Use Of Ca2 Research Articles

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.

Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content

The calA, calC, and calD mutants of Escherichia coli are known to be sensitive to Ca2+ (R. N. Brey and B. P. Rosen, J. Bacteriol. 139:824-834, 1979). In the absence of any added stimuli for chemotaxis, both the calC and the calD mutants swam with a tumbly bias. Both the calC and the calD mutants were defective in chemotaxis as measured by computer analysis, use of swarm plates, and capillary assays. The calA mutant was only slightly defective in motility and only slightly impaired in chemotaxis. Chemotactically wild-type cells had an intra-cellular free-Ca2+ level of about 105 nM. The intracellular free-Ca2+ levels of the mutants, as determined by use of the fluorescent Ca2+ indicator dye fura-2 or fluo-3, were about 90, about 1,130, and about 410 nM for calA, calC, and calD, respectively. Lowering the intracellular free-Ca2+ levels in wild-type cells and in the tumbly cal mutants by use of Ca2+ chelators promoted running (smooth swimming). Overexpression of CheZ (which causes dephosphorylation of CheY-phosphate) in the wild type and in the tumbly cal mutants decreased the level of tumbliness (which is caused by CheY-phosphate). The calA mutant was 4- to 10-fold more resistant than the wild type to the inhibitory effect of omega-conotoxin on chemotaxis. omega-Conotoxin had no effect on Ca2+ extrusion by wild-type E. coli; that result suggests that omega-conotoxin affects Ca2+ transport at the point of entry instead of exit.

Insulin-Stimulated Glucose Transport Inhibits Ca 2+ Influx and Contraction in Vascular Smooth Muscle

Insulin attenuates serotonin-induced Ca2+ influx, the intracellular Ca2+ transient, and contraction of cultured vascular smooth muscle cells from dog femoral artery. These studies were designed to test whether insulin-induced glucose transport was an early event leading to the inhibitory effects of insulin on Ca2+ influx, intracellular Ca2+ concentration, and contraction in these cells. Insulin 1 nmol/L stimulated the 30-minute uptake of [3H]2-deoxyglucose in these cells via a phloridzin-inhibitable mechanism. Contraction of individual cells was measured by photomicroscopy, intracellular Ca2+ concentration was monitored by measuring fura 2 fluorescence by use of Ca(2+)-sensitive excitation wavelengths, and Ca2+ influx was estimated by the rate of Mn2+ quenching of intracellular fura 2 fluorescence when excited at a Ca(2+)-insensitive wave-length. In the presence of 5 mmol/L glucose, preincubation of cells for 30 minutes with 1 nmol/L insulin inhibited 10(-5) mol/L serotonin-induced contraction of individual cells by 62% (P < .01) and decreased the serotonin-stimulated component of Mn2+ influx by 78% (P < .05). Removing glucose from the preincubation medium or adding 1 mmol/L phloridzin completely eliminated these effects of insulin. Insulin lowered the serotonin-induced intracellular Ca2+ peak by 37% (P < .05), and phloridzin blocked this effect of insulin. When glucose uptake was increased to the insulin-stimulated level by preincubation of the cells for 30 minutes with 25 mmol/L glucose in the absence of insulin, serotonin failed to stimulate Mn2+ influx, the serotonin-induced Ca2+ peak was decreased by 46% (P < .05), serotonin-induced contraction was inhibited by 60% (P < .01), and addition of insulin did not further inhibit contraction. Since the effects of insulin on serotonin-stimulated Ca2+ transport, intracellular Ca2+ concentration, and contraction were dependent on glucose transport and were duplicated when glucose transport was stimulated by high extracellular glucose concentration rather than insulin per se, it is concluded that insulin-stimulated glucose transport is an early event that leads to decreased Ca2+ influx and contraction in vascular smooth muscle.

Calcium Stimulation of Ammonium Absorption and Growth by Beet

AbstractInefficiency in N use by plants contributes to environmental degradation. We examined the use of Ca2+ as a means of increasing NH+4 absorption in beet (Beta vulgaris cv. Detroit dark red). Ammonium absorption was measured in greenhouse and field studies. In the greenhouse, beet plants received full‐strength nutrient solutions with Ca2+: NH+4 molar ratios (MR) of 0, 0.25, 0.5 and 1.0 (Ca2+:NH+4) and a NO‐3 check. Absorption of 15NH+4 was monitored over a 30‐h period. The field production trials occurred over 3 yr on a calcareous Saneli silty clay loam [clayey over sandy or sandy‐skeletal, montmorillonitic (calcareous), thermic Vertic Torrifluvent]. The 15NH+4 absorption study showed the greatest plant weight at the molar ratio (MR) of 1.0 in tops (except in seedlings) and at 0.5 in bulbs. In seedlings, a biomass reallocation occurred in the root‐bulbs from the tops. In both the intermediate and mature growth stages the weight of roots, tops and bulbs greatly increased. In the 15NH+4 absorption studies N concentrations were unchanged or decreased as the Ca2+ concentrations increased, but total N increased. Seedling bulb‐root weight increases were apparenlly metabolite transfer from tops. The intermediate and mature beet plants showed increases in total N. In the greenhouse growth test, vegetative matter increased in excess of 300% at the MR of 0.5 over the check (MR:0). Growth of beet in the 0.5 MR treatment exceeded that on the NO‐3 comparison by 14%. In field testing at a MR of 0.25 (CaCl2:urea), yields increases averaged 26% over 3 yr when compared with urea alone.

Mechanisms by which endothelin-1 stimulates increased cytosolic free calcium ion concentrations in single rat Sertoli cells

The regulation by endothelin-1 (ET-1) of cytosolic free calcium ion concentrations ([Ca2+]i) was investigated in single immature rat (testicular) Sertoli cells. [Ca2+]i was estimated in individual gonadal cells by digital imaging videomicroscopy using the calcium indicator dye fura-2/AM. Two concentration-dependent types of ET-1-induced [Ca2+]i signals were observed. Responses to high ET-1 concentrations (1.0-1000 nM) were characterized by a biphasic, rapid, and transient [Ca2+]i rise (spike) within 10 sec, followed by an exponential decrease toward a new steady state level (plateau phase) in 98% of responsive cells. At low concentrations of ET-1 (0.001 or 0.1 nM), the [Ca2+]i increase was slower, reaching peak values 40-100 sec after stimulation and remaining elevated for 2-3 min of observation. There was cell-cell heterogeneity in the amplitude and kinetics of the [Ca2+]i response to the same concentration of ET-1. However, there was a significant ET-1 concentration-dependent increase in the total percentage of cells responding to ET-1. Removal of extracellular Ca2+ or use of Ca2+ channel blockers (verapamil or cobalt) did not affect the ET-1-induced [Ca2+]i spike phase, but abolished the plateau phase, suggesting that ET-1 induces the mobilization of Ca2+ from internal stores, followed by calcium influx from extracellular sources. In cell population experiments, ET-1 attenuated FSH-stimulated cAMP and estradiol accumulation by Sertoli cells. These inhibitory effects were mimicked by phorbol 12-myristate 13-acetate, an activator of protein kinase-C, suggesting that ET-1 action on Sertoli cells might be linked to the protein kinase-C pathway. In conclusion, the present investigations demonstrate that ET-1 activates an intracellular signaling pathway involving [Ca2+]i in single rat Sertoli cells. The sources of the biphasic [Ca2+]i response include mobilization of Ca2+ from internal stores, followed by Ca2+ influx via verapamil- and cobalt-sensitive Ca2+ channels. Increasing ET-1 concentrations recruit an increasing number of individual Sertoli cells responding with a spike-plateau [Ca2+]i signal, thus offering a mechanism at the single cell level for the ET dose-response curve.

Mechanisms by which endothelin-1 stimulates increased cytosolic free calcium ion concentrations in single rat Sertoli cells.

The regulation by endothelin-1 (ET-1) of cytosolic free calcium ion concentrations ([Ca2+]i) was investigated in single immature rat (testicular) Sertoli cells. [Ca2+]i was estimated in individual gonadal cells by digital imaging videomicroscopy using the calcium indicator dye fura-2/AM. Two concentration-dependent types of ET-1-induced [Ca2+]i signals were observed. Responses to high ET-1 concentrations (1.0-1000 nM) were characterized by a biphasic, rapid, and transient [Ca2+]i rise (spike) within 10 sec, followed by an exponential decrease toward a new steady state level (plateau phase) in 98% of responsive cells. At low concentrations of ET-1 (0.001 or 0.1 nM), the [Ca2+]i increase was slower, reaching peak values 40-100 sec after stimulation and remaining elevated for 2-3 min of observation. There was cell-cell heterogeneity in the amplitude and kinetics of the [Ca2+]i response to the same concentration of ET-1. However, there was a significant ET-1 concentration-dependent increase in the total percentage of cells responding to ET-1. Removal of extracellular Ca2+ or use of Ca2+ channel blockers (verapamil or cobalt) did not affect the ET-1-induced [Ca2+]i spike phase, but abolished the plateau phase, suggesting that ET-1 induces the mobilization of Ca2+ from internal stores, followed by calcium influx from extracellular sources. In cell population experiments, ET-1 attenuated FSH-stimulated cAMP and estradiol accumulation by Sertoli cells. These inhibitory effects were mimicked by phorbol 12-myristate 13-acetate, an activator of protein kinase-C, suggesting that ET-1 action on Sertoli cells might be linked to the protein kinase-C pathway. In conclusion, the present investigations demonstrate that ET-1 activates an intracellular signaling pathway involving [Ca2+]i in single rat Sertoli cells. The sources of the biphasic [Ca2+]i response include mobilization of Ca2+ from internal stores, followed by Ca2+ influx via verapamil- and cobalt-sensitive Ca2+ channels. Increasing ET-1 concentrations recruit an increasing number of individual Sertoli cells responding with a spike-plateau [Ca2+]i signal, thus offering a mechanism at the single cell level for the ET dose-response curve.

Calcium-dependent inactivation of RNA polymerase III transcription.

Extracts from whole oocytes of Xenopus laevis are widely used as an efficient in vitro system for the transcription of cloned genes by RNA polymerase III. We have found that these extracts no longer support RNA polymerase III transcription in response to a brief incubation in the presence of Ca2+. However, when transcription complexes were first formed on the genes, a subsequent incubation in the presence of Ca2+ had little effect. Fractionation of extracts was used to show that transcription factors (TF) IIIC and, to a lesser extent, TFIIIB, but not RNA polymerase III, were targets of the Ca(2+)-dependent inactivation process. An additional component (not present in fractionated TFIIIC or TFIIIB) was required for the Ca(2+)-dependent destruction of transcription factor activity. The Ca(2+)-dependent inactivation process was blocked by protease inhibitors that inhibit known Ca(2+)-dependent proteases called calpains. These results suggest that TFIIIC and TFIIIB are inactivated by an endogenous calpain. The common use of Ca2+ as a second messenger and the widespread distribution of calpains suggest that the proteolytic degradation of transcription factors may be a general mechanism for the regulation of gene expression.

High efficiency transfection of monkey kidney COS-1 cells

Methods are described for achieving high efficiency transient transfection of COS-1 cells. A transfection solution of vector DNA and DEAE-dextran in phosphate buffered saline is added to the cells, followed by treatment with culture medium containing chloroquine, resulting in a maximum efficiency of about 50%. The high efficiency obtained is primarily dependent on the use of Ca2+- or Mg2+-free buffer solutions for transfection, because the addition of these ions greatly reduces efficiency. Shocking the cells with dimethylsulfoxide does not increase transfection efficiency. COS-1 cells are monkey kidney cells that have a genomic insertion of the SV40 T antigen gene, allowing plasmid expression vectors bearing an SV40 replication origin to be amplified in these cells. Therefore this transfection method is useful for optimizing transient expression of genes in a mammalian cell system.

The solubility of CaMoO4(c) and an aqueous thermodynamic model for Ca2+-MoO 4 2 -ion-interactions

The solubility of powellite [CaMoO4(c)] was studied in aqueous Na2MoO4, CaCl2 and Ca(NO3)2 solutions ranging in concentrations from 1×10−4M to 1.0M and over equilibration times extending to 36 days. Our experimental data were interpreted using the aqueous ion-interaction model of Pitzer and coworkers. The Ca2+−MoO 4 2− ion-interactions were found to be analogous to Ca2+−SO 4 2− . The use of Ca2+−MoO 4 2− ion-interactions parameters (β(0)=0.2, β(1) = 3.1973 and β(2)) and a logK sp of −7.93 gave excellent predictions of all of the experimental data. Commonion ternary interaction parameters such as MoO 4 2− −Cl− or MoO 4 2− −NO 3 − were not required.

