Changes In Extracellular Calcium Concentration Research Articles

Fast calcium transients translate the distribution and conduction of neural activity in different regions of a single sensory neuron.

In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1-10mM NiCl2 and 10-500µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

Molecular and Clinical Aspects of the Target Therapy with the Calcimimetic Cinacalcet in the Treatment of Parathyroid Tumors

Parathyroid tumors are almost invariably associated with parathormone (PTH) hypersecretion resulting in primary (PHPT) or secondary (SHPT) hyperparathyroidism. PHPT is the third most common endocrine disorder with a prevalence of 1-2% in post-menopausal women; SHPT is a major complication of chronic kidney failure, the prevalence of which is increasing. The calciumsensing receptor (CASR) is the key molecule regulating PTH synthesis and release from the parathyroid cells in response to changes in extracellular calcium concentrations. A potent calcimimetic, cinacalcet, has been developed in the last ten years and made available for medical treatment of both PHPT and SHPT. Cinacalcet has been demonstrated to be effective in inhibiting PTH secretion, though the drug fails to normalize PTH release, both in PHPT and SHPT patients with different degrees of disease severity, including patients with parathyroid carcinomas and with MEN1-related parathyroid tumors. Here we reviewed the molecular aspects of CASR target therapy and the effect of the CASR gene single nucleotide polymorphisms. Clinical data concerning the efficacy and safety of cinacalcet in controlling hyperparathyroidism are reported, focusing on the treatment of the different types of parathyroid tumors. Finally, limits of this target therapy are analyzed, pointing out the lack of efficacy in improving kidney and bone morbidities in PHPT and cardiovascular diseases in SHPT. Though cinacalcet is a target therapeutic option for parathyroid tumors, further approaches are warranted to fully control these metabolic disorders and the underlying tumors.

The role of calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) in human osteoclast formation and resorption.

Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorption. In this study, we sought to determine the effect of [Ca(2+)]e and NAADP, a potent calcium mobilising messenger that induces calcium uptake, on human osteoclast formation and resorption. CD14+ human monocytes were cultured with M-CSF and RANKL in the presence of different concentrations of calcium and NAADP and the effect on osteoclast formation and resorption evaluated. We found that the number of TRAP+ multinucleated cells and the extent of lacunar resorption were reduced when there was an increase in extracellular calcium and NAADP. This was associated with a decrease in RANK mRNA expression by CD14+ cells. At high concentrations (20mM) of [Ca(2+)]e mature osteoclast resorption activity remained unaltered relative to control cultures. Our findings indicate that osteoclast formation is inhibited by a rise in [Ca(2+)]e and that RANK expression by mononuclear phagocyte osteoclast precursors is also [Ca(2+)]e dependent. Changes in NAADP also influence osteoclast formation, suggesting a role for this molecule in calcium handling. Osteoclasts remained capable of lacunar resorption, even at high ECF [Ca(2+)]e, in keeping with their role in physiological and pathological bone resorption.

Calcium-sensing in the kidney

Changes in extracellular calcium concentration affect several functions of the renal tubule. The calcium-sensing receptor (CaSR), initially identified in the parathyroid gland cells, is also expressed in the kidney and was assumed to mediate all effects of extracellular calcium on the renal tubule. The purpose of this review is to critically review the evidence supporting this assumption. Recent results confirm that, in the kidney, the CaSR is mainly expressed in the thick ascending limb of the loop of Henle. There, it is involved in the control of calcium reabsorption, independently of its action on parathyroid hormone secretion, through an effect on the paracellular pathway permeability. Although extracellular calcium affects transports other than that of calcium, the direct evidence that CaSR is involved in these effects is still lacking in many instances. As the CaSR in the kidney controls calcium reabsorption and excretion and subsequently affects blood calcium concentration, agonists and antagonists of the CaSR could be used to control blood calcium concentration in patients who have lost their ability to regulate parathyroid hormone secretion. In addition, more work is needed to further decipher the molecular mechanisms through which CaSR determines calcium transport in the loop of Henle.

Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

BackgroundFamilial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding protein (G-protein)–coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia.MethodsWe performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells.ResultsThe kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2–associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2–associated mutations increased cell sensitivity.ConclusionsGα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.)

