Ca Ionophore A23187 Research Articles

Enhanced calcium-sensitivity of erythrocytes in hypertension--calcium-induced changes of erythrocyte osmotic fragility in essential hypertension.

To investigate the Ca-sensitivity of the erythrocyte membrane in hypertension, the changes of the osmotic fragility of erythrocytes by Ca-loading and the effects of Ca-channel blockers or calmodulin-antagonist were observed in patients with essential hypertension. Erythrocytes were obtained from untreated patients with essential hypertension and age-matched normotensive subjects. Treatment of erythrocytes with Ca-ionophore A23187 and Ca in bathing medium caused the reduction of the osmotic fragility of erythrocytes dose-dependently on Ca-concentration. The degree in alteration of the osmotic fragility of erythrocytes was greater in essential hypertension than that in normotensive controls. In addition, Ca-induced changes of erythrocyte osmotic fragility was inversely correlated with the plasma renin activity in essential hypertension. In the presence of Ca-antagonists (verapamil, diltiazem) or calmodulin-antagonist (trifluoperazine), the reduction of the osmotic fragility of erythrocytes by Ca-loading was inhibited, and the differences of the osmotic fragility of erythrocytes between the hypertensives and the normotensive controls were abolished by these drugs. These results suggest that the greater changes of the osmotic fragility of the erythrocytes by Ca-loading in essential hypertension might be due to the abnormality of Ca-handling of the cell membranes causing an increase in the intracellular Ca concentration, contributing at least partially to the pathogenesis of hypertension.

New Class of 5-Lipoxygenase Inhibitors: Correlation Between Inhibition of LTB4 Production and Chemiluminescence of Human Polymorphonuclear Granulocytes.

The effect of several series of compounds on the biosynthesis of leukotriene B4 in human polymorphonuclear granulocytes (PMNL) was measured after stimulation of the cells by the Ca(2+) ionophore A23187. Phenylhydrazone derivatives and some pyrrole derivatives strongly inhibited 5-lipoxygenase activity at 10 µM. In contrast, 4-aminopyrrolopyrimidines and 4-oxopyrrolopyrimidmes did not exhibit any effect. The compounds examined for inhibition of 5-lipoxygenase were also tested in a chemiluminescence assay for potential effects on the emission of photons as a measure of activated oxygen species generated by the stimulated granulocytes. There was a good correlation between lipoxygenase inhibition and suppression of chemiluminescence; however, some derivatives with no inhibitory activity against lipoxygenase still reduced chemiluminescence, which suggests that an alternative inhibitory mechanism of chemiluminescence must be present. Cyclooxygenase inhibitors such as aspirin did not suppress chemiluminescence of granulocytes. It is, therefore, unlikely that cyclooxygenase is required in the chemiluminescence reaction. In contrast, LTB4 seems to play an important role in the zymosan-induced chemiluminescence of PMNL.

Localization of 5-lipoxygenase within human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) stimulated with the Ca-ionophore A23187 or opsonized zymosan not only release marker enzymes of specific granules but secrete 5-lipoxygenase activity as well. In the presence of BSA cells incubated with [ 14C]AA were able to synthetize 5-HPETE but failed to produce 5-HETE, LTB 4, and its ω-oxidation metabolites. Subcellular fractionation studies by differential and isopycnic equilibrium density centrifugation demonstrated main lipoxygenase activity in particulate fractions consisting of specific granules, but not in cytosolic fractions. These results suggest the association of 5-lipoxygenase with specific granules. 5-lipoxygenase released from the cells upon appropriate stimulation reached its peak activity after 10 min and was then rapidly inactivated. It appears that the intermediate 5-HPETE may be generated extracellularly but has to re-enter the intracellular space for further metabolization.

Activated human eosinophils generate SRS-A leukotrienes following IgG-dependent stimulation.

Eosinophils, a class of granular leukocytes, are prominent in many inflammatory processes, particularly in asthma, certain allergic diseases and during infections with helminthic parasites. Following incubation with the Ca ionophore A23187 (refs 1-4) (a non-physiological agent which circumvents membrane calcium-gating mechanisms), eosinophils generate large amounts of sulphidopeptide leukotrienes, potent inducers of smooth muscle constriction and mucus production. These are now known to represent the activity previously termed 'slow-reacting substance of anaphylaxis' (SRS-A) but attempts to identify a physiological stimulus for SRS-A production by eosinophils have so far been unsuccessful. The cells contain recognized receptors for IgG (Fc) and it is known that they adhere to, and can be activated by, contact with the surface of large organisms such as helminthic larvae. We show here that eosinophils, particularly when activated, produce sulphidopeptide leukotrienes after contact with large particles coated with IgG.

Potassium movements associated with amino acid and sugar transport in enterocytes isolated from rabbit jejunum.

Active transport of amino acids and the sugar, alpha-methyl-D-glucoside (alpha-MG) caused an increase in the rate of K efflux from isolated rabbit enterocytes. These effects were inhibited by apamin (5 X 10(-7) M), quinidine (10(-3) M), Ba (5 X 10(-3) M) and trifluoperazine (5 X 10(-5) M) but not by the loop diuretic furosemide (10(-4) M). None of these drugs affected the basal rate of K efflux. The stimulatory effects of amino acids or alpha-MG on K efflux are too great to be explained in terms of an increase in the electrical driving force across the plasma membrane of these cells and a change in membrane permeability is envisaged. An apparent Ca-dependent K permeability in isolated enterocytes can be demonstrated using the Ca ionophore A23187. The effect of the ionophore on K efflux is abolished by apamin or Ba. It is proposed that Ca-dependent K channels mediate the sugar and amino acid induced increases of K efflux. Under control conditions there is a decrease in intracellular K concentration during accumulation of alanine or alpha-MG. Ba by itself does not alter K concentration but it did produce a marked increase when used in conjunction with alanine or alpha-MG. The accumulation of alpha-MG was inhibited in the presence of Ba. This is consistent with an interference with the driving force for sugar accumulation. It is suggested that the increase in K permeability described has a role in both maintaining ion homoeostasis during Na-coupled transport and contributing to the driving force for sugar and amino acid absorption.

1041 REDUCED PRODUCTION OF HISTAMINE RELEASE ENHANCING FACTOR (HREF) BY CORD BLOOD MONONUCLEAR CELLS

The liberation of histamine and other inflammatory mediators constitute a major component of host defense. HREF is a unique cytokine produced by PHA stim. mononuclear cells (MC) that may be an important mediator of inflammatory responses. Incubation of granulocytes with HREF enhanced subsequent basophil histamine release (BHR) induced by anti-IgE, f-Met peptide, and the Ca ionophore A23187 (124±43%, 139±40%, and 66±29% enhancement). HREF production was dependent on the number of cells cultured, PHA cone., and duration of culture. Conditioned media containing HREF was active after heat (70°C × 30 min), absorption by activated lymphocytes, and thyroglobulin removal of PHA. HREF produced by cord blood MC of 19 newborns produced significantly less enhancement of anti-IgE induced BHR than adult controls (39±30% vs 53±31% enhancement, p<.05). This was not related to impaired PHA responsiveness. Both stim. and unstim. cord MC incorporated greater 3H thymidine than controls (p <.001). T cell enriched (TCE) populations produced greater HREF than unfrac. or T-depleted (TD) populations (TCE 73±32% enhancement; unfrac. 42±20%; TD 43±25%; N=13, p <0.05). Functional cellular immune deficiency in newborns and deficient lymphokine synthesis by cord lymphocytes have been previously reported. HREF is a potentially important determinant of histamine release and reduced capacity for its production may contribute to the relative immunodeficiency of newborns.

