptors (GPR). Transcripts for the receptor are expressed in parathyroid and other tissues that sense Ca2+o (viz., kidney and thyroidal C-cells) as well as those that have no known role in extracellular Ca2+ homeostasis, such as the brain. The availability of the cDNA clone for the Ca2+o-sensing receptor made it possible to test the hypothesis that mutations in the gene encoding the human homolog of the receptor cause inherited disorders of mineral ion metabolism. Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are, in fact, caused by mutations that reduce the activity of the receptor when they are present in the heterozygous and homozygous states, respectively. In contrast, we have subsequently discovered a family in which a form of autosomal dominant hypocalcemia results from an activating mutation in the receptor gene. The Ca2+-sensing receptor, therefore, permits Ca2+o to play a "hormonelike" role as an extracellular first messenger in addition to its well described role as an important intracellular second messenger.

Calcium-Ion–Sensing Cell-Surface Receptors

Calcium ions are essential for a wide variety of biologic functions, including vital extracellular processes, such as blood clotting, intercellular adhesion, and skeletal integrity, as well as intracellular processes, such as the regulation of hormonal secretion, cell division, and cell motility.1–4 Complex, free-living terrestrial organisms must maintain a near-constant concentration of extracellular calcium. How the body maintains and regulates extracellular calcium levels is a complex puzzle that has intrigued researchers for decades.3–5 One large piece of this puzzle that is falling into place concerns a cell-surface receptor that responds to, or “senses,” extracellular calcium-ion concentrations in parathyroid, kidney, . . .

The Cloning of Extracellular Ca2+-Sensing Receptors from Parathyroid and Kidney: Molecular Mechanisms of Extracellular Ca2+-Sensing

The parathyroid cell detects changes in the extracellular ionized calcium concentration (Ca2+o) with exquisite sensitivity, but the mechanisms through which it senses Ca2+o have remained obscure. Recently, we isolated a cDNA encoding a Ca2+o-sensing receptor from bovine parathyroid using expression cloning in Xenopus laevis oocytes. The expressed receptor stimulates phospholipase C and has a pharmacological profile almost identical to that of the native receptor. Furthermore, its deduced amino acid sequence confirms that it belongs to the superfamily of G-protein-coupled receptors. Receptor transcripts are present in parathyroid and other tissues sensing Ca2+o (e.g., kidney and thyroidal C-cells) as well as those not known to be involved in Ca2+o homeostasis (viz., in the brain). We have also shown that mutations in the receptor cause three inherited disorders of calcium metabolism: Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) result from inactivating mutations, when present in the heterozygous and homozygous states, respectively, whereas an autosomal dominant form of hypocalcemia is due to an activating mutation. Thus this Ca2+o-sensing receptor permits Ca2+o to act as an extracellular, first messenger in addition to its better known role as an intracellular second messenger.

Sensing of extracellular Ca 2+ by parathyroid and kidney cells: Cloning and characterization of an extracellular Ca 2+-sensing receptor

The ability of the parathyroid cell to sense minute fluctuations in the extracellular ionized calcium concentration (Ca 2+ o) is essential for maintaining mineral ion homeostasis. However, the mechanisms(s) through which the parathyroid cell and other cells recognize and respond to changes in Ca 2+ o has remained unclear. We recently isolated a cDNA encoding a Ca 2+ o-sensing receptor from bovine parathyroid using expression cloning in Xenopus laevis oocytes. The receptor shows pharmacologic properties that are almost identical to those of the receptor on the parathyroid cell and, like the latter, stimulates phospholipase C in a G-protein-dependent manner. The amino acid sequence of the cloned receptor deduced from this cDNA predicts a protein with a molecular mass of 121 kd, which has three principal structural domains. The first is a 613 amino acid, putatively extracellular amino terminus which has several regions rich in acidic amino acids that may potentially be involved in binding Ca 2+ and other polycationic agonists. The second comprises seven membrane-spanning segments that are characteristic of the superfamily of G-protein-coupled receptors, and the third is a 222 amino acid cytoplasmic tail. Transcripts for this Ca 2+ o-sensing receptor are present in the parathyroid as well as in the kidney, thyroid, and brain. We next investigated the hypercalcemic disorders, familial hypocalciuric hypercalcernia and neonatal severe hyperparathyroidism, as possible examples of inherited abnormalities in this Ca 2+ o-sensing receptor, since both disorders show abnormal Ca 2+ o-sensing and/or handling in the kidney and parathyroid. Indeed, further studies documented the presence of mutations in the coding region of the receptor gene in affected members of three familial hypocalciuric hypercalcemia families, including one homozygote with neonatal severe hyperparathyroidism, who had two copies of the abnormal allele. A receptor construct that introduced one of these mutations into the wild-type receptor showed over a 90% reduction in its activity when expressed in Xenopus laevis oocytes. Thus, this form of Ca 2+ o-sensing receptor belongs to the superfamily of G-protein-coupled receptors, and inactivating mutations appear to result in mild extracellular Ca 2+ “resistance” in individuals heterozygous for the mutation and much more severe resistance in homozygotes. In addition, this Ca 2+ o-sensing receptor may mediate some of the long-recognized, yet poorly characterized actions of extracellular calcium on certain aspects of renal function as well as on other cell types, permitting Ca 2+ o to act, in effect, as an extracellular first messenger in addition to carrying out its more widely recognized role as an intracellular second messenger.

Calcium dynamics in the central nervous system

Calcium ions are critically important in many functions of the nervous system from neurotransmitter release to intracellular signal transduction. The large difference between intracellular and extracellular calcium ion concentration ([Ca 2+]) highlights the importance of the mechanisms controlling influx and efflux of this ion. Loss of the regulatory ability of these mechanisms and the subsequent increased intracellular calcium levels may be involved in pathological events of brain trauma, stroke, epilepsy and other diseases. Ca 2+ dynamics in the CNS ranging from ‘waves’ to ‘spirals’ are being studied because of the availability of fluorescent indicators of Ca 2+ combined with confocal microscopy. Cellular mechanisms of Ca 2+ signal transduction have been extensively reviewed (Tsien and Tsien, 1990; Carafoli, 1992; Berridge, 1993; Berridge and Dupont, 1994; Pozzan et al., 1994; Clapham, 1995; Ghosh and Greenberg, 1995). The aim of this review is to present the types of Ca 2+ dynamics observed in the CNS thus far, both in normal brain function as well as in response after injury.

Nicotinic antagonist‐produced frequency‐dependent changes in acetylcholine release from rat motor nerve terminals.

1. The frequency (0.5-150 Hz) and calcium dependence (0.5-2.0 mM) of the effects of the nicotinic antagonist tubocurarine (0.2 microM) on acetylcholine (ACh) liberation from motor nerve terminals has been examined using binomial analysis of quantal transmitter release. 2. At an extracellular calcium ion concentration ([Ca2+]o) of 2.0 mM, tubocurarine produced a decrease in the endplate current (EPC) quantal content of approximately 30% at high frequencies of motor nerve stimulation (50-150 Hz). In contrast, at low frequencies of stimulation (0.5-1.0 Hz), tubocurarine enhanced the EPC quantal content by approximately 20%. 3. The enhancement of EPC quantal content produced by tubocurarine at low frequencies of motor nerve stimulation was [Ca2+]o dependent, being abolished when [Ca2+]o was lowered from 2.0 to 0.5 mM. In contrast, the decrease in quantal content produced by tubocurarine at high frequencies of motor nerve stimulation was independent of [Ca2+]o, being approximately 30% at all calcium ion concentrations studied. 4. In direct contrast to tubocurarine, the nicotinic antagonist vecuronium (1.0 microM) produced no increase in EPC quantal content at low frequencies of nerve stimulation. However, at high frequencies of nerve stimulation it decreased EPC quantal content to a similar extent to 0.2 microM tubocurarine. The frequency-dependent decrease in EPC quantal content produced by 1.0 microM vecuronium in 2.0 mM [Ca2+]o was very similar to that seen with 0.2 microM tubocurarine in 0.5 mM [Ca2+]o. 5. Binomial analysis revealed that all the changes in EPC quantal content associated with both nicotinic antagonists were due to changes in the size of the pool of quanta in the nerve terminal available for immediate release with no effect on the probability of release of an individual quantum. 6. The results are interpreted in terms of two separately identifiable prejunctional actions of the nicotinic antagonists, both involving an action at nicotinic ACh receptors situated on the motor nerve terminal. Thus, at high frequencies of motor nerve stimulation tubocurarine and vecuronium produce a [Ca2+]o-independent decrease in ACh release, probably through an inhibitory action on a positive-feedback prejunctional nicotinic autoreceptor closely related to the muscle-type nicotinic ACh autoreceptor. However, at low frequencies of motor nerve stimulation we suggest that tubocurarine, but not vecuronium, produces a [Ca2+]o-dependent increase in ACh release through an action at a negative-feedback prejunctional neuronal-type nicotinic ACh autoreceptor.