Localization, Distribution, and Function of the Calcium‐Sensing Receptor in Sperm

The intracellular movement of calcium, through calcium channels, plays a major role on sperm cell function. The calcium-sensing receptor (CaSR), a molecular mechanism by which many cells detect changes in extracellular calcium concentration, has not been described in spermatozoa. The aim of this study was to investigate the expression of the CaSR in testicular tissue and sperm cells and the functional consequences of spermatozoid CaSR activation by calcimimetics. CaSR mRNA and protein were identified both in rat testicular tissue and in rat spermatozoa using real-time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. Functionality of CaSR was evaluated by studying the influence of calcimimetic AMG 641 on rat and pig sperm motility. Treatment with AMG 641 100 nM for 1 hour increased rat sperm motility from a score of 1.0 ± 0.1 to 3.8 ± 0.3 (P < .05). AMG 641 also resulted in a modest but significant increase in the pig sperm motility parameters evaluated by computer-assisted sperm analysis. AMG 641 was effective in a wide range of concentrations but resulted in a more marked effect at 50-100 nM. In addition, AMG 641 did not have any negative effect on sperm viability, which was measured by flow cytometry. In conclusion, our results demonstrate the expression of functional CaSR in testicular tissue and sperm, which can be activated by calcimimetic AMG 641.

Abstract 909: Calcium intake, CASR polymorphisms and phenotype, and the risk of colorectal adenoma: Results from Tennessee Colorectal Polyp Study

Abstract Background: A number of cohort studies and randomized trials suggest that high calcium consumption may confer a reduced risk of colorectal cancer. Results, however, have been inconsistent. It is possible that the calcium effect may differ by polymorphisms in the genes involved in calcium regulation. Calcium-sensing receptor (CASR) tightly regulates systemic calcium levels by sensing minor changes in extracellular calcium concentration. Studies have found that CaSR is expressed in the human colon epithelium and regulates epithelial proliferation and differentiation. It was also shown that luminal calcium inhibited colorectal tumors through CaSR. Four studies have thus far investigated the associations of CASR polymorphisms and haplotypes and their interactions with calcium intakes in relation to risk of colorectal cancer or adenoma. However, these studies have generated inconsistent results. Objectives: In the current study, we first screened whether CASR variants or their interactions with calcium intake were associated with urinary calcium phenotype among 233 normal controls. SNPs which were associated with phenotype were further evaluated for their independent association and interaction with calcium intake on colorectal adenoma risk. Methods: Included in the study were participants of the Tennessee Colorectal Polyp Study (TCPS), an ongoing colonoscopy-based case control study conducted in Nashville, TN. Genotyping was performed using the Affymetrix Human Mapping 500K array set for 666 cases and 751 controls. Twenty two tagging single-nucleotide polymorphisms (SNPs) for CASR were analyzed. Linear regression models (SAS MIXED procedure) were used to estimate association between genotypes and urinary calcium level. Multivariate unconditional logistic regression models were used to estimate the odds ratio (OR) and 95% confidence intervals (CI) for each SNP. Multiplicative interactions for gene-diet interactions were evaluated in logistic regression models by using likelihood ratio tests. Results: Eleven SNPs were significantly associated with urinary calcium levels. Among these 11 SNPs, 4 SNPs significantly interacted with calcium intake in association with risk of adenoma. The association of the genotypes with adenoma risk differed by high or low level of calcium intake. We are conducting additional genotyping assays and will present the updated results in the AACR meeting. Conclusions: These findings suggest that some genetic variations in CASR and/or their interactions with dietary calcium intake have significant functional impact on calcium reabsorption, in turn, affect the risk of colorectal adenoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 909.

Extracellular calcium modulates in vitro bone marrow-derived Flk-1+ CD34+ progenitor cell chemotaxis and differentiation through a calcium-sensing receptor

Angiogenesis is a complex process regulated by many cell types and a large variety of biochemical signals such as growth factors, transcription factors, oxygen and nutrient diffusion among others. In the present study, we found out that Flk-1+ CD34+ progenitor cells (bone marrow resident cells with an important role in angiogenesis) were responsive to changes in extracellular calcium concentration through a membrane bound, G-protein-coupled receptor sensitive to calcium ions related to the calcium-sensing receptor (CaSR). Calcium was able to induce progenitor cell migration in Boyden chamber experiments and tubulogenesis in Matrigel assays. Addition of anti-CaSR antibodies completely blocked the effect, while CaSR agonist Mg2+ produced a similar response to that of calcium. Real time RT-PCR for a wide array of angiogenesis-related genes showed increased expression of endothelial markers and signaling pathways involved in angiogenesis. These results suggest calcium could be a physiological modulator of the bone marrow progenitor cell-mediated angiogenic response.

