Dissociation of Ca2+ accumulation from protein release in calcium paradox: effect of barium

Experiments were undertaken to establish whether Ba2+ can substitute for Ca2+ in maintaining the structural and functional integrity of the glycocalyx. Adult rat hearts were perfused with Ca2+-free buffer at 37 degrees C, with and without added Ba2+. Ten minutes later Ba2+-free, Ca-containing medium was reintroduced. Hearts that had been perfused with Ca2+-free medium exhibited a distorted glycocalyx. On reperfusion with Ca2+-containing medium these hearts released protein (P less than 0.001), gained Ca2+ (P less than 0.001), and depleted their tissue stores of ATP (P less than 0.001) and CP (P less than 0.001). Hearts that had been perfused with Ca2+-free, Ba2+-containing medium retained an apparently intact glycocalyx. On reperfusion with Ca2+-containing. Ba2+-free medium they gained Ca2+ but did not lose protein. These results suggest that Ba2+ only partially replaces Ca2+ in maintaining the integrity of the cell surface. It is also concluded that the absence of protein release does not necessarily exclude the occurrence of severe changes in ionic permeability after cell injury.     

Effect of nitroprusside on smooth muscle and adrenergic nerve terminals in isolated blood vessels

Experiments were designed to assess the mode of action of nitroprusside on isolated blood vessels and its relative potency on venous and arterial smooth muscle. Strips from dog blood vessels were mounted in an organ bath for isometric tension recording. Sodium nitroprusside (10(-5) M) depressed the contraction of saphenous vein strips caused by electric stimulation, tyramine, K+, Ba++, norepinephrine and acetylcholine. The depression of the norepinephrine-induced contractions also occurred in a Ca++- free medium and when Ca++ influx was inhibited by verapamil. Nitroprusside reduced the frequency of the spontaneous contractions of strips of portal-mesenteric veins. It depressed the contraction caused by norepinephrine in tibial artery strips more than in saphenous vein strips. Saphenous vein strips were incubated with (3H)norepinephrine and mounted for superfusion and isometric tension recording. Sodium nitroprusside (10(-5) M) had no effect on the basal efflux of 3H compounds. During electric stimulation, it did not change the output of (3H)norepinephrine but increased the outflow of deaminated and O-methylated metabolites. Thus sodium nitroprusside 1) has a direct effect on the smooth muscle cells which is independent of Ca++ influx, 2) depresses contractions of different types of vascular smooth muscle and 3) does not inhibit the release of norepinephrine from the nerve endings.     

Alteration by phosphatidyl serine of tension responses and 45Ca distribution in aortic smooth muscle

The effects of phosphatidyl serine (PS) on 45Ca distribution, 45Ca movements and contractions were examined in rabbit aortic smooth muscle. Contractile responses to submaximal concentrations of norepinephrine and histamine were potentiated by prior exposure to PS, but equivalent responses to potassium were unaffected. Addition of PS to the incubation solution decreased 45Ca uptake; exposure of aortic strips to PS during washout of either 45Ca or promethium (147Pm) resulted in maintained increases in efflux. These PS-induced alterations in net loss of 45Ca or 147Pm can be attributed to a decreased membrane reuptake and/or rebinding. However, the presence of PS during the washout significantly reduced the increases in 45Ca efflux rate elicited with either 0.05 mM concentrations of Ca++ or ethylenediamine tetraacetic acid. Thus, in rabbit aortic smooth muscle, exogenous PS can alter the availability and/or exchangeability of a membrane-bound Ca++ fraction. By specifically increasing the affinity for Ca++ at relevant membrane sites or stores. PS may enhance the ability of vascular smooth muscle to respond to stimulatory agents that mobilize Ca++ from these sites and, in this manner, potentiate contractile responses.     

Clinical utility of Lens culinaris agglutinin-reactive alpha-fetoprotein in small hepatocellular carcinoma: special reference to imaging diagnosis

