Calcium Concentration In Medium Research Articles

Effects of cholinergic agents on the vasopressin-mediated water transport in the isolated toad bladder

The aim of this work was to study the effect of some pharmacological cholinergic agents on the events that follow the interaction of arginine vasopressin with toad bladder membrane receptors related to synthesis of 3′5′ cAMP. The water flow through the membrane was measured gravimetrically in sac preparations of the membrane. In the absence of arginine vasopressin (AVP), carbachol induced a significant increase in the water flow (37%) related to the basal (Ringer's solution). On the other hand, when carbachol and AVP were associated, a significant decrease of AVP hydrosmotic activity occurred (23%). The inhibitory effect of carbachol on the AVP action was almost completely abolished by the cholinergic antagonists atropine, pirenzepine, 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP) and the calcium antagonist lanthanum. Similarly, when carbachol and 3′5′ cyclic adenosine monophosphate (3′5′ cAMP) were associated, a decrease of nucleotide hydrosmotic activity was observed (12.80%). This effect was partially restored by the addition of pirenzepine or 4-DAMP in the bath solution. These results suggest a role for muscarinic receptors of sub-type M 1 and M 3, which are involved in the intracellular calcium release. The increase of calcium concentration in the intracellular medium acts as a negative modulator in the hydrosmotic action of antidiuretic hormone.

Calmodulin-Like Skin Protein: A New Marker of Keratinocyte Differentiation

The expression of the calmodulin-like skin protein, a recently discovered new skin-specific calcium binding protein, was studied in cultured keratinocytes, reconstructed human epidermis, and normal human skin. Using a calmodulin-like skin protein specific polyclonal antibody and Western blot analysis we could show that in cultured keratinocytes calmodulin-like skin protein expression is strongly induced after stimulating cell differentiation by increasing the medium calcium concentration. Known modulators of epidermal differentiation such as sodium butyrate and the synthetic retinoid CD 367 strongly affected calmodulin-like skin protein expression. A more than 10-fold increase was observed in the presence of sodium butyrate, whereas CD 367 abolished almost completely calmodulin-like skin protein expression already at nanomolar concentrations. Calmodulin, another calcium binding protein that is expressed throughout the living layers of the epidermis, is not affected by these modulators. In normal human skin, calmodulin-like skin protein expression is restricted to the stratum granulosum and the lower layers of the stratum corneum. From these results we conclude that calmodulin-like skin protein is a new marker of late keratinocyte differentiation with a role distinct from calmodulin.

Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts.

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.

Phosphatidylinositol 3-Kinase is required for adherens junction-dependent mammary epithelial cell spheroid formation

Adherens junctions facilitate and maintain epithelial cell-cell adhesion. This is true of mammary epithelial cells, both in two dimensional monolayers and in three-dimensional basement membrane cultures. Using the immortalized, functional mouse mammary epithelial scp2 cell line, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PI3-kinase) disrupted adherens junctions. In monolayers, this disruption was associated with decreased E-cadherin and β-catenin at sites of cell-cell contact and decreased association of both proteins with the cytoskeleton. Changes in the distribution of f-actin after PI3-kinase inhibition suggest that this disruption of adherens junctions may be mediated by alterations to the cytoskeleton. In basement membrane cultures, PI3-kinase inhibition reversibly prevented adherens junction-dependent spheroid formation and differentiative milk protein gene expression, both in scp2 cells and in a second mouse mammary epithelial cell line, EpH4. Decreasing the calcium concentration in the culture medium produced similar, although less dramatic, phenotypic effects. These data indicate that adherens junctions contribute, at least in part, to the efficient induction of basement membrane-dependent differentiation of mammary epithelial cells.

Calcium-level responsive controlled drug delivery from implant dosage forms to treat osteoporosis in an animal model

The effects of plasma calcium levels on estradiol release from a self-setting apatite bone cement containing 0.5% estradiol and on the bone mineral density (BMD) of ovariectomized rats were investigated. Apatite cement consisting of an equimolar mixture of tetracalcium phosphate, dicalcium phosphate dihydrate and 0.5% β-estradiol was prepared. The in vitro release profiles from the cements in simulated body fluid containing 0, 5 and 10 mg/100 ml calcium indicated that estradiol release rate decreased with increasing calcium concentration in the dissolution medium. After subcutaneous implantation of the cement, in vivo estradiol release in diseased rats (ovariectomized rats on a low calcium diet) was significantly higher than that in normal rats. The diseased rats maintained a low calcium level during drug release. The bone mass of the recovery model rat was greater after the experiment than before. The results suggested that the severity of osteoporosis in this animals can be reduced by the implantation of this estradiol-loaded apatite cement.

1α,25-Dihydroxyvitamin D3 Induced Differentiation of Cultured Human Keratinocytes is Accompanied by a PKC-Independent Regulation of AP-1 DNA Binding Activity

1alpha,25(OH)2D3 and its analogs are potent mediators of keratinocyte differentiation in vitro. The precise mechanism of this action is still unknown. The nuclear transcription factor activator protein 1 seems to play an important role in keratinocyte differentiation. The purpose of this study was to investigate the effect of 1alpha,25(OH)2D3 on activator protein 1 DNA binding activity in cultured human keratinocytes. In a time-course study of human keratinocytes incubated with 1alpha,25(OH)2D3 (10-7-10-11 M) a significant dose-dependent increase in activator protein 1 DNA binding activity as determined by electrophoretic mobility shift assay was seen after 36 h. This increase was followed by a significant dose-dependent decrease in activator protein 1 DNA binding activity after 72 h. When differentiation was induced by raising the calcium concentration in the culture medium from 0.09 to 0.3 mM a similar increase in activator protein 1 DNA binding activity was observed after incubation for 48 h. Pharmacologic down-modulation of the protein kinase C activity with GF 109203X reversed the calcium-induced increase in activator protein 1 DNA binding activity and abolished keratinocyte differentiation as determined by a transglutaminase assay. In contrast, activator protein 1 DNA binding activity and keratinocyte differentiation were not affected when protein kinase C activity was down-modulated in the experiments with 1alpha,25(OH)2D3. The activator protein 1 complex in human keratinocytes consists of dimers of Fra-1, Fra-2, c-Jun, JunD, and c-Fos. Our results demonstrate that 1alpha, 25(OH)2D3- and calcium-induced keratinocyte differentiation are accompanied by changes in activator protein 1 DNA binding activity. Protein kinase C activation appears to be essential for the calcium-dependent induction of keratinocyte differentiation, whereas a protein-kinase-C-independent activation of activator protein 1 DNA binding and keratinocyte differentiation is responsible for the 1alpha,25(OH)2D3-induced effects.

Osteoclastic responses to various calcium phosphates in cell cultures.

Disks made of hydroxyapatite, beta-tricalcium phosphate, carbonate apatite, tetracalcium phosphate, alpha-tricalcium phosphate, dicalcium phosphate dihydrate, and octacalcium phosphate were incubated in osteoclastic cell cultures for 2 days. The first five salts were sintered and the last two were compressed before incubation. Osteoclasts resorbed only the sintered carbonate apatite disks. However, osteoclasts were able to resorb octacalcium phosphate disks that were preincubated for 1 day in medium without cells, indicating that surface conditioning was important for osteoclastic resorption of this calcium phosphate. Although resorption did not occur, medium calcium and phosphorus changed to an appreciable extent after a 2-day incubation of beta-tricalcium phosphate, tetracalcium phosphate, alpha-tricalcium phosphate, and dicalcium phosphate dihydrate. These changes in the medium calcium and phosphate concentrations could explain why osteoclasts appeared to have lost their activity on these calcium phosphate disks and were not capable of resorbing them. With hydroxyapatite disks no changes were observed in the medium calcium and phosphorus before and after incubation. Moreover, the osteoclasts appeared to be essentially the same as with the sintered carbonate apatite disks and with bone slices used as a control. Nevertheless, no pits or lacunae were observed on the hydroxyapatite disks, indicating that sintered carbonate apatite should be superior to sintered hydroxyapatite as a bioresorbable bone substitute.