Depressing effects of caffeine at crayfish neuromuscular synapses I. Dosage response and Ca++ gradient effects.

The response of crayfish synaptic terminals to drugs began to be studied to characterize the terminal's physiological characteristics. Caffeine, the first drug to be studied, was selected to enhance synaptic transmission because of its ability to increase calcium release from internal stores.1. The largest excitor neuron to the superficial flexor muscle system of Procambarus clarkii was stimulated at 10 Hz while recording junction potentials from several lateral muscle fibers.2. Caffeine unexpectedly decreased synaptic transmission in this system in a dosage-dependent manner. The depressing effect of caffeine was observed at 5 mM caffeine and junction potentials disappeared completely at 50 mM. Washing the preparation in fresh control Ringers did not restore the amplitudes of the junction potentials.3. Changes in extracellular calcium concentrations delayed or depressed the caffeine effect depending on the calcium gradient across the membrane or the caffeine dosage. The data suggest that calcium is involved in caffeine's response in this system in a way yet to be determined.

The Effects of Changes in Extracellular Calcium Concentrations on the Electrical Properties of Command Neurons after Acquisition of a Defensive Conditioned Reflex in Snails

Studies of the electrical characteristics of the command neurons of a defensive reflex showed that the membrane potential showed no significant changes in response to changes in the calcium concentration in the perfusing solution in either intact or trained snails. Increases in the calcium ion concentration in intact snails were accompanied by increases in the threshold potential, from 14 +/- 0.7 mV at 2.5 mM Ca2+ to 21.8 +/- 0.9 mV at 20 mM Ca2+. The threshold potential in trained snails decreased in response to both increases and decreases in calcium concentrations, from 16.8 +/- 0.6 mV (physiological saline containing 10 mM Ca2+) to 13.3 +/- 0.6 mV at 20 mM Ca2+ and 11.8 +/- 0.8 mV at 2.5 mM Ca2+. The critical depolarization level changed correspondingly: in intact snails, this decreased with increases in calcium concentration, while in trained snails it increased in response to both increases and decreases in the calcium concentration.

Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane

The calcium-sensing receptor (CaSR) is a class III G-protein-coupled receptor (GPCR) that responds to changes in extracellular calcium concentration and plays a crucial role in calcium homeostasis. The mechanisms controlling CaSR trafficking and surface expression are largely unknown. Using a CaSR tagged with the pH-sensitive GFP super-ecliptic pHluorin (SEP-CaSR), we show that delivery of the GPCR to the cell surface is dependent on receptor-activity-modifying proteins (RAMPs). We demonstrate that SEP-CaSRs are retained in the endoplasmic reticulum (ER) in COS7 cells that do not contain endogenous RAMPs whereas they are delivered to the plasma membrane in HEK 293 cells that do express RAMP1. Coexpression of RAMP1 or RAMP3, but not RAMP2, in COS7 cells was sufficient to target the CaSR to the cell surface. RAMP1 and RAMP3 colocalised and coimmunoprecipitated with the CaSR suggesting that these proteins associate within the cell. Our results indicate that RAMP expression promotes the forward trafficking of the GPCR from the ER to the Golgi apparatus and results in mature CaSR glycosylation, which is not observed in RAMP-deficient cells. Finally, silencing of RAMP1 in the endogenously expressing HEK293 cells using siRNA resulted in altered CaSR traffic. Taken together, our results show that the association with RAMPs is necessary and sufficient to transfer the immature CaSR retained in the ER towards the Golgi where it becomes fully glycosylated prior to delivery to the plasma membrane and demonstrate a role for RAMPs in the trafficking of a class III GPCR.

The calcium-sensing receptor regulates mammary gland parathyroid hormone-related protein production and calcium transport.