We sought to determine whether insulin/insulin-like growth factor-1 (IGF-1) and an insulin-sensitizing agent, troglitazone, have additive vasodilatory effects and the possible involvement of intracellular Ca2+ ([Ca2+]i) and/or glucose utilization in these effects. Contractile responses to norepinephrine (NE) and potassium chloride (KCl), as well as relaxation to endothelium-dependent (acetylcholine [Ach]) and -independent (sodium nitroprusside [NaNP]) agents, were examined in rat tail artery rings in the presence of insulin/IGF-1 and/or troglitazone. Endothelium-intact tail artery rings stretched to 1 g tension were preincubated with troglitazone (3 micromol/L) and/or insulin/IGF-1 (100 nmol/L) prior to addition of graded doses of NE and KCI. A 90-minute exposure to troglitazone attenuated the maximal contraction to graded doses of NE and KCI (P<.0001). Incubation in glucose-free medium decreased the responses only to NE; troglitazone further attenuated the NE-induced contraction (P = .001). In submaximally precontracted endothelium-intact rings, troglitazone increased the relaxation both to NaNP (P<.0001) and to Ach (P = .001). Contraction experiments in depolarizing KCI (25 mmol/L) or Ca2+ -free buffer showed that troglitazone and insulin have a similar Ca2+ dependency. In conclusion, troglitazone, like insulin/IGF-1, attenuates responses to vasoactive agonists through a Ca2+ -dependent mechanism that may require the presence of glucose but is independent of insulin action and nitric oxide (NO) production.     

Characterization of postsynaptic alpha-1 and alpha-2 adrenoceptors in the feline saphenous and femoral veins [corrected

Experiments were designed to characterize the effects mediated by alpha-1 and alpha-2 adrenoceptors in saphenous and femoral veins of the cat. Ring segments of saphenous and femoral veins were mounted for isometric tension recording in modified Krebs-bicarbonate solution, gassed with 95% O2-5% CO2 and maintained at 37 degrees C. Norepinephrine (a mixed alpha 1 and alpha 2 agonist), phenylephrine (a preferential alpha 1 agonist) and clonidine (a preferential alpha 2 agonist) caused dose (concentration)-dependent contractions in saphenous and femoral veins. The maximal contractions produced by clonidine were significantly less than those produced by norepinephrine or phenylephrine in both veins. However, threshold dose and EC50 values indicated that clonidine was more potent than norepinephrine and phenylephrine. Contractile responses to these agonists were attenuated when the veins were pretreated with alpha 1-or alpha 2-adrenoceptor antagonists, prazosin and yohimbine, respectively. The contractile responses to norepinephrine and tyramine were inhibited to a greater extent by yohimbine than by prazosin in both saphenous and femoral veins, suggesting that norepinephrine released from perivascular nerve terminals activates preferentially postsynaptic alpha 2-adrenoceptors. Further examination of alpha-adrenoceptor subtypes was achieved by comparing pA2 values of prazosin and yohimbine from Arunlakshana and Schild plots. Chronic sympathetic denervation by removing lumbar sympathetic chain significantly reduced the contractile responses evoked by tyramine. Denervation did not significantly affect the concentration-response curve to phenylephrine but significantly augmented the contractile responses evoked by clonidine in both veins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of relaxant action of nicardipine, a new Ca++-antagonist, on isolated dog cerebral and mesenteric arteries

In helically-cut strips of cerebral and mesenteric arteries contracted with prostaglandin (PG) F2 alpha, carbocyclic thromboxane A2 (cTxA2) or K+, the addition of nicardipine caused a dose-related relaxation. Nicardipine-induced relaxation was greater in cerebral than in mesenteric arteries when contracted with PGF2 alpha and cTxA2, but did not appreciably differ in the arteries contracted with K+. Cerebral arteries contracted with hemolysate and PGF2 alpha relaxed in response to nicardipine to a similar extent. The contractile response to PGF2 alpha was attenuated by pretreatment with nicardipine, the attenuation being greater in cerebral than in mesenteric arteries. Ca++-induced contractions in cerebral and mesenteric arteries previously exposed to Ca++-free media and depolarized by excess K+ were attenuated by nicardipine to a similar extent. PGF2 alpha-induced contractions of cerebral arteries exposed to Ca++-free media were attenuated by nicardipine, whereas those of mesenteric arteries were unaffected. Attenuations by nicardipine of the Ca++-induced contraction in PGF2 alpha-treated cerebral arteries were greater than those seen in mesenteric arteries. It may be concluded that nicardipine produces a greater relaxation of cerebral arteries than mesenteric arteries, possibly due to a greater inhibition of the Ca++-influx and to a decrease in the release of Ca++ from intracellular storage sites in cerebral arteries. As far as the concentrations used are concerned, nicardipine appears to attenuate the inward movement of Ca++ across cell membrane in mesenteric arterial smooth muscle, but not the release of intracellularly stored Ca++.     

Hypothalamic Na+ and Ca++ ions and temperature set-point: new mechanisms of action of a central or peripheral thermal challenge and intrahypothalamic 5-HT, NE, PGEