A morphological study of the effects of indomethacin,flufenamate, salicylate and calcium ions on rabbit peritoneal neutrophil polymorphs.

Rabbit peritoneal neutrophil polymorphs have been examined by transmission electron microscopy to determine the effects of removing and replacing extracellular calcium. Degranulation, disruption of the cell membrane and vesiculation are all more marked in the presence of calcium than in its absence. Cooling the cells to 4 degrees has a protective effect. The addition of indomethacin, flufenamate or salicylate to a calcium-free incubating medium decreases degranulation, protects the membrane and reduces vesiculation, particularly at 4 degrees. When extracellular calcium is replaced at 37 degrees indomethacin and salicylate slightly reduce the amount of degranulation; flufenamate and salicylate significantly reduce the signs of damage. Higher concentrations of indomethacin and flufenamate cause considerable degranulation and damage. When extracellular calcium is present in the incubating medium throughout, the addition of indomethacin, flufenamate or salicylate shows varying effects. High concentrations of all these drugs, however, cause extensive degranulation and damage.     

Study on intracellular calcium localization by potassium antimonate technique in rabbit VX2 carcinoma

It is well known that a rabbit having VX2 carcinoma, which derived from virus-induced papillomas, shows hypercalcemia from the early stage of its growth. Ultrathin sections of the VX2 carcinoma were examined for the evaluation of calcium localization using a potassium antimonate technique and X-ray microanalysis. The calcium antimonate complex was shown to be most prevalent in the euchromatin and mitochondria.     

The effects of salicylate on temperature regulation in the rabbit

1. A stable pyrexia has been produced in rabbits by intravenous injection of leucocyte pyrogen followed by a continuous infusion.2. Intravenous sodium salicylate, given 4 hr after the start of pyrogen infusion, induced rapid and progressive defervescence. The rate of fall of temperature was dose dependent.3. Intravenous injection of sodium salicylate had no effect on the temperature of afebrile rabbits.4. The intraventricular injection of small doses of sodium salicylate, given 4 hr after the start of a pyrogen infusion, caused a rapid, dose-dependent defervescence, but had no significant effect on the temperature of afebrile rabbits.5. Salicylates exert at least part of their antipyretic activity through an action on the central nervous system.     

Diacylglycerols modulate human polymorphonuclear neutrophil responsiveness: effects on intracellular calcium mobilization, granule exocytosis, and superoxide anion production

The synthetic diacylglycerols (DG), sn-1,2-dihexanoylglycerol (diC6), sn-1,2-dioctanoylglycerol (diC8), and 1-oleoyl-2-acetylglycerol (OAG) stimulated the release of granule constituents from and superoxide anion (O2-) generation by human polymorphonuclear neutrophils (PMN). The DGs did not induce a rise in the cytosolic-free calcium concentration ([Ca2+]i), as monitored by the fluorescence of PMNs loaded with the fluorescent CA2+ indicator, Fura-2. DiC6, diC8, and OAG inhibited PMN degranulation elicited with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), and 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and the calcium ionophore, A23187. In contrast to their inhibitory effects on granule exocytosis, diC6, diC8 and OAG enhanced FMLP-, AGEPC-, LTB4 and A23187-stimulated O2- production. Activation of the respiratory burst with phorbol 12-myristate 13-acetate (PMA) was unaffected by the DGs. DiC8 inhibited the rise in [Ca2+]i elicited with FMLP, LTB4, and AGEPC; this effect, as well as the DG-mediated suppression of degranulation, could be reversed with the protein kinase C (PKC) inhibitor, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7). These data indicate that in addition to possessing the intrinsic capacity to activate PMNs, DG may function in a PKC-mediated autoregulatory mode to influence PMN activation in a response-specific manner by affecting certain components of receptor-coupled and receptor-independent signal transduction systems in a stimulus-specific manner.     

A morphological study of the effects of 5-hydroxytryptamine and indomethacin on rat mesenteric venules.

A method is described for preparing venules of the rat mesentery for electron microscopy after the application of 5-hydroxytryptamine (5-HT) and pretreatment with indomethacin. Local application of 5-HT caused the leakage of colloidal carbon and the emigration of leucocytes into the venule wall. 5-HT also caused endothelial cells to bulge and their nuclei to contort. It increased the number of protrusions on both the luminal and abluminal surfaces of the endothelium and increased the width of the subendothelial space, and the degree of vesiculation in the endothelial cells. Systemic treatment with indomethacin significantly decreased the amount of carbon passing through the endothelium after the local application of 5-HT, but enhanced some of the other effects of 5-HT. Thus it increased the bulging of endothelial cells and contortion of their nuclei, and further increased the number of surface protrusions and the subendothelial space. It had no effect on the emigration of leucocytes resulting from the application of 5-HT.     

A comparative study of the antipyretic effects of indomethacin and dipyrone in rats

OBJECTIVE: Compare the antipyretic effects of dipyrone and indomethacin. MATERIALS AND METHODS: Fever was induced in rats by i. v. LPS or i. c. v. interleukins (IL), prostaglandins (PG), arachidonic acid (AA), pre-formed pyrogenic factor (PFPF), tumour necrosis factor-alpha (TNF-alpha) or corticotrophin releasing hormone (CRH). Dipyrone and indomethacin were administered i.p., arginine vasopressin V1-receptor antagonist, d(CH2)5 Tyr(Me)AVP, into the ventral septal area. Cyclooxygenase (COX-1/-2) blocking activity was assessed in transfected COS-7 cells. CRH release from isolated hypothalami was determined by ELISA. RESULTS: Indomethacin or dipyrone reduced LPS, IL-1beta, IL-6 or TNF-alpha induced fever and CRH release from rat hypothalamus. Only dipyrone inhibited IL-8, PFPF or PGF2alpha fever. Only indomethacin inhibited fever induced by AA or IL-1beta, plus AA. Neither antipyretic affected fever caused by PGE2 or CRH. d(CH2)5Tyr(Me)AVP only blocked antipyresis induced by indomethacin. Dipyrone at a very high concentration (10 mM) inhibited only COX-1, while indomethacin (0.1 microM) blocked COX-1 and COX-2 in COS-7 cells. CONCLUSION: The antipyretic effect of dipyrone differs from that of indomethacin in that it does not depend on AVP release or inhibition of PG synthesis.     

Suppressive effects of nonsteroidal anti-inflammatory drugs diclofenac sodium, salicylate and indomethacin on delayed rectifier K(+)-channel currents in murine thymocytes

Lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since nonsteroidal anti-inflammatory drugs (NSAIDs), the most commonly used analgesic and antipyretic drugs, exert immunomodulatory effects, they would affect the channel currents in lymphocytes. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of diclofenac sodium, salicylate and indomethacin on the channel currents in murine thymocytes and the membrane capacitance. Diclofenac sodium and salicylate significantly suppressed the pulse-end currents of the channel. However, indomethacin suppressed both the peak and the pulse-end currents with a significant increase in the membrane capacitance. This study demonstrated for the first time that NSAIDs, such as diclofenac sodium, salicylate and indomethacin, exert inhibitory effects on thymocyte Kv1.3-channel currents. The slow inactivation pattern induced by indomethacin was thought to be associated with microscopic changes in the plasma membrane surface detected by the increase in the membrane capacitance.     

Suppressive effects of nonsteroidal anti-inflammatory drugs diclofenac sodium,salicylate and indomethacin on delayed rectifier K+-channel currents in murine thymocytes

Lymphocytes predominantly express delayed rectifier K⁺-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since nonsteroidal anti-inflammatory drugs (NSAIDs), the most commonly used analgesic and antipyretic drugs, exert immunomodulatory effects, they would affect the channel currents in lymphocytes. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of diclofenac sodium, salicylate and indomethacin on the channel currents in murine thymocytes and the membrane capacitance. Diclofenac sodium and salicylate significantly suppressed the pulse-end currents of the channel. However, indomethacin suppressed both the peak and the pulse-end currents with a significant increase in the membrane capacitance. This study demonstrated for the first time that NSAIDs, such as diclofenac sodium, salicylate and indomethacin, exert inhibitory effects on thymocyte Kv1.3-channel currents. The slow inactivation pattern induced by indomethacin was thought to be associated with microscopic changes in the plasma membrane surface detected by the increase in the membrane capacitance.     

In vitro effects of Ω-3 fatty acids on neutrophil intracellular calcium homeostasis and receptor expression for FMLP and LTB4

Diets enriched in-3 fatty acids exert antiinflammatory properties by suppressing some neutrophil (PMN) functions. Changes in cytosolic Ca²⁺ concentration, [Ca²⁺]_i, are important for PMN activation and are in part regulated by membrane Ca²⁺ ATPases. Since membrane proteins are influenced by their lipid environment, we investigated the in vitro effects of the-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the [Ca²⁺]_i of PMNs in response to f-Met-LeuPhe (FMLP), leukotriene B₄ (LTB₄), and ionomycin. The resting [Ca²⁺]_i of PMNs (in high Ca²⁺ environment) was increased after pretreatment (37C, 2 h) with DHA, but not with EPA, or the other fatty acids, oleic acid (OA), or linolenic acid (LA). The stimulated [Ca²⁺]_i by either FMLP or LTB₄ was suppressed in a high Ca²⁺ environment after pretreatment with either EPA or DHA but not with OA or LA. The stimulated [Ca²⁺]_i rise by ionomycin was augmented after pretreatment with DHA but not with EPA, OA, or LA. Pretreatment of PMNs with either EPA or DHA reduced the receptor expression for both FMLP and LTB₄. Since -3 fatty acids inhibit the expression of receptors for two activators of PMNs, FMLP and LTB₄, as well as the [Ca²⁺]_i rise in response to those two stimuli, we propose that the antiinflammatory properties of EPA and DHA may be attributed, at least in part, to alteration in membrane activation of phagocytes.     

A chromatic horizontal cell in the Xenopus retina: intracellular staining and synaptic pharmacology.

1. We identified a chromatic-type horizontal cell (C-cell) in the Xenopus retina by intracellular dye injection with Lucifer yellow or horseradish peroxidase (HRP). C-cells hyperpolarized in response to blue light and depolarized in response to red light. 2. In either photopic or mesopic states, moderate-intensity blue and red stimuli evoked responses that were inverted with respect to each other but of similar waveform and latency. In the presence of a bright green adapting field, the maximal voltage (Vmax) of the hyperpolarizing and depolarizing response component approached 30 mV; the kinetics of both waveforms were fast, and the hyperpolarizing response was followed by a small depolarizing overshoot at light OFF. Thus the blue-sensitive photoreceptor is capable of initiating large visual signals under photopic conditions when transmission from green-sensitive rods is suppressed. Under mesopic conditions (no adapting field) the kinetics of both waveforms were slower. The Vmax of the hyperpolarizing response reached 30-40 mV, whereas the cone-mediated depolarization saturated at 15 mV. 3. Both response components of the C-cell showed large receptive fields with no center-surround antagonism. 4. The C-cell perikaryon was located in the distal inner nuclear layer. It emitted four to seven long, tapering processes that ran horizontally for 90-100 microns. Two kinds of terminal dendrites, short and long, extended from the tapering processes toward the layer of photoreceptor bases. 5. Glycine (5-10 mM) completely eliminated the depolarizing response of the C-cell, whereas the hyperpolarizing component was unaffected. In contrast, gamma-aminobutyric acid (GABA; 5-10 mM) had no obvious effect on either component. 6. The C-cell light response was modified in two stages by cis-2,3-piperidine dicarboxylic acid (cis-PDA; 0.5-5 mM): first the depolarizing response disappeared; then the membrane potential hyperpolarized concomitant with a large reduction or elimination of the hyperpolarizing light response. In contrast, DL-2-amino-4-phosphonobutyric acid (APB) had no obvious effect on either response component or the membrane potential of the cell. 7. Our pharmacological findings are consistent with the view that the hyperpolarizing response in the C-cell is mediated by direct synaptic input from a blue-sensitive photoreceptor. The depolarizing response mediated by the red-sensitive cone could be explained by a direct synapse from the red cone or an indirect pathway involving luminosity (L-type) horizontal cells.     

Dual mechanism in induction of human neutrophil cytotoxicity: activation of protein kinase C and elevation in intracellular calcium.