Calcium requirement for protoplast transfection mediated by polyethylene glycol of Lactobacillus casei by PL-1 Phage DNA.

The effects of some divalent cations on protoplast transfection mediated by polyethylene glycol of Lactobacillus casei ATCC 27092 by PL-1 phage DNA in 50 mM Tris-maleate buffer (pH 6.0) were investigated. The efficiency of transfection increased about 30 times in the presence of 10 mM Ca2+. Sr2+ increased the transfection rate as well, but Ba2+, Mn2+, and Mg2+ did not. Co2+ and Zn2+ inhibited transfection. The simultaneous use of Ca2+ and Mg2+ increased the transfection efficiency. Impairment of transfection caused by lack of Ca2+ could not be reversed by the addition of Ca2+ later. A decrease in the Ca2+ concentration to an ineffective level before transfection ended immediately inhibited transfection. Protoplasts were transfected with a phage adsorption mutant resistant to PL-1, also, and these metal ions had the same effect. Multiplication of phages in the transfected protoplasts was independent of the presence or absence of calcium ions. Calcium ions seemed to be involved in the entry of PL-1 DNA into the host protoplasts.

Calcium Stimulation of Ammonium Absorption in Onion

AbstractThe inefficiency of N use in soils has been studied widely, producing varied suggestions for increased effectiveness. The object of this research was to examine the use of Ca2+ as a means to increase NH+4 absorption, using onion (Allium cepa L.) as a test crop. Ammonium absorption in the presence of variable Ca2+ concentrations was measured in short‐term greenhouse studies as well as longterm greenhouse studies and field‐production trials. The greenhouse 15NH+4 absorption study was conducted in 4‐L pots containing 3.5 kg of calcareous Gila sandy loam (coarse‐loamy, mixed, calcareous, thermic Typic Torrifluvents). The long‐term growth study was conducted in calcareous quartz sand. Irrigation solutions consisted of half‐strength nutrient solutions (5 mol m−3 in NH+4) with Ca2+:NH+4 molar proportions of 0, 0.25, 0.50, 1.00, and 2.00. The 15NH+4 absorption study was conducted during a 30‐h period on 60‐d onion. The long‐term growth experiment received the same nutrient solutions until mature onion bulbs were produced (about 5 cm in diameter). A 2‐yr field production trial used single rows on 1‐m wide beds. The soil was a calcareous Saneli silty clay loam [clayey over sandy or sandy‐skeletal, montmorillonitic (calcareous), thermic Vertic Torrifluvent]. In 15NH+4 studies, NH+4 absorption increased with added Ca by as much as 189% in onion bulbs and by 67% in onion tops relative to absorption with no Ca added. These maximum increases were in association with solution Ca2+:NH+4 molar proportions (MP) of 0.5 in onion bulbs and 1.0 in onion tops. Total plant dry weights with Ca added increased as much as 100% in bulbs and 25% in tops in 30‐h 15NH+4 absorption tests over the 0 Ca2+ controls. In greenhouse growth tests, vegetative matter increased as much as 40% with increasing Ca2+ concentrations at constant NH+4 levels compared to the 0 Ca2+ control. This translated to increased total N absorption. In 2 yr of field testing with a MP of 0.25, mature bulbs from both years increased 64% over yields with urea alone. Thus the addition of Ca2+ with urea produced significantly improved plant N‐use efficiencies (greater plant yield at the same level of N application). These results were suggested from both the short‐term greenhouse 15NH+4 absorption experiment and from the long‐term greenhouse growth trial.

Modification of the Clastogenic Effects of Cobalt by Calcium in Bone Marrow Cells of Mice in vivo.