The involvement of cytoskeleton in the regulation of transbilayer movement of phospholipids in human blood platelets

Activation of human platelets by different activators resulted in a different extent of degradation of the cytoskeletal proteins actin-binding protein and myosin, as well as of the non-cytoskeletal protein P235. The highest extent of proteolysis was observed with Ca-ionophore A23187 and decreased on going from A23187 > collagen plus thrombin > collagen > thrombin = ADP. The same order of potency has been found previously ((1983) Biochim. Biophys. Acta 736, 57–66) for the ability of platelet activators to induce exposure of aminophospholipids in the outer leaflet of the platelet plasma membrane, and to stimulate platelets to become procoagulant. Degradation of cytoskeletal proteins as a result of platelet stimulation by collagen plus thrombin was prevented in the presence of dibutyryl cAMP or EDTA but not in the presence of aspirin. This also runs in parallel with platelet procoagulant activity. Moreover, platelets from a patient with a partial deficiency in platelet procoagulant activity revelaed a diminished extent of degradation of cytoskeletal proteins upon platelet stimulation with collagen plus thrombin. It is concluded that alterations in cytoskeletal organization upon platelet stimulation may lead to alterations in the orientation of (amino)phospholipids in the plasma membrane, and may therefore play a regulatory role in the expression of platelet procoagulant activity.

708 BILE SALTS FUNCTION AS CALCIUM IONOPHORES

It has been previously demonstrated that bile salts (BS) induce calcium (Ca) dependent hemolysis which is directly proportional to Ca influx into red blood cells. To examine how BS's might facilitate Ca movement across membranes, we investigated Ca uptake by unilamellar phospholipid vesicles (PV)prepared from soy phosphatidates. PV's were incubated with 5.0 mM 45Ca either alone (control) or with added 750 μM BS - taurodeoxycholate (TDC) or tauroeholate (TC) (Hepes buffer, pH 7.0, 37°C). After 10 min, Ca uptake was 1.88 ± .08 nmoles by the control PV's, 3.28 ± .04 nmoles by the PV's with TC, and 5.06 ± .03 nmoles by the PV's with TDC. 45Ca uptake was also compared to sodium-22 (22Na) or rubidium-86 (86Rb) uptake in the presence of TDC. After 5 min of incubation, TDC-associated Ca uptake was nearly 2 times greater than those of either Na or Rb. Finally, PV's were exposed either to 25 nM valinomycin (V) which induced an electrochemical gradient across PV membranes, or to 25 nM nigericin (N) which induced a pH gradient. TDC without V or N increased Ca uptake 1.67 ± .08 times over controls. Exposure to V increased TDC-associated Ca uptake 1.45 ± .05 times, and exposure to N increased it 1.90 ± .03 times. Conclusions: (1) BS's induce cation uptake by PV's that is more specific for Ca than for Na or Rb; (2) as was true of red blood cells, TDC induces greater Ca uptake than TC; (3) pH gradients promote greater BS-indueed Ca uptake than electrochemical gradients. Hypothesis: The mechanism of BS-induced Ca uptake may be similar to that of Ca ionophore A23187 whose activity is similarly promoted by pH gradients.