Human neutrophils can damage the non-immunized K562 cell line when stimulated by phorbol myristate acetate (PMA). The combination of the activation of protein kinase C by phorbol and the increase of free intracellular calcium by ionophore potentiates the lytic reaction. The OKT9-immunized target cells are not able to induce by themselves the lytic response in neutrophils, but by combining the two signals, the antigenic stimulus and PMA, a high level of cytolytic response is attained. The addition of EGTA does not affect neutrophil cytotoxicity against antibody-coated targets, while it markedly reduced the lytic reaction against non-immunized targets; in contrast, the addition of EGTA together with the ionophore ionomycin completely suppresses the lysis of immunized and non-immunized targets. The treatment of neutrophils with the protein kinase C inhibitor H-7 causes a dose-related inhibition of the lytic functions that is greater on unsensitized K562. Thus the interaction of Fc receptors with immunized targets is required for reaching the maximal cytolysis. The enhanced lytic activity that occurs in the presence of immunized targets is mediated by calcium flux, as detected by using the monoclonal antibody AB8.28 which binds to FcIII receptors (FcRIII), thus supporting that both signals are involved.     

Glucocorticoid effects on rabbit fetal lung maturation in vivo: an ultrastructural morphometric study.

Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animals were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant-associated protein, SP-A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body cross-sectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissue cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell differentiation, and a concomitant increase in epithelial-mesenchymal interactions.     

Acute effects of taurine on intracellular calcium in normal and diabetic cardiac myocytes

The present study investigated the acute effects of taurine on intracellular Ca²⁺ ([Ca²⁺]_i) in normal and diabetic cardiac myocytes. [Ca²⁺]_i was monitored using fura-2 in single myocytes isolated from control or streptozotocin-treated rats and paced at frequencies between 0.33 Hz and 2.0 Hz in the absence or presence of 20 mM taurine. Increasing stimulus frequency resulted in significant increases in resting and peak [Ca²⁺]_i, and amplitude of the Ca²⁺ transient in both control and diabetic myocytes. The amplitude of the Ca²⁺ transient and the extent of its increase with increasing frequency was, however, significantly lower in the diabetic myocytes. Taurine significantly increased resting [Ca²⁺]_i, peak [Ca²⁺]_i, and the amplitude of the Ca²⁺ transient in both control and diabetic myocytes at all stimulus frequencies examined. The degree of potentiation of the Ca²⁺ transient decreased with increasing stimulus frequency in control cells but not in diabetic cells. In the absence of taurine the decay of the Ca²⁺ transient was significantly slower in diabetic than control myocytes. Taurine was without significant effect on the time course of the Ca²⁺ transient decay in control cells, however, in diabetic cells it significantly accelerated the rate of decay. The data demonstrate directly that taurine is able to increase [Ca²⁺]_i and the amplitude of the Ca²⁺ transient in both normal and diabetic cardiac myocytes. In addition several of the effects of taurine appeared to be more pronounced in diabetic than control cells. Received: 13 January 1999 / Received after revision: 12 March 1999 / Accepted: 14 April 1999     

A quick and easy method for the staining of reticulocytes.

This report presents a simplification of the conventional method for the staining of reticulocytes that is easier, faster and requires no extraneous equipment. Blood is applied directly to a spot of dried stain on a microscope slide. An identical slide is placed over the first and the blood and stain are mixed for about a minute. The slides are held apart by paper labels at each end. The slides are then slid apart to produce a smear on each slide.     

Dendritic and axonic fields of purkinje cells in developing and X-irradiated rat cerebellum. a comparative study using intracellular staining with horseradish peroxidase

Intracellular staining of cerebellar Purkinje cells with horseradish peroxidase was achieved in normal developing rats (8–13 days old), in normal adult rats and in adult rats in which the cerebellum had been degranulated by X-ray treatment. The mono- and multiple innervation of Purkinje cells by climbing fibres was electrophysiologically determined and correlated with their dendritic pattern and axonal field.In immature rats, considerable variations in dendritic arborization were observed between cells at the same age, according to their position in the vermis. In adult X-irradiated animals, a large variety of dendritic shapes was found, confirming previous anatomical data, but no obvious correlation was found between the morphology of the dendrites of Purkinje cells and their synaptic investment by climbing fibres.As regards the axonal field, the adult branching pattern of recurrent axon collaterals was almost established by postnatal day 8, except for some cells which exhibited richer recurrent collaterals. On the other hand, in X-irradiated animals, profuse plexuses were the rule and they originated either from one collateral stem, or from several collaterals, also independently of the number of afferent climbing fibres. The existence of these enlarged recurrent collateral plexuses can be explained by the persistence of an immature stage, and certainly also by collateral sprouting following the largely impaired innervation of the terminal field during development.These results emphasize the role of the cellular interactions that occur during Purkinje cell growth in the formation of both its axonal and dendritic fields.     