THE INTERACTION BETWEEN COBALTOUS CHLORIDE AND CALCIUM CHLORIDE WAS OBSERVED USING AS ENDPOINTS MITOTIC INDEX AND FREQUENCY OF CHROMOSOMAL ABNORMALITIES. THE TWO SALTS WERE ADMINISTERED ORALLY TO LABORATORY-BRED MALE MICE IN VIVO SINGLY OR ONE FOLLOWED BY THE OTHER OR BOTH SIMULTANEOUSLY. CHROMOSOMES WERE STUDIED FROM BONE MARROW PREPARATIONS AFTER 24 HR. IN ALL CASES, THE ADMINISTRATION OF CACL2 TWO HOURS BEFORE COCL2 PROTECTED AGAINST THE CLASTOGENIC EFFECTS OF THE LATTER TO A SIGNIFICANT EXTENT. SIMULTANEOUS FEEDING OF THE TWO SALTS REDUCED THE DAMAGE BY COCL2 ONLY WHEN CACL2 WAS GIVEN IN A HIGHER CONCENTRATION. THE REDUCTION OF CLASTOGENICITY OF CATIONIC COBALT BY CALCIUM HAS BEEN ATTRIBITED TO THE COMPETITION BETWEEN THE TWO IONS. THIS REPORT OF THE USE OF CA(2+) IN REDUCING THE CLASTOGENICITY OF CO(2+) IS A NEW ONE.

Agonist-induced vascular tone.

The cellular mechanisms underlying the agonist-induced sustained contraction of the vascular smooth muscle are reviewed in the light of the use of Ca2+ and the change of Ca2+ sensitivity of the contractile apparatus. It is generally accepted that the main trigger for contraction of vascular smooth muscle is the elevation of intracellular Ca2+ concentration. However, the measurement of intracellular Ca2+ concentration during the sustained phase of agonist-induced contraction is reported to be lower than that of high K+ stimulation or the value obtained by the experiments with chemically skinned smooth muscle preparations. These observations indicate that a second regulatory system may exist. One possible mechanism is the effectiveness of Ca2+ use. Agonist-induced Ca2+ influx may be more effective in raising the intracellular Ca2+ in the bulk of the cytoplasm than is Ca2+ entry induced by depolarization by the inhibition of a putative sarcoplasmic reticulum buffer barrier. Another possibility is the change of Ca2+ sensitivity of the contractile apparatus. Although the survey of the recent literature concerning the phorbol ester-induced vasoconstriction tends to support a role for protein kinase C in the change of Ca2+ sensitivity of the contractile proteins, it fails to establish a clear link between receptors, protein kinase C, and myofilaments. By using new methods for permeabilizing smooth muscle fibers, which retain the function of receptors and signal transduction systems, we now provide direct evidence that the activation of G protein by norepinephrine or guanosine 5'-0-(3-triphosphate) (GTP-gamma-S), nonhydrolyzable GTP analogue, enhances myofilament sensitivity to Ca2+.

Lack of effect of nicardipine and diltiazem on glucose- and arginine-induced insulin release in obese subjects.

The metabolic effects of calcium channels blockers have already been studied both in normal and diabetic humans and results were quite controversial, depending on the drug used, the dose administered, and the type of patient. Little information exists on the use of Ca2+ antagonists in obese people, even if these persons are a population risk group for developing diseases in which these drugs may be requested for treatment. Thus, we evaluated, in obese humans, the metabolic effects of two Ca2+ antagonist drugs recently made commercially available to treat diseases such as hypertension and ischemic heart disease: nicardipine and diltiazem. Sixteen obese subjects were submitted to an intravenous glucose tolerance test (0.33 g/kg) (IVGTT) and an arginine test tolerance (30 g in 30 minutes) (ATT) before and after a week of oral treatment with nicardipine (60 mg/day) or diltiazem (360 mg/day). Plasma values of glucose, insulin, and C-peptide during IVGTT, and of glucose, insulin and glucagon during ATT did not show any modification during treatment with either drug. Thus the Ca2+ antagonists, nicardipine and diltiazem, at therapeutic doses in obese subjects do not significantly affect glucose tolerance or insulin and glucagon release.

Prostaglandin production by the guinea-pig endometrium: is calcium necessary?