884 CALCIUM-INDUCED ECHINOCYTOSIS(E)IN HUMAN RED BLOOD CELLS FROM NEONATES(N) AND ADULTS(A)

Echinocytosis in RBC's from A & N were quantitated by l)photo-microscopy; 2)frequency histograms of the 6 stages of disc-sphere transformation, scored from 0-5; and 3)morphologic index (MI), the mean score for 200 cells. One hr. after adding Ca ionophore A23187 to cells in isotonic media with 100 μM free Ca and 90 mM Kex, the MI was 1.7 ± 0.5 (S.D., 5 donors) for N and 4.2 ± 0.2(6) for A (P<0.0005). Under equivalent conditions, net 45Ca uptake, cell deformabi1ity as measured by the Ektocytometer, and ATP decline were comparable for A & N. In contrast to Ca, the potencies of 2 echinocytogenic drugs(phloridzin & dinitrophenol) were similar for A & N. While Ca causes a progressive shape change with heterogeneous cell populations, the 2 drugs caused an immediate and stable E with homogeneous cell populations. E caused by the 2 drugs and by Ca were all reversible by the cupping agent, chlorpromazine. Preservation of ATP by 0.5 mM vanadate showed low ATP was not required for Ca-induced E. Vanadate also abolished the inhibition of Ca-induced E in cells from N. In cells treated with Ca and A23187, Mg suppressed E without vanadate, but had no effect with vanadate. These observations are consistent with a model of Ca-induced E whereby Ca-activated phospholipase(s) is counterbalanced by a Mg and vanadate-sensitive repair process, with more active repair in red cells of N as compared with A. The results define a new difference between red cells from A & N, and suggest that cells from N are a useful variant for understanding the mechanism of Ca-induced E. (Supported in part by AHA/Upstate New York Chapter).

Calcium-activated potassium channels in isolated presynaptic nerve terminals from rat brain.

86Rb efflux was examined in isolated presynaptic nerve terminals (synaptosomes) from rat brain in a study designed to assess K permeability (PK) changes sensitive to alterations in internal Ca activity. Rb efflux from 86Rb-loaded synaptosomes into nominally Ca-free physiological saline (PSS) containing 5 mM-K was about 0.3-0.4%/s. Raising extracellular K concentration [( K]o), to depolarize the synaptosomes, stimulated the 86Rb efflux. Addition of Ca to the 5 mM-K PSS had no effect, but Ca did further stimulate 86Rb efflux into K-rich solutions. The effect of Ca was graded, with apparent half-maximal activation, KA approximately equal to 0.5 mM-Ca. These data fit the view that, during depolarization, Ca enters the terminals through voltage-regulated Ca channels, and that the rise in intracellular Ca concentration opens certain (Ca-activated) K channels. The Ca-dependent stimulation of 86Rb efflux was greatest during the initial seconds of incubation (component CT), and then declined to a much lower rate (component CS). Much of this change in rate could be attributed to inactivation of voltage-regulated Ca channels and reduced entry of Ca. The Ca-dependent increase in 86Rb efflux was completely inhibited by 100 microM-La. In the presence of Ca, but not in its absence, the Ca ionophore A23187 stimulated 86Rb efflux both in 5 and 100 mM-K PSS. The effect in 100 mM-K was quantitatively greater, perhaps because of the increased outward driving force on Rb in depolarized synaptosomes. When synaptosomes were suspended in media containing the voltage-sensitive fluorescent dye, DiS-C3-(5) (1,1'-dipentyl-2,2'-thiocarbocyanine), the addition of Ca+ A23187 decreased the fluorescence intensity (= synaptosome hyperpolarization) when the media contained 5 mM-K but not 100 mM-K. This implies that in the presence of Ca + A23187, PK was increased, and the membrane potential moved closer to the K equilibrium potential, EK. Quinine sulphate, a blocker of Ca-activated K channels, reduced the Ca-stimulated 86Rb efflux with high affinity (apparent half-maximal inhibition, KI approximately equal to 1 microM). Tetraethylammonium chloride, another agent known to block Ca-activated K channels, was also a relatively potent inhibitor of Ca-stimulated 86Rb efflux (KI approximately equal to 0.2 mM). The K-channel blocker, 4-aminopyridine, partially inhibited Ca-stimulated 86Rb efflux at concentrations below 0.5 mM, but stimulated this efflux at concentrations greater than or equal to 1 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Alteration of surfactant function due to protein leakage: special interaction with fibrin monomer