An in vivo study of the effects of ischaemia on uterine contraction, intracellular pH and metabolites in the rat.

There are no data concerning the functional or metabolic effects of hypoxia in vivo in smooth muscle. We have therefore used 31P-NMR spectroscopy and intra-uterine pressure measurements to examine simultaneously, in vivo, the effect of ischaemia on uterine metabolites, intracellular pH (pHi) and force. A 1-2 cm portion of uterus from day 1 postpartum anaesthetized rats was exteriorized and an NMR surface coil placed on it. A balloon catheter in the uterine lumen recorded intra-uterine pressure changes from the same area. Reversible occluders were placed around the uterine artery. Occlusion produced a decrease and then abolition of contractions, within 10 min. In four of five animals contraction was abolished within 2 min. Upon reperfusion force was rapidly restored (1 min), in all preparations. The mean level of force was significantly above control (pre-occlusion) 20-30 min after reperfusion. The NMR data showed a significant fall in [ATP] (28%) and [phosphocreatine] (34%) during occlusion. Inorganic phosphate doubled in concentration during this period. Metabolites recovered slowly upon reperfusion, taking 20-30 min to return to pre-occlusion levels. The mean pHi fell from 7.32 to 7.00 upon occlusion and was rapidly reversed upon reperfusion. The changes in pHi closely correlated with the changes in uterine force. Decreases of pHi of a similar magnitude in vitro have previously been shown to abolish contractions; thus it is suggested that during ischaemia in vivo the depression of contraction is caused by the large fall in pHi.     

Possible reflection of intracellular calcium binding in the divergent pattern of relaxation in rat and rabbit uterus

The mechanical responses to potassium of the rabbit and rat uterus were different. The force of a potassium-induced contracture showed a rapid decline in the rabbit uterus, whereas it was fully maintained for a long period in the rat uterus. The increase in 45Ca influx following exposure to high KCl was substantial and similar (0.3 mmol kg-1) in both rat and rabbit uterus. Calcium uptake in post nuclear supernatant (PNS) from the rat myometrium was significantly lower than that from the rabbit myometrium in both the rate and capacity of uptake. This uptake was reduced to about 10% in the presence of sodium azide in PNS from both rat and rabbit myometria. Both the rate and capacity of calcium uptake by mitochondria isolated from PNS of rat myometrium were considerably lower than that by rabbit myometrial mitochondria. Microsomes isolated from rat and rabbit PNS had very low capacity for calcium uptake compared with mitochondria. No difference was found in the rate or the capacity of uptake between the rat and the rabbit myometrial microsomes. The data support the argument for a role of mitochondria in myometrial relaxation and further provide evidence for a considerable species difference in calcium uptake by myometrial mitochondria.     

The effects of inhibition of the sarcolemmal Ca-ATPase on systolic calcium fluxes and intracellular calcium concentration in rat ventricular myocytes

 The effects of carboxyeosin, an inhibitor of the sarcolemmal Ca-ATPase, were studied on intracellular Ca and membrane currents in isolated rat ventricular myocytes. In the absence of carboxyeosin, 150-ms-duration depolarizing pulses from –40 to 0 mV resulted in an L-type Ca current on depolarization and a Na-Ca exchange ”tail” current on repolarization. The calculated entry of Ca on the L-type current was 1.3 times greater than the efflux via the Na-Ca exchange. The addition of carboxyeosin (20 μM) resulted in either an increase of the Na-Ca exchange current or a decrease of the L-type Ca current such that the Ca entry and efflux were exactly equal. These results suggest that, under control conditions, a carboxyeosin-sensitive Ca-ATPase contributes about 24% of the total Ca efflux from the cell and, therefore, that the sarcolemmal Ca-ATPase has a significant role in regulation of sarcolemmal Ca fluxes. Received: 9 December 1998 / Received after revision: 1 February 1999 / Accepted: 2 February 1999