The output of prostaglandin (PG) F2 alpha from guinea-pig endometrium obtained on day 15 of the oestrous cycle and maintained in tissue culture was significantly (P less than 0.05) reduced by the use of Ca2+-depleted medium, EGTA (a Ca2+ chelator), 8-(N,N-diethyl-amino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8; an intracellular Ca2+ antagonist), trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7; both calmodulin antagonists). Nifedipine inhibited PGF2 alpha output at a concentration (100 mumol/l) much greater than that usually required to block Ca2+ channels. Verapamil had a small but significant (P less than 0.05) inhibitory effect on PGF2 alpha output at 10-100 mumol/l. The outputs of PGE2 and, to a lesser extent, 6-keto-PGF1 alpha (the hydrated product of PGI2) were also reduced by using Ca2+-depleted medium. EGTA reduced the outputs of PGE2 and 6-keto-PGF1 alpha on day 1 of culture, but stimulated 6-keto-PGF1 alpha output on day 3 of culture. The outputs of PGE2 and 6-keto-PGF1 alpha were increased by TMB-8 (100 mumol/l) on day 3 of culture and by TFP and, to a smaller extent, by W-7 on all 3 days of culture. Nifedipine (100 mumol/l by not 1 or 10 mumol/l) reduced the outputs of PGE2 and 6-keto-PGF1 alpha on all 3 days of culture, whereas verapamil (100 mumol/l but not 1 or 10 mumol/l) increased the outputs of these two prostaglandins on days 2 and 3 of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of intracellular calcium mobilization

Publisher Summary This chapter describes the use of Ca2+-sensitive fluorescent dye technology with the extremely precise technology of flow cytometry for the single-cell analysis of changes in intracellular Ca2+ levels. The model cell involves the antireceptor stimulation of mouse splenic B lymphocytes, although the techniques described are readily adaptable to any cell type, which can be analyzed by flow cytometry and is not incompatible with the quin 2 or indo-1. The chapter also presents the relevant principles of flow cytometry. It is important to be familiar with the fundamental principles of flow cytometry to understand the techniques to be described and to permit their adaptation to the various instruments currently in use. The basic principle of the analytic system is to pass a monodisperse cell suspension (loaded with fluorescent dye) through a quartz flow cell, which is intersected by a focused laser beam of appropriate excitation wavelength. The chapter provides an overview of materials and explains the optimization of laser alignment.

On the nature of calcium ion binding between phosphatidylserine lamellae

Ca2+ binding between phosphatidylserine (PS) lamellae gives rise to a phase with the composition Ca(PS)2. When aqueous Ca2+, hydrated PS, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant. At Ca2+ concentrations below this critical value, no binding of Ca2+ to PS is detected. Above this value, Ca2+ binds to PS to form Ca(PS)2. The invariant Ca2+ concentration is 0.14 microM for palmitoyloleoylphosphatidylserine (POPS) and 3.0 microM for dioleoylphosphatidylserine (DOPS). For the mixed acyl chain PS derived from bovine brain (BBPS) this Ca2+ concentration ranges from 0.25 to 0.7 microM. The observed phase behavior is described by the phase rule for the three-component system of water, Ca2+, and PS, with temperature and pressure constant. In order for Ca2+ to bind between PS lamellae to form the Ca(PS)2 phase, the aqueous Ca2+ concentration must be supersaturated. The equilibrium Ca2+ concentration is determined by dissolving Ca(PS)2 by use of Ca2+ chelators.

Ca2+i:K+i ratio and total intracellular calcium in essential and renal hypertension.

The calcium ion (Ca2+) and potassium ion (K+) content in the ashed material from red blood cells was determined by flame photometry in 61 essential hypertensives, 11 renal hypertensives and in 47 normotensive controls, and intracellular K+ concentration was measured in the haemolysate. The ratio between Ca2+ and K+ content in ashed red blood cells (Ca2+i:K+i) was 2.07 +/- 0.91 X 10(-3) in normotensives, 4.91 +/- 2.17 X 10(-3) in essential hypertensives (P less than 0.01) and 3.48 +/- 2.04 X 10(-3) in renal hypertensives (P less than 0.05). Intracellular K+ concentration was 94.3 +/- 3.1 mmol/l in normotensives, 94.7 +/- 3.8 mmol/l in essential hypertensives and 93.8 +/- 3.9 mmol/l in renal hypertensives. Therefore intracellular total Ca2+ concentration is increased in the red blood cells from essential hypertensives and, to a lesser extent, in the red blood cells from renal hypertensives. The use of Ca2+i:K+i ratios in red blood cells may thus be useful in assessing cellular Ca2+ content in hypertension.