In isolated rabbit lungs standardized amounts of edema were induced. Stimulation with the Ca ionophore A23187, leukotriene C4, Pseudomonas aeruginosa cytotoxin and human serum (activated complement) all resulted in protein leakage into the alveolar space with no change in the total phospholipid content. The pressure-volume characteristics of the lungs and the characteristics of the lavage surfactant (Wilhelmy balance) were markedly altered, correlating to the lavage protein content. The surfactant alterations were reproduced by addition of perfusion fluid protein to control surfactant in vitro. All changes were far less expressed or even missing in isolated lungs developing the same amount of edema due to omittance of proteins from the perfusion liquid. Different proteins added to control surfactant in the Wilhelmy balance showed a marked rank order of potency in interfering with surfactant function: immunoglobulins G and M and elastin less than albumin less than fibrinogen less than fibrin monomers. The fibrin monomer effect was reproduced by addition of thrombin to a surfactant fibrinogen mixture and was partly reversed by subsequent incubation with plasmin. In conclusion, high-permeability edema induced by different means results in alterations of lung mechanics and surface activity of lavaged surfactant, presumably due to protein surfactant interaction. Among different proteins, fibrin monomers are especially effective in interfering with surfactant function.

Formation of leukotrienes and prostaglandins in human lung tissue, measured by HPLC and RIA

Human parenchymal lung tissue, obtained from adults after lobectomy on account of tumours, was chopped and labelled with 14C-arachidonic acid in the presence of glutathione and Ca-ionophore A23187. The formation of leukotrienes (LTs) and other lipoxygenase products was measured by high-performance liquid chromatography (HPLC). The quantities of both the unlabelled and radioactive compounds were determined. Prostaglandins (PGs) were measured by radioimmunoassay (RIA) after separation by HPLC. 3H labelled LTs and PGs were used as markers and standards for recovery calculations. In the identification of arachidonic acid (AA) products by means of 3H labelled compounds, a decrease in retention times, compared with the identical 14C labelled compounds and the unlabelled compounds measured by absorption at 280 nm, was observed. This may be a source of errors. Relatively large amounts of LTB 4 and smaller ones of LTC 4 and LTD 4/LTE 4 were formed. These amounts are given in the table below. A difference occurred in the specific activities of these compounds. This may indicate that the substances are not formed from the same AA pool. Recently it has been shown that human alveolar macrophages produce LTB 4, and that allergen challenge of chopped human lung tissue elicits contraction that correlates with the release of both LTs (C 4, D 4 and E 4) and PGs (1). Godard et al. have shown that the eosinophil count in bronchoalveolar lavage fluid from allergic asthmatics was increased and that stimulation of these macrophages by Zymosan leads to a two fold increase in the release of PGs (2). In further studies the relationship between LTs/PGs in alveolar macrophages and lung tissue of asthmatics will be investigated. LTs B 4, C 4, D 4 and E 4 were gifts of Dr. J. Rokach (Merck Frosst, Canada) and H LTs were obtained from Amersham, U.K.

Spontaneous [3H]noradrenaline release from the main pulmonary artery of the rabbit induced by sodium-pump inhibition.

Inhibition of Na pump either by ouabain (10(-4) M) or by K removal increased the [3H]noradrenaline [( 3H]NA) release from the isolated main pulmonary artery of the rabbit in the presence of neuronal (cocaine, 3 X 10(-5) M) and extraneuronal (corticosterone, 5 X 10(-5) M) uptake blockers. The ouabain-evoked [3H]NA release began after a delay of about 30 min and peaked after 66 min of ouabain application. Both times were shortened by omission of K from the external medium. About 90% of ouabain-evoked [3H]NA release proved to be external Ca concentration ([Ca]o) dependent and the peak effect was delayed by about 80 min in Ca-free (+ 1 mM EGTA) solution. In the presence of external Ca (2.5 mM) the [3H]NA-releasing effect of 'K-free' treatment was much less pronounced than that of 10(-4) M ouabain, the initial delay in transmitter release was shorter (10-15 min) and the peak effect developed earlier (at 42 min). On readmission of K the [3H]NA release recovered quickly to the original value. Ca removal did not antagonize the transmitter release observed in K-free solution, but the peak release was delayed by about 90 min. A low concentration of ouabain (10(-5) M) failed to produce transmitter release in the presence of normal external K, but markedly increased the release in K-free solution. The release was much bigger than the sum of their separate effects, and the rate of rise was faster than when 10(-4) M ouabain was applied in normal solution. Excess Ca (7.5; 15 mM) inhibited the [3H]NA release observed in K-free solution. 7.5 mM-Ca also delayed the transmitter-releasing action of 10(-4) M ouabain, an effect antagonized by omission of K from the external medium. The mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10(-5) M) significantly increased the [3H]NA release in Ca-free, 1 mM EGTA-containing solution, and enhanced the effects of ouabain (10(-4) M). The Ca ionophore A23187 (10(-5) M) also significantly increased the [3H]NA release in the absence of external Ca and in the presence of 1 mM EGTA. Again, in the presence of A23187 the effects of 10(-4) M ouabain in releasing neurotransmitter were enhanced. When CCCP and A23187 were applied together in Ca-free, EGTA solution the [3H]NA releasing action of ouabain was still apparent. Veratridine (10(-4) M) enhanced the transmitter release in the absence of external Ca in a tetrodotoxin (TTX)-sensitive manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of effect of dibutyryl cyclic adenosine 3',5'-monophosphate on canine renal renin release.