Calcium Stores in Symbiosomes from Yellow Lupin Root Nodules

Summary The PPA technique was used for the visualization of calcium in the symbiosomes of yellow lupin root nodule cells at the ultrastructural level both in situ and in vitro. Nodulated plants were grown in pots containing 6 kg of sand with nutrients and various calcium concentrations. At relatively low calcium level (without added calcium or 2 mmol of endogenous calcium per pot) the pyroantimonate calcium precipitates were detected on the cytoplasmic side of the PBM, in the bacteroid cytoplasm, to a much lesser extent within the PBS, and, in addition, in some other subcellular compartments of the infected cells. The observed features in symbiosome calcium distribution became much more profound after increasing the calcium concentration (up to 3 and 6 mmol/pot) in the nutrient medium. In addition, under these conditions numerous electron opaque particles associated with the inner surface of the PBM appeared as well. With the same technique it was found that isolated lupin symbiosomes incubated in the presence of ATP, Mg2+ and Ca2+ are able to accumulate Ca2+, as judged by clearly observed deposits of large size in the PBS in this case. In the experiments with calcium- and ΔpH-sensitive dyes (arsenazo III and acridine orange, respectively), the existence of a calcium pool within the symbiosomes was confirmed by their ability to release calcium ions into the suspension medium after permeabilization of the PBM with the Ca 2+ ionophore A23187. These results provide direct evidence for the hypothesis that the symbiosomes behave as calcium stores in infected cells and, in addition, may be involved in calcium homeostasis in the plant cytosol.

Survival and exopolysaccharide production in Sinorhizobium meliloti WSM419 are affected by calcium and low pH.

Cells of Sinorhizobium meliloti WSM419 showed an adaptive acid-tolerance response when grown at pH 5.8 instead of pH 7.0. Increasing concentrations of calcium in the exposure medium significantly decreased the death rate of WSM419 cells under conditions of acid stress (pH 4.0). The effect of calcium on survival at pH 4.0 however, appears unconnected to exopolysaccharide (EPS), since a strain with a mutation in exoY (Rm0540) responded to calcium in the exposure medium in the same way as its wild-type parent (Rm2011). The concentration of calcium in the growth medium also affected subsequent survival at pH 4.0, and the effect varied with pH. In cells grown at pH 5.8, higher calcium concentrations also markedly increased the rate of synthesis of EPS; this was not seen in cells grown at pH 7.0. 1H NMR spectra for isolated EPS from WSM419 cultures grown at pH 5.8 and pH 7.0 showed that low pH markedly lowered the degree of substitution with acetyl and pyruvyl groups, but not the degree of substitution with succinyl groups; calcium concentration did not affect the pattern of substitution at either pH. For EPS to be involved in the effect of calcium concentration in the growth medium on survival would imply a deleterious effect of the EPS produced at low pH.

Juvenile hormone biosynthesis in larval and adult stick insects, Carausius morosus

Corpora allata (CA) from adult egg-carrying Indian stick insects, Carausius morosus, synthesise and release juvenile hormone (JH) III in vitro. No JH biosynthesis was observed in larvae, young adults, and old adult females that do not carry sclerotised eggs. In females, which bear sclerotised eggs, a consistent JH biosynthesis was observed. Supplementation of precursors of JH biosynthesis (farnesol, mevalonic acid lactone) greatly enhanced JH biosynthesis in a stage-, age-, and dose-dependent manner, but CA from the last larval instar retained the biosynthesised JH within the gland. Elevated calcium concentration in the incubation medium stimulated JH biosynthesis by CA from older adults but had either no or a poor effect on CA from young adults and larvae. The results obtained with farnesol, mevalonic acid lactone, and calcium indicate that the rate-limiting steps of JH biosynthesis very likely occur before the formation of mevalonic acid and that these early steps cannot be stimulated by elevated calcium concentrations in larvae and young adults. In older adults, in which spontaneous JH biosynthesis occurs, elevated calcium concentration can markedly stimulate JH biosynthesis. A pre-purified extract from brains of adult females had a stimulating effect on JH biosynthesis by CA from adult females. The results indicate that JH biosynthesis in C. morosus may require food-derived farnesol and may be regulated by allatotropic signals from the brain, possibly triggered by sclerotised oocytes in the ovary.

Endogenous Adenosine Reduces the Occurrence of Ischemia-induced Ventricular Fibrillation in Rat Heart

The aim of this study was to determine whether endogenous adenosine has antiarrhythmic effects on ischemia-induced ventricular tachyarrhythmias. We therefore modulated the effect of endogenous adenosine in isolated rat hearts using four different approaches. First, interstitial adenosine was elevated by metabolic inhibition with either EHNA (erythro-9-(2-hydroxy-3-nonly)adenine) or acadesine [5-amino-1-β-D-imidazole-4-carboxamide). Second, cardiac effects of A1adenosine receptors were allosterically enhanced with PD81,723 (2-amino-4,5-dimethyl-3-thienyl)[3-(trifluoromethyl)phenyl]-methanone. Third, endogenous adenosine release was suppressed with NBMPR (S-(4-nitrobenzyl)-6-thioinosine), and fourth, adenosine receptor subtypes were blocked with antagonists of different selectivity. Regional ischemia, induced by coronary artery ligation, caused ventricular fibrillation of a reproducible kind in about 20% of untreated hearts with a low calcium concentration in the perfusion medium (0.80 mmol/l CaCl2) and in about 75% with high calcium (1.85 mmol/l) within an observation period of 30 min. At high calcium, EHNA (1 and 10μmol/l) and acadesine (500μmol/l) suppressed the occurrence of ventricular fibrillation from 68% (controls) to 47%, 33% and 38%, respectively. Conversely, PD81,723 (10μmol/l) did not influence the occurrence of ventricular fibrillation. At low calcium, NBMPR (0.1 and 1μmol/l) resulted in a concentration-dependent rise of ventricular fibrillation from 13% (controls) to 40% and 57%, respectively. The adenosine receptor antagonists theophylline (100μmol/l), XAC (Xanthine Amine Congener; 1μmol/l) and 8-PT (8-phenyltheophylline; 1μmol/l) caused a rise in the occurrence of ventricular fibrillation from 25%, 15% and 18% (controls) to 57%, 39% and 44%, respectively, and the selective A2areceptors antagonist CSC (8-(3-chlorostyryl)caffeine; 5μmol/l) from 20% to 56%. Conversely, the selective A1receptor blocker DPCPX (8-cyclopentyl-1,3-dipropyl-xanthine; 1μmol/l) was ineffective. NBMPR or EHNA concentration-dependent suppressed or increased ischemia-induced adenosine overflow, respectively, in a concentration-dependent manner, whereas the adenosine receptor antagonists did not influence adenosine overflow. We conclude that endogenous adenosine is an antiarrhythmic mediator accumulating in acute ischemic myocardium to a level which effectively decreases the occurrence of ventricular fibrillation by an A2adenosine receptor activation in the isolated rat heart.

Regulation of mitochondrial ATP synthase activity in human myocardium.

1. In previous studies regulation of the F1F0-ATPase of mitochondrial complex V (ATP synthase) has been demonstrated in rat cardiomyocytes, canine mycocardium and skeletal muscle from children. The aim of the present study was to examine regulation of ATP synthase in human myocardium in response to different metabolic states. 2. Biopsy material was obtained from 10 children undergoing cardiac surgery. Mitochondria in the post-nuclear supernatant were incubated under different metabolic conditions for 15 min and then broken by sonication. ATP synthase was measured spectrophotometrically using a coupled enzyme assay. 3. ATP synthase can be rapidly measured in sonicated preparations of heart mitochondria from children. We show that direct regulation at the level of ATP synthase occurs in these mitochondria. ATP synthase capacity is decreased in response to blocking of the respiratory chain by cyanide (mimicking anoxia) or uncoupling of mitochondria, falling to 76% and 66% of control values respectively. Upregulation of ATP synthase can be demonstrated in heart mitochondria when the calcium concentration in the incubation medium is increased to 5 microM (130% of control). 4. ATP synthase is actively regulated in heart mitochondria from children. The enzyme is upregulated in response to increased calcium. This transition may reflect the increased energy demand when cardiac workload is increased.