Desulfated Hirugen (Hirudin 54-65) Induces Endothelium-Dependent Relaxation of Porcine Pulmonary Arteries

Desulfated hirugen (hirudin 54-65) at concentrations from 0.1 to 2 microM was found to relax PGF2 alpha-precontracted ring segments of porcine pulmonary arteries with intact endothelium. The relaxation was associated with a pronounced increase in cGMP in the vessels. This endothelium-dependent relaxant effect depended on the extracellular calcium ion concentration and was probably due to the release of endothelium-derived NO as indicated by its susceptibility to blockade of the NO synthesis by NG-nitro-L-arginine. In the presence of indomethacin (3 microM) the maximum hirugen effect was significantly diminished by about 25%. In contrast, neither the sulfated hirugen nor recombinant desulfato hirudin at equimolar concentrations exerted endothelium-dependent relaxation. Hence, the relaxant effect did not correspond to the anticoagulant activity. Desulfated hirugen can be assigned to the group of well-known peptides causing vasodilatation via an endothelium-dependent mechanism.

Factors affecting nerve excitability and conduction as a basis for desensitizing dentine

Nerve excitability is principally determined by the state of membrane sodium channels, which can be influenced by factors such as drugs or the ionic composition of the extracellular fluids. The excitability of isolated nerves is increased by lowered extracellular calcium and hydrogen ion concentrations, and similar effects are seen on intradental nerve excitability. This is of interest as some agents used to desensitize hypersensitive dentine are believed to act by reducing intradental nerve excitability. A variety of ions, including potassium and divalent cations, have been shown to reduce intradental nerve excitability when applied in relatively high concentrations to dentine close to the pulp. However, these conditions do not mimic the clinical situation, where agents are applied to outer dentine, up to 2 mm from the pulp. Also, when agents are applied to dentine it is difficult to quantify the precise changes in extracellular fluid composition, and there is the additional complication of possible interactions between the agents and dentine. These problems can be minimized by using isolated nerves, which allow better control over the conditions and ionic concentrations necessary to achieve nerve conduction block. Such methods can provide a convenient way to screen potential desensitizing agents before evaluation in vivo or in clinical trials.

Spontaneous Oscillations of Cytoplasmic Free Calcium Ion Concentration in Cultured Smooth Muscle Cells from Guinea Pig Ileum

The cytoplasmic free calcium ion concentration ([Ca2+]i) of cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent [Ca2+]i indicator, fura-2, was measured by digital ratio imaging microscopy. Spontaneous [Ca2+]i oscillations were observed in 25% to 80% of the cells, which differed with the batches of the cultured cells after 5 to 8 days in culture. The frequency and amplitude of the [Ca2+]i oscillations in each individual cell were usually regular, but heterogeneity between neighboring cells was observed. The spontaneous [Ca2+]i oscillations were also observed even after incubation of the cells under a serum-free condition for 72 hr. Exchange of extracellular solution to Ca(2+)-free solution containing EGTA or BAPTA immediately stopped the [Ca2+]i oscillations. The ratio of the oscillating cells was dependent on the extracellular calcium ion concentration ([Ca2+]o); and heterogeneity in the range of the [Ca2+]o to generate the [Ca2+]i oscillations was observed. An inorganic Ca(2+)-antagonist, LaCl3, immediately suppressed the [Ca2+]i oscillations, but the treatment with verapamil or nicardipine, Ca(2+)-channel blockers, did not have any effect on the [Ca2+]i oscillations. An inhibitor of the intracellular Ca2+ pump, thapsigargin, induced a transient increase in [Ca2+]i and then inhibited the spontaneous [Ca2+]i oscillations. Neomycin, a compound known to inhibit phosphoinositide turnover, inhibited the [Ca2+]i oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory action of parathyroid hormone on renin secretion in vitro: a study using isolated rat kidney, isolated rabbit glomeruli and superfused dispersed rat juxtaglomerular cells.

1. We have previously reported that pharmacological concentrations (125nmol/l) of parathyroid hormone may stimulate renin release in the stable recirculating and non-filtering isolated rat kidney. 2. In the present study we have attempted to extend these initial observations by examining the concentration-related response of renin release to parathyroid hormone, using the same model of isolated kidney, and determining whether the effect of parathyroid hormone on renin release can be demonstrated by more direct approaches. Thus, the effects of parathyroid hormone on renin secretion were investigated in two other renal preparations: isolated rabbit glomeruli and isolated rat juxtaglomerular cells. 3. In the isolated kidney, rat parathyroid hormone significantly stimulated renin accumulation in the perfusate in a concentration-related manner with a threshold of 1 nmol/l. 4. In both glomeruli and juxtaglomerular cells bovine [Nle8,18,Tyr34]parathyroid hormone-(1-34)amide effectively and repeatedly stimulated renin release. These results imply that there is a direct stimulatory effect of parathyroid hormone on renin release. 5. We also examined the effect of [Nle8,18,Tyr34]parathyroid hormone-(1-34)amide during extracellular calcium buffering in the glomeruli. [Nle8,18,Tyr34]parathyroid hormone-(1-34)amide was uneffective in calcium-free medium. Increasing the extracellular ionized calcium concentration to 2.5 mmol/l increased the extent of stimulation in accordance with the reported ability of parathyroid hormone to block calcium channels and relax vascular smooth muscle cells. 6. These results provide further support for the role of parathyroid hormone as a direct mediator of renin secretion; moreover, the renin-stimulating action of parathyroid hormone may be mediated through the inhibition of calcium influx.

Hemodynamic interactions when combining verapamil, acute changes in extracellular ionized calcium concentration and enflurane, halothane or isoflurane in chronically instrumented dogs.

To assess the hemodynamic interactions when combining verapamil, acute changes in extracellular ionized calcium concentration [Ca2+] and enflurane (2.5%), halothane (1.2%) or isoflurane (1.6%), seven dogs were chronically instrumented to measure heart rate (HR), aortic, left atrial and left ventricular (LV) pressures, and cardiac output (CO). [Ca2+] was lowered 0.35 mmol.l-1 by citrate infusion and then increased 0.35 mmol.l-1 above control level by CaCl2 infusions. Verapamil was infused at 3 micrograms.kg-1 x min-1 (loading dose 200 (awake), 150 (isoflurane) or 100 (enflurane and halothane) micrograms.kg-1), giving mean verapamil concentrations around 75 (range of means: 66-84 ng.ml-1). Verapamil produced mostly minor changes in the cardiovascular effects of changing [Ca2+] in both awake and anesthetized dogs, indicating mostly additive effects. Verapamil induced a decrease in HR at high [Ca2+] and abolished an increase in mean aortic pressure at both low and high [Ca2+] awake. Verapamil exaggerated the decrease in CO and stroke volume (SV) induced by low [Ca2+] during enflurane anesthesia and abolished the increase in CO induced by low [Ca2+] and exaggerated the increase in SV and LV dP/dtmax induced by high [Ca2+] during halothane anesthesia.

Excitatory amino acid-induced phosphoinositide turnover in guinea pig cerebral cortical slices: selective enhancement by spermine of the response to DL-1-aminocyclopentane-trans-1,3-dicarboxylate.

In the presence of 1 mM spermine, accumulations of 3H labelled inositol phosphates elicited by quisqualate (100 microM) and 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD, 300 microM) were significantly enhanced by 21 and 26%, respectively, without a significant alteration in the accumulation elicited by L-glutamate (10 mM) or DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionate (10 microM). Analysis of concentration-response data indicated that the presence of spermine led to an increase in the maximal response to t-ACPD without altering the EC50 value. The stimulatory effect of spermine on the accumulation of t-ACPD-elicited 3H-inositol phosphates was not reversed by ifenprodil or diethylenetriamine (putative polyamine site antagonists), by agents that activate or inhibit protein kinase C, or by calcium channel blockade, but was abolished in the presence of elevated extracellular calcium ion concentration. We conclude that spermine enhances the phosphoinositide turnover in guinea pig cerebral cortical slices elicited by the "metabotropic" excitatory amino acid receptor. The site through which the action of spermine is mediated remains to be defined, but it is apparently distinct from that suggested to modulate N-methyl-D-aspartate receptor activity.

Effects of isoflurane and halothane on the calcium ion-tension curve in rat myocardium.

In order to clarify the interaction of volatile anesthetics and extracellular calcium ion on the myocardial contraction, effects of both isoflurane (1.0%) and halothane (0.5%) on the extracellular calcium ion concentration ([Ca(2+)](O))-tension curve were studied. Increasing [Ca(2+)](O) enhanced the myocardial contraction response, and the maximal response was obtained at [Ca(2+)](O) of 3.0 mM. Halothane depressed the maximal value of the tension development in response to increasing [Ca(2+)](O), while isoflurane did not ( P < 0.01). The probit response of the developed tension to the changes in [Ca(2+)](O) indicated that isoflurane increased the median effective concentration (EC(50)) of [Ca(2+)](O) significantly from 0.484 +/- 0.051 (mean +/- SEM) to 0.870 +/- 0.056 mM ( P = 0.001), but halothane did not ( P = 0.018). Therefore, 1.0% isoflurane was concluded to move the [Ca(2+)](O)-tension curve to the right, while a downwards shift occurred with 0.5% halothane.

Transmembrane-mediated changes in [Ca2+] are involved in the signaling pathway leading to macrophage cytocidal differentiation: implications of localized changes in intracellular [Ca2+] and of interferon priming on Ca2+ utilization.

Macrophage cytocidal activation requires the sequential impingement on the macrophage of a priming stimulus (interferon [IFN] alpha, beta, or gamma) and a triggering stimulus (such as polyinosinic acid:polycytidylic acid [poly [I:C]] or bacterial lipopolysaccharide). The mechanism of progression from the IFN-primed state to the cytocidal state is poorly understood. By quantifying the level of expression of a gene product (complement component factor B [Bf]) associated with cytocidal activation and through the use of phenotypically distinct populations of macrophages (unprimed and IFN-primed), we have investigated the functional necessity of changes in intracellular concentration of free calcium ions ([Ca2+]i) in signaling the transition from the primed to the cytocidal state. Elevating the [Ca2+]i by incubation of unprimed macrophages with the calcium ionophore, ionomycin, failed to induce the expression of Bf. By contrast, Bf was expressed at high levels when IFN-primed macrophages were exposed to ionomycin, suggesting that priming induced within the macrophages the capacity to respond to a nonspecific change in [Ca2+]i. Quantification of the [Ca2+]i in response to exposure to ionomycin revealed an initial transient elevation, followed by a secondary sustained component. No differences in these changes were observed between unprimed and IFN-primed macrophages. We therefore questioned if changes in [Ca2+]i were also implicated in the transition between the primed and the cytocidal state using the ligand, poly [I:C]. In contrast to ionomycin, incubation of IFN-primed macrophages with poly [I:C] did not sustain measurable increases in [Ca2+]i, yet fully stimulated the transition from the IFN primed to the cytocidal state. However, incubation of IFN-primed macrophages with poly [I:C] in the presence of 1) a Ca2+/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffer calculated to clamp the extracellular concentration of free calcium ions to a value approximately equal to the resting [Ca2+]i; 2) the calcium channel blocker verapamil; or 3) the intracellular Ca2+ antagonists (W-7, W-13, and TMB-8) substantially inhibited the induction of Bf. Collectively, these data support the following conclusions. First, that changes in [Ca2+]i comprise an important element in the induction of progression from the IFN-primed to the cytocidal state. Second, the failure to detect global changes in [Ca2+]i in response to the ligand, poly [I:C], suggests that changes in [Ca2+]i or Ca2+ movement may occur in either a spatially restricted or in an asynchronous cyclical fashion and are not detected by population fluorescence measurements. Third, the source of the relevant Ca2+ is extracellular. Fourth, our findings suggest that priming influences macrophage functional responses at a locus that is distal to the changes in [Ca2+]i, thereby potentially allowing signaling processes to be utilized to initiate different cellular responses.

Cardiovascular effects of acute changes in extracellular ionized calcium concentration induced by citrate and CaCl2 infusions in conscious, chronically instrumented dogs and their interactions with ganglionic blockade.

To assess the hemodynamic effects of acute changes in extracellular ionized calcium concentration, [Ca2+], seven dogs were chronically instrumented to measure heart rate, aortic, left atrial, and left ventricular (LV) pressures, cardiac output, and coronary and renal blood flows. [Ca2+] was lowered 0.35 mmol.l-1 by citrate infusion and then increased 0.35 mmol.l-1 above control level by CaCl2 infusions. This protocol was performed in the conscious dogs with and without ganglionic blockade (chlorisondamine 2 mg.kg-1). LV dP/dtmax decreased at low [Ca2+] and increased at high [Ca2+] during all conditions. The other hemodynamic variables measured were only slightly changed by changing [Ca2+] without ganglionic blockade and surprisingly even less with ganglionic blockade. Therefore, the lesser hemodynamic effects induced by acute changes in [Ca2+] in the conscious compared with anesthetized dogs cannot be explained by the depressant effects of the anesthetics upon the autonomic nervous system. We have suggested that the binding of Mg2+ to citrate may be of importance for the minor hemodynamic effects in the conscious dogs.

Two possibly distinct prostaglandin E1 receptors in N1E-115 clone: one mediating inositol trisphosphate formation, cyclic GMP formation, and intracellular calcium mobilization and the other mediating cyclic AMP formation.

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.