We evaluated the effect of intrarenal arterial infusion of dibutyryl cyclic AMP on renal renin release. Dibutyryl cyclic AMP (300 micrograms/kg X min) produced significant and reversible increases in renin secretion rate, renal blood flow and urinary Na+ excretion. In the non-filtering kidneys, dibutyryl cyclic AMP increased renin secretion rate and renal blood flow. In indomethacin-treated dogs, dibutyryl cyclic AMP also produced significant and reversible increases in renin secretion rate and renal blood flow but had no effect on Na+ excretion. During infusion of dibutyryl cyclic AMP, infusion of the Ca ionophore A23187 failed to inhibit renin release. These findings suggest that the effect of dibutyryl cyclic AMP on renin release is not mediated by prostaglandins or intracellular Ca and does not involve the macula densa. We conclude that dibutyryl cyclic AMP has a direct effect on the juxtaglomerular cells.

Direct demonstration of increased intracellular concentration of free calcium as measured by quin-2 in stimulated rat peritoneal mast cell.

Rat mast cells, passively sensitized with monoclonal mouse IgE antibody, were stimulated with multi-valent antigen, and an increase in cytoplasmic Ca2+ was determined by using the fluorescent probe quin-2. The increase in quin-2 fluorescence reached maximum within 20 sec after the antigen challenge and then gradually declined. A substantial increase in quin-2 fluorescence was observed in the presence of EGTA, indicating that bridging of cell-bound IgE antibody molecules by antigen induced not only Ca2+ influx but also mobilization of intracellular Ca2+. Phosphatidylserine added to the medium enhanced both the antigen-induced histamine release and the increase in quin-2 fluorescence and slowed the rate at which the quin-2 signal returned to basal levels. Both the antigen-induced increase in quin-2 fluorescence and histamine release were inhibited by pretreatment of mast cells with inhibitors of methyltransferases, theophylline, or cromoglycate. It was also found that methyltransferase inhibitors and theophylline inhibited not only stimulus-dependent calcium influx but also release of bound calcium from intracellular stores. Other secretagogues, compound 48/80 (1 microgram/ml) and Ca ionophore A23187 (0.1 microM), induced a rapid increase in cytoplasmic Ca2+ in rat mast cells and subsequent histamine release. In contrast, the cocarcinogenic compound phorbol 12-myristate 13-acetate caused histamine release without increasing the quin-2 fluorescence.

A voltage-gated hydrogen ion current in the oocyte membrane of the axolotl, Ambystoma.