Differentiation of cultured keratinocytes promotes the adherence of Streptococcus pyogenes.

Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections.

The influence of sintering temperature on the proliferation of fibroblastic cells in contact with HA-bioceramics.

HA-ceramics used in human surgery as osteoconductive surfaces show a great variety of characteristics. Certain characteristics such as grain size, porosity, and surface area, are controlled by the sintering temperature of the slurry. We grew L-929 fibroblast cells on HA-ceramic disks that had been sintered at different temperatures ranging from 850 degrees-1350 degrees C. The cell line growth rate was lower on ceramic disks than on the culture-grade polystyrene used as a negative control. Cell growth correlated with the ceramic sintering temperature although no significant difference in the cell adhesion to the different ceramics was shown. Growth rate on ceramics sintered at low temperatures (850 degrees and 950 degrees C) was negative whereas it was positive on disks sintered at higher temperatures. When the cells were separated from the disks by a polycarbonate membrane, the growth rate was negative on those membranes in contact with low-temperature sintered disks and positive on the high-temperature sintered disks. The calcium and phosphorus concentration in the culture medium in contact with ceramics sintered below 1050 degrees C decreased during the culture period. Ceramics sintered between 1100 degrees and 1250 degrees C brought about an increase in Ca and P concentrations while ceramics sintered at higher temperatures did not induce any changes. SEM examination of the 850 degrees and 1200 degrees C sintered ceramics showed that the 850 degrees C sintered ceramics consisted of small grains with pores between them and the 1200 degrees C sintered ceramics were made of larger grains without any visible pores, thereby decreasing the surface of material in contact with the culture medium. This difference in surface area was confirmed by the fact that the amount of albumin absorbed onto the ceramic was dependent on the sintering temperature. In conclusion, the modification of the culture medium brought about by high-surfaced ceramics could influence the growth of cells with which such ceramics come in contact.

CeReS-18, a novel cell surface sialoglycopeptide, induces cell cycle arrest and apoptosis in a calcium-sensitive manner.

Very few growth inhibitors have been identified which can inhibit the proliferation of a broad spectrum of human breast cancer cell lines. CeReS-18, a novel cell surface sialoglycopeptide growth inhibitor, can reversibly inhibit the proliferation of both estrogen receptor positive (MCF-7) and negative (BT-20) human breast cancer cell lines. In addition, at concentrations above those required for the reversible inhibition of cell proliferation, CeReS-18 can also induce cell death in MCF-7 cells. Changes in nuclear and cytoplasmic morphology, characteristic of apoptosis, were detected in MCF-7 cells treated with a cytotoxic concentration of CeReS-18, and internucleosomal DNA cleavage was also observed. The sensitivity of MCF-7 and BT-20 cells to the biological properties of CeReS-18 could be influenced by altering the calcium concentration in the extracellular growth medium, such that when the calcium concentration in the environment was decreased, and increased sensitivity to CeReS-18-induced growth inhibition and cytotoxicity were observed. The addition of the calcium chelating agent EGTA to MCF-7 cells, cultured in a normal calcium environment, could mimic the increased sensitivity to the biological effects of CeReS-18 observed under reduced calcium conditions.

The cultured human cervical epithelium: a new model for studying paracellular transport.

Human cervical cells form confluent cultures on filters with a relatively high degree of paracellular permeability. We sought to determine the extent to which acute and reversible changes in permeability in responses to modulators of paracellular resistance affect the resistance of the lateral intercellular space (RLIS), or of the tight junctions (RTJ). The question was approached experimentally by measuring the effects of combinations of physical/chemical conditions whose actions on the paracellular resistance are relatively well understood (e.g., changes in hydrostatic gradients, medium tonicity, and medium calcium concentration). Changes in paracellular permeability were determined relative to changes in transepithelial electrical resistance (TER) and to the epithelial permeability to the trisulfonic acid pyranine (Ppyr). Changes in RTJ were determined relative to changes in the transepithelial cation selectivity, as determined from changes in dilution potential. 1) Low extracellular calcium abrogates the TER and decrease transepithelial cation selectivity (i.e., decreases RTJ). 2) Changes in TER induced by changes in hydrostatic gradients and medium tonicity modulate TER but have no effect on cation selectivity (i.e., the effect was on RLIS). 3) Low-calcium-related decreases in TER are additive to those of hydrostatic and hypertonic gradients and hypotonic conditions. 4) Subluminal to luminal hydrostatic and hypertonic gradients decrease TER, but the effects are nonadditive. These results indicate that RLIS and RTJ can be selectively modulated. Based on quantitative analysis of the data it is predicated that RLIS and RTJ constitute the TER of the cultured cervical epithelium, and contribute about 25 and 75%, respectively, to the total resistance.

Plasma Calcium Level‐responsive Oestradiol Release From Self‐setting Apatitic Bone Cement in Ovariectomized Rats

The dependency of in-vitro and in-vivo drug release from a self-setting bioactive calcium phosphate cement containing 0.5% oestradiol on the plasma calcium level in rats was investigated. The in-vitro release profiles from drug-loaded calcium phosphate cements with various calcium levels in simulated body fluid, indicated that the release rate decreased with increasing calcium concentration in dissolution media. After subcutaneous implantation of drug-loaded cement in healthy and vitamin D-deficient ovariectomized rats, oestradiol released in the diseased rat, with low plasma calcium level, was significantly higher than that in the healthy rat, indicating that oestradiol release from the cement depended on the plasma calcium level.

Stimulation versus Inhibition of Keratinocyte Growth by 1,25-Dihydroxyvitamin D3: Dependence on Cell Culture Conditions

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) inhibits proliferation of keratinocytes in vitro and psoriatic epidermal cells in vivo and is considered to be a negative regulator of keratinocyte growth. It has been recently observed, however, that 1,25(OH)2D3 and its active analogs stimulate epidermal proliferation after topical application in mice. In this study we show that 1,25(OH)2D3, depending on the culture conditions, can either stimulate or inhibit DNA synthesis in human keratinocytes. In cells cultured with 0.15 mM calcium in the absence or with low levels (0.1 ng/ml) of epidermal growth factor, exposure to 10(-11) - 10(-6) M 1,25(OH)2D3 imposed cell cycle block in the late G1 phase. When keratinocytes were cultured in the presence of high extracellular calcium concentration (1.8 mM), 1,25(OH)2D3 in concentrations of 10(-11) - 10(-9) M stimulated cell growth by increasing the proportion of cells entering S phase. 1,25(OH)2D3 also stimulated growth of keratinocytes cultured in low calcium concentrations when the cells were previously suspended for a short time in a semisolid medium. Growth stimulation was absent in the presence of the anti-E-cadherin antibody, which is known to inhibit calcium-dependent differentiation. These results suggest that keratinocytes committed to terminal differentiation by an elevation of calcium concentration or suspension in a semisolid medium respond to 1,25(OH)2D3 with an increase in DNA synthesis. In contrast, proliferating undifferentiated keratinocytes may be the main target for the anti-proliferative activity of 1,25(OH)2D3.

Effect of temperature on the contractile response of isolated rat small intestine to acetylcholine and KCl: calcium dependence.

The influence of temperature on phasic and tonic components of isometric tension of the isolated rat small intestine was investigated at two temperatures (15 degrees C and 21 degrees C) where spontaneous movements were not evident. Warming did not shift acetylcholine-response curve whereas it enhanced the amplitude of maximal tension. The rate of tension development varied with acetylcholine concentration and increased with temperature. Warming enhanced and accelerated responses to single doses of high KCl. The effect of temperature on acetylcholine responses was mimicked by lowering calcium concentration in the bathing medium but in zero-calcium medium responses were higher at 21 degrees C than at 33 degrees C. Discussion of previous reports underlines that opposite observations on the effect of temperature on smooth muscle contraction are mainly explained by experimental protocols. In turn, it is suggested that the obtained effects of temperature might be related to the regulation of intracellular calcium concentration.

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.