The transfer of calcium from mother to milk during lactation is poorly understood. In this report, we demonstrate that parathyroid hormone-related protein (PTHrP) production and calcium transport in mammary epithelial cells are regulated by extracellular calcium acting through the calcium-sensing receptor (CaR). The CaR becomes expressed on mammary epithelial cells at the transition from pregnancy to lactation. Increasing concentrations of calcium, neomycin, and a calcimimetic compound suppress PTHrP secretion by mammary epithelial cells in vitro, whereas in vivo, systemic hypocalcemia increases PTHrP production, an effect that can be prevented by treatment with a calcimimetic. Hypocalcemia also reduces overall milk production and calcium content, while increasing milk osmolality and protein concentrations. The changes in milk calcium content, milk osmolality, and milk protein concentration were mitigated by calcimimetic infusions. Finally, in a three-dimensional culture system that recapitulates the lactating alveolus, activation of the basolateral CaR increases transcellular calcium transport independent of its effect on PTHrP. We conclude that the lactating mammary gland can sense calcium and adjusts its secretion of calcium, PTHrP, and perhaps water in response to changes in extracellular calcium concentration. We believe this defines a homeostatic system that helps to match milk production to the availability of calcium.

Numerical Analysis of Ca 2+ Depletion in the Transverse Tubular System of Mammalian Muscle

Calcium currents were recorded in contracting and actively shortening mammalian muscle fibers. In order to characterize the influence of extracellular calcium concentration changes in the small unstirred lumina of the transverse tubular system (TTS) on the time course of the slow L-type calcium current ( I Ca), we have combined experimental measurements of I Ca with quantitative numerical simulations of Ca 2+ depletion. I Ca was recorded both in calcium-buffered and unbuffered external solutions using the two-microelectrode voltage clamp technique (2-MVC) on short murine toe muscle fibers. A simulation program based on a distributed TTS model was used to calculate the effect of ion depletion in the TTS. The experimental data obtained in a solution where ion depletion is suppressed by a high amount of a calcium buffering agent were used as input data for the simulation. The simulation output was then compared with experimental data from the same fiber obtained in unbuffered solution. Taking this approach, we could quantitatively show that the calculated Ca 2+ depletion in the transverse tubular system of contracting mammalian muscle fibers significantly affects the time-dependent decline of Ca 2+ currents. From our findings, we conclude that ion depletion in the tubular system may be one of the major effects for the I Ca decline measured in isotonic physiological solution under voltage clamp conditions.

L-type Calcium Channel Blockers and EGTA Enhance Superoxide Production in Cardiac Fibroblasts

Since the recognition of the importance of calcium ions to cardiac contractility, the effects of alterations in calcium homeostasis on cardiac myocyte function have attracted immense attention. However, the possibility that changes in extracellular calcium concentration or the administration of calcium channel blockers may exert significant effects on cardiac fibroblasts has not hitherto been explored. This communication presents evidence, for the first time, that EGTA, calcium-free incubation and L-type calcium channel blockers increase endogenous superoxide production in adult rat cardiac fibroblasts. A combination of ryanodine and EGTA was found to have an even greater effect. The observations indicate that extracellular calcium levels influence endogenous superoxide production in cardiac fibroblasts and support the postulation that myocardial fibroblasts may contribute to the cardiac effects of calcium channel blockers and alterations in extracellular calcium concentration.

Calcium-Mediated Cox-2 Expression and Pge 2 Production in the Rat Medullary Thick Ascending Limb(mTAL) Is Tnf α -Dependent

P89 Severe hypercalcemia often is associated with polyuria in patients. We have shown that mTAL cells express cyclooxygenase-2(COX-2) when challenged with tumor necrosis factor-alpha(TNF α ), an effect that was linked to the decrease in TNF α -mediated 86 Rb + uptake, an in vitro correlate of natriuresis. The mTAL expresses calcium sensing receptor(CaR), a G-protein coupled receptor that senses small changes in extracellular calcium concentration([Ca 2+ ] o ) and ultimately increases intracellular calcium concentration([Ca 2+ ] i ) and protein kinase C(PKC) activity. We assessed the effects of [Ca 2+ ] o on COX-2 and TNF α protein expression as calcium increases TNF α synthesis in certain cell types, and PMA, a PKC activator, induces COX-2 gene expression in mTAL cells. Western blot analysis showed that COX-2 protein expression was increased two-fold after challenge for 9 hours with 1.7 mM CaCl 2 . ELISA analysis revealed that PGE 2 formation was increased from 11 ± 1.5 to 68 ± 21 pg/μg protein (p 2 ; no effect was observed when cells were exposed to 3.4 mM NaCl suggesting that the increase in PGE 2 synthesis was a function of increasing [Ca 2+ ] o . TNF α synthesis increased from 1.3 ± 0.1 to 5.4 ± 0.4 pg/μg protein (p 2 . CaR agonist poly-L-arginine (100nM) increased PGE 2 production from 15 ± 0.1 to 66.3 ± 4.4 pg/μg protein (p α production from 0.7 ± 0.1 to 3.7 ± 0.3 pg/μg protein after a 9 hr incubation period. Pretreatment of cells with 1 μg/ml neutralizing TNF α antibody reduced COX-2 protein expression and PGE 2 production by approximately 40%. These data indicate that Ca 2+ o , as well as CaR selective agonists, increase mTAL COX-2 expression, PGE 2 formation, and TNF α production in primary cultures of mTAL cells. We conclude that the increases in COX-2 expression and PGE 2 production are, in part, TNF α -dependent, suggesting that Ca 2+ o may be a modulator of extracellular fluid volume regulation via a cytokine-mediated autocrine mechanism.