Membrane currents in the immature oocyte of the urodele amphibian Ambystoma were studied using the two-micro-electrode voltage-clamp technique. A current carried by H ions (IH) constituted the major portion of outward current activated by depolarizations from the resting voltage (about -60 mV). Net inward current was not observed at this developmental stage. The reversal potential for IH measured from tail currents obtained in two step voltage-clamp experiments shifted by 54 mV per unit change in external pH between pH 6.9 and 8.4. The reversal potential at pH 7.4 was not affected by changes in external K or Cl concentrations. A small change in reversal potential was observed with removal of external Na. The amplitude of IH was not affected by removal of external Ca (Mg or Sr substitution). Ca ionophore A23187 shifted the current-voltage relation towards negative voltages. Activation of IH did not appear to depend on Ca influx. The instantaneous current-voltage relation for IH, measured from tail currents at approximately equal internal and external H ion concentrations, was linear between -40 and +30 mV. The steady-state conductance-voltage relationship was sigmoidal with membrane voltage, and, at pH 7.4, was one-half maximum at about +15 mV (V1/2). The time courses of activation and deactivation were proportional to 1-exp (-t/tau). A plot of time constant (tau) against voltage was bell-shaped, with a maximum near V1/2. These results suggested that the activation of IH is voltage dependent. Increases in the external H ion concentration shifted the conductance-voltage and time constant-voltage relations in parallel towards positive voltages. The magnitude of these shifts showed a lower saturation near pH 9. Low concentrations of external Cd (10-300 microM) reduced current amplitude by shifting the current-voltage relation in the positive direction. Cd also reduced the limiting slope conductance. These effects were partially reversible.

Rabbit proximal colon: a distinct transport epithelium.

Rabbit proximal colon (PC), examined in vitro, exhibits transport characteristics distinct from distal colon. Unlike distal colon, PC does not demonstrate amiloride-inhibitable electrogenic Na absorption. Additionally, neither amphotericin nor "stimulatory" anions induce Na absorption in PC. Electrical measurements and radioisotopic flux studies under short-circuit conditions indicate that PC is a moderately leaky epithelium with a conductance (9.0 +/- 0.2 mS) midway between ileum and distal colon; under basal conditions PC has Na and Cl transport rates near zero, and 5.5 microM epinephrine (Epi) stimulates electrically silent Na-Cl coupled absorption. The mechanism of this cotransport was investigated further: Cl substitution with either sulfate or gluconate did not substantially alter Epi-enhanced Na absorption. The Epi stimulation of Cl absorption in a Na-free solution was diminished. Amiloride, 10(-3) M, inhibited Epi-enhanced Na absorption by approximately 50%. The effects of cAMP-mediated (theophylline or 8-bromo-cAMP) and Ca-mediated (ionophore A23187) secretory stimuli were examined. In the basal state, none of these agents had a consistent effect on ion transport. However, after stimulation of Na and Cl absorption by Epi, both of the cAMP-related secretagogues had a marked antiabsorptive effect on Na and Cl transport. The antiabsorptive effects of Ca ionophore A23187 were less marked. These results suggest that rabbit proximal colon a) does not share the transport properties characteristic of distal colon, b) possesses an Epi-sensitive, Na-Cl-coupled absorptive pathway, and c) responds to secretory stimuli in an antiabsorptive manner rather than by electrogenic secretion.

MEMBRANE LIPID FLUIDITY AND FILTERABILITY OF HUMAN RBC'S FROM ADULTS &amp; NEWBORNS

Red Blood Cells (RBC's) of human newborns have a number of different characteristics from those of adults. In order to increase understanding of these differences, membrane lipid fluidity (MLF) as indicated by the fluorescence polarization of diphenylhexatriene (DPH) and filterability(F) as a gross measure of cell deformability were compared in the presence and absence of calcium. DPH fluorescence polarization in fresh intact cells from adults was 0.282±.011(S.D.n=14), significantly less than 0.327±0.010(S.D.,n=7) in newborns. While quantitative estimates from DPH fluorescence polarization are subject to uncertainties, the results suggest decreased fluidity in the hydrophobic core of red cell membranes of newborns. This is consistent with the greater proportion of saturated fatty acids in RBC membranes from newborns.Treating RBC's from adults with 1μM Ca ionophore A23187 at 5 mMCaCl2, decreases F by 49±12%(S.D. ,n=4). In contrast MLF of RBC's is unchanged with 1μM A23187 and 5 mMCaCl2. RBC's from newborns exhibit decreased F at O mM and 5 mMCa in comparison with RBC's from adults. Experiments with Ca demonstrate conditions under which F and MLF are independent and uncorrelated parameters. When Ca induces echinocytosis and decreases F it does so without causing bulk changes in the hydrophobic core of the membrane. Ca must exert its effects either on the polar lipid headgroups or on the cytoskeletal proteins.