Role of Calcium Ion Antagonists of the Bladder Detrusor Muscle: In Vitro and in Vivo Study

Calcium (Ca2+) ions play an important role in the contractility of the detrusor. Most of the neurotransmitters and modulators act on the detrusor through altering the final intracellular concentration of Ca2+. We studied three commonly used Ca2+ antagonists on their effect of detrusor contractility in vitro: verapamil, nifedipine and segontin. All three inhibited the detrusor-induced contraction in a dose-dependent fashion. Verapamil showed noncompetitive inhibition. Segontin showed a competitive inhibition on both phasic and tonic contractions of the detrusor strips. Nifedipine selectively inhibited the phasic contraction noncompetitively but competitively suppressed the tonic contraction. The in vivo application of verapamil on the bladder of rabbits with multiple-sclerosis-like disease showed a significant increase in bladder capacity. The study shows the possibility of the potential use of Ca2+-antagonist to suppress the problem of bladder instability.

Regulation of protein kinase C activity by natural inhibitors

The CaZ+ and phospholipid-dependent protein kinase (protein kinase C) is believed to couple extracellular messenger-induced polyphosphoinositide turnover with diverse cellular events, e.g. thrombin-induced aggregation of, and neurotransmitter release from, platelets (Berridge, 1984; Berridge & Irvine, 1984; Nishizuka, 1984). Recent reports indicate that protein kinase C may function in the negative feedback regulation of this induced polyphosphoinositide turnover (Leeb-Lundberg et al., 1985; Orellina et al., 1985; Watson & Lapetina, 1985). Walsh et al. (1984) reported the use of Ca2+ -dependent hydrophobic interaction chromatography (using phenylSepharose CL4B) in the purification of protein kinase C and a Ca2+ -binding protein of M , 21 000 (21K CaBP) from bovine brain. The 21K CaBP was subsequently purified to homogeneity using heat treatment and hydroxyapatite chromatography. However, bulk purification of this calciprotein necessitated an additional fractionation step due to contaminating proteins. DEAESephacel chromatography yielded not only the 21K CaBP but also two previously undescribed Ca2+ binding proteins of M , 18 400 and 17 000 (McDonald & Walsh, 1985~). The 17K CaBP was found to be a potent inhibitor of protein kinase C (McDonald & Walsh, (l985a,b). We now describe a modified purification procedure which facilitates the isolation of not only the 17K CaBP with increased yields but also a novel potent protein kinase C inhibitor, an M , 12000Ca2+ -binding protein (12K CaBP). Bovine brain 100 OOOg supernatant (obtained from 750 g of tissue) was fractionated on phenyl-Sepharose exactly as described b Walsh et al. (1984). We have found the yield of CaL-binding proteins to be higher using fresh rather than regenerated phenyl-Sepharose. Fractions eluted from this column by chelation of Ca2+ with EGTA were split into 21KCaBPand 17KCaBPrich pools which were heat-treated, dialysed and freezedried. After being redissolved in 20 mM-Tris/HCI (pH 7.5)/1 mM-EGTA/I mMdithiothreitol, both protein mixtures were fractionated separately on DEAE-Sephacel (McDonald & Walsh, 1985~). Detection of the eluted proteins by SDS/polyacrylamide-gradient slab-gel electrophoresis revealed the presence of not only the 21 K and 17KCaBPs, but also a 12KCaBP. The 17K and 12K CaBPs inhibited protein kinase C-catalysed phosphorylation of histone 111-S by an average of 75% at 2 . 9 ~ ~ 17K CaBP and 85% at 4.1 ~ M I ~ K CaBP. The inhibitory effects of both calciproteins were not due to interaction with substrates or cofactors nor to phosphatase activity, as neither CaBP dephosphorylated substrates which had been previously phosphorylated by protein kinase C, even after a prolonged incubation period. The inhibitory effect was shown to be a property of these proteins since trypsin digestion destroyed the inhibitory effects of both calciproteins (Fig. I ) .