Calcium-Mediated Cox-2 Expression and Pge 2 Production in the Rat Medullary Thick Ascending Limb(mTAL) Is Tnf α -Dependent

P89 Severe hypercalcemia often is associated with polyuria in patients. We have shown that mTAL cells express cyclooxygenase-2(COX-2) when challenged with tumor necrosis factor-alpha(TNF α ), an effect that was linked to the decrease in TNF α -mediated 86 Rb + uptake, an in vitro correlate of natriuresis. The mTAL expresses calcium sensing receptor(CaR), a G-protein coupled receptor that senses small changes in extracellular calcium concentration([Ca 2+ ] o ) and ultimately increases intracellular calcium concentration([Ca 2+ ] i ) and protein kinase C(PKC) activity. We assessed the effects of [Ca 2+ ] o on COX-2 and TNF α protein expression as calcium increases TNF α synthesis in certain cell types, and PMA, a PKC activator, induces COX-2 gene expression in mTAL cells. Western blot analysis showed that COX-2 protein expression was increased two-fold after challenge for 9 hours with 1.7 mM CaCl 2 . ELISA analysis revealed that PGE 2 formation was increased from 11 ± 1.5 to 68 ± 21 pg/μg protein (p&lt;0.05) by 1.7 mM CaCl 2 ; no effect was observed when cells were exposed to 3.4 mM NaCl suggesting that the increase in PGE 2 synthesis was a function of increasing [Ca 2+ ] o . TNF α synthesis increased from 1.3 ± 0.1 to 5.4 ± 0.4 pg/μg protein (p&lt;0.05) after challenge with 2 mM CaCl 2 . CaR agonist poly-L-arginine (100nM) increased PGE 2 production from 15 ± 0.1 to 66.3 ± 4.4 pg/μg protein (p&lt;0.0001), and increased TNF α production from 0.7 ± 0.1 to 3.7 ± 0.3 pg/μg protein after a 9 hr incubation period. Pretreatment of cells with 1 μg/ml neutralizing TNF α antibody reduced COX-2 protein expression and PGE 2 production by approximately 40%. These data indicate that Ca 2+ o , as well as CaR selective agonists, increase mTAL COX-2 expression, PGE 2 formation, and TNF α production in primary cultures of mTAL cells. We conclude that the increases in COX-2 expression and PGE 2 production are, in part, TNF α -dependent, suggesting that Ca 2+ o may be a modulator of extracellular fluid volume regulation via a cytokine-mediated autocrine mechanism.

Nonuniformity of calcium efflux from pancreatic acinar cells and its analysis by mathematical model of calcium diffusion and buffering in extracellular solution

We studied spatial and temporal patterns of Ca2+ extrusion from pancreatic acinar cells evoked by acetylcholine(ACh)-induced activation of plasma membrane calcium pumps. Using a modification of an earlier developed model, we estimated the time course of extracellular calcium concentration changes near the basal pole of a cell in the case, when calcium ions are released from the same site on the cell surface, and in the case when they are extruded from the apical pole and diffuse to the basal one. It is concluded that at the first stage of ACh-induced Ca2+ extrusion the appearance of Ca2+ elevation near the basal pole of the cells cannot be explained as a result of diffusion, but is mainly determined by Ca2+ efflux from this pole. The results also show that there are plasma membrane calcium pumps in both apical and basal parts of pancreatic acinar cells, but the activity of the pumps is substantially higher in the apical region.

A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor.

The calcium-sensing receptor regulates the secretion of parathyroid hormone in response to changes in extracellular calcium concentrations, and mutations that result in a loss of function of the receptor are associated with familial hypocalciuric hypercalcemia. Mutations involving a gain of function have been associated with hypocalcemia in two kindreds. We examined the possibility that the latter type of mutation may result in a phenotype of familial hypocalcemia with hypercalciuria. We studied six kindreds given a diagnosis of autosomal dominant hypoparathyroidism on the basis of their hypocalcemia and normal serum parathyroid hormone concentrations, a combination that suggested a defect of the calcium-sensing receptor. The hypocalcemia was associated with hypercalciuria, and treatment with vitamin D resulted in increased hypercalciuria, nephrocalcinosis, and renal impairment. Mutations in the calcium-sensing-receptor gene were identified by DNA-sequence analysis and expressed in human embryonic kidney cells (HEK-293). Five heterozygous missense mutations (Asn118Lys, Phe128Leu, Thr151Met, Glu191Lys, and Phe612Ser) were detected in the extracellular domain of the calcium-sensing-receptor gene and shown to cosegregate with the disease. Analysis of the functional expression of three of the mutant receptors in HEK-293 cells demonstrated shifts in the dose-response curves so that the extracellular calcium concentrations needed to produce half-maximal increases in total inositol phosphate in the cells were significantly (P=0.02 to P<0.001) lower than those required for the wild-type receptor. Gain-of-function mutations in the calcium-sensing receptor are associated with a familial syndrome of hypocalcemia with hypercalciuria that needs to be distinguished from hypoparathyroidism.

Human Keratinocytes Regulate Their Expression of B7/BB-1 Antigen by a Unique, Calcium-Dependent Mechanism

Previous studies of normal human keratinocytes have indicated that these cells express BB-1 antigen, an important adherence molecule usually associated with "professional" antigen-presenting cells. We studied freshly isolated epidermal cells and noted that the frequency of BB-1-positive cells in normal human skin varied from 2.6 to 7.4% of total epidermal cells. Two-color flow cytometry confirmed that keratinocytes were the major cell in the epidermis that expressed BB-1, because less than 10% of total epidermal Langerhans cells were positive for BB-1. Northern blot analysis of RNA extracted from normal human epidermis revealed low levels of 1.7-kb B7-1 transcripts, which were independent of the presence of epidermal Langerhans cells, again indicating that such transcripts were derived from keratinocytes. Keratinocytes cultured in medium containing low concentrations of extracellular calcium (0.07 mM) expressed low levels of cell-surface BB-1. However, keratinocytes cultured in medium with higher levels of extracellular calcium (1.5 mM) lost cell-surface expression of BB-1. Similarly, low-calcium keratinocytes expressed the 1.7- and 2.9-kb B7-1 transcripts, whereas high-calcium keratinocytes expressed only the 1.7-kb transcript. Studies of the cell-surface expression of BB-1 by plastic adherent monocytes indicated that such cells do not respond to similar changes in extracellular calcium concentrations. Calcium-induced differentiation of keratinocytes regulates the expression of BB-1 antigen as well as transcripts, which is a novel mechanism for the regulation of this molecule.

Changes of extracellular calcium concentration induced by application of excitatory amino acids in the human neocortex in vitro

The influence of the glutamate subreceptor agonists N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on cortical field potentials and on changes in extracellular free calcium concentration ([Ca 2+] o) was tested on human neocortical slices (eleven from nine different patients). The tissue used was a small portion of that which is normally removed for the treatment of a brain tumor. [Ca 2+] o and field potentials were measured by Ca 2+-selective microelectrodes. Local pressure-microejection of NMDA (100 μmol/1)- and AMPA (1 mmol/1)-induced negative field potentials with maximal amplitudes of 0.9 ± 0.1 mV (11 slices, mean ± S.E.M.) and 1.0 ± 0.1 mV (nine slices), respectively. The negative field potentials induced by NMDA were accompanied by monophasic decreases of [Ca 2+] o (0.8 ± 0.1 mmol/l, nine slices). AMPA elicited no (three slices) or only minor decreases of [Ca 2+] o (0.2 ± 0.1 mmol/l, five slices). The responses to the glutamate subreceptor agonists NMDA and AMPA were reversibly depressed by adding their specific antagonists DL-2-amino-5-phosphonovalerate (APV, 100 μmol/1, six slices) and 6-cyano-7-nitroquinoxalin-2,3-dion (CNQX, 5 μmol/1, four slices), respectively. The results correspond to findings in animal experiments and are consistent with the interpretation that in the human neocortex the Ca 2+ permeability of channels gated by NMDA is higher than those gated by AMPA.