Thiol-dependent and non-thiol-dependent stimulations of insulin release.

The effects of reduced glutathione (GSH) and diamide (an oxidant of GSH) on insulin release induced by glucose, glyceraldehyde, leucine, tolbutamide, glibenclamide, Ca-ionophore A-23187, isoprenaline, and dbcAMP were studied using isolated rat pancreatic islets. In the absence or presence of low glucose (5.6 mM) neither GSH (0.1 mM) nor diamide (0.1 mM) affected insulin release. Insulinotropic action of glucose (11.1 mM) and glyceraldehyde (11.1 mM), and that of tolbutamide (0.1 mg/ml) and leucine (10 mM) both in the presence of 11.1 mM glucose was further augmented by GSH and inhibited by diamide. GSH (0.05-1 mM) and diamide (0.1 mM) failed to affect the insulin secretion evoked by glibenclamide (5 micrograms/ml) + glucose (11.1 mM), Ca-ionophore A-23187 (50 micrograms/ml) + glucose (5.6 mM), isoprenaline (1 microM) + glucose (5.6 mM), and db-cAMP (1 mM) + glucose (5.6 mM). The data suggest that the insulin-releasing capacity of glucose, glyceraldehyde, tolbutamide, and leucine depends on the redox state of islet thiols, whereas the insulin-releasing effect of glibenclamide, Ca-ionophore, isoprenaline, and db-cAMP does not. The possibility that thiol dependency is associated with those compounds increasing Ca uptake but not with compounds acting as Ca-ionophores or only by increasing intracellular cAMP is discussed.

Sodium/calcium exchange in smooth-muscle microsomal fractions.

The existence of Na+ -dependent Ca2+ transport was investigated in microsomal fractions from the longitudinal smooth muscle of the guinea-pig ileum and from the rat aorta, and its activity was compared with that of the plasmalemmal ATP-dependent Ca2+ pump previously identified in these preparations. The rate of Ca2+ release from plasmalemmal vesicles previously loaded with Ca2+ through the ATP-dependent Ca2+ pump was transiently faster in the presence of 150 mM-NaCl in the medium than in the presence of 150 mM-KCl or -LiCl or 300 mM-sucrose. Na+-loaded vesicles took up Ca2+ when an outwardly directed Na+ gradient was formed across the membrane. The Ca ionophore A23187 induced a rapid release of 85% of the sequestered Ca2+, whereas only 15% was displaced by La3+. Ca2+ accumulated by the Na+-induced Ca2+ transport was released by the addition of NaCl, but not KCl, to the medium. Ca2+ uptake in Na+-loaded vesicles was inhibited in the presence of increasing NaCl concentration in the medium. Half-maximum inhibition was observed with 28 mM-NaCl. Data fitted the Hill equation, with a Hill coefficient (h) of 1.9. Na+-induced Ca2+ uptake was a saturable function of Ca2+ concentration in the medium. Half-maximum activity was obtained with 18 microM-Ca2+ in intestinal-smooth-muscle microsomal fraction and with 50 microM-Ca2+ in aortic microsomal fraction. The results suggest that in these membrane preparations a transmembrane movement of Ca2+ can be driven by a Na+ gradient. However, the Na+-induced Ca2+ transport had a lower capacity, a lower affinity and a slower rate than the ATP-dependent Ca2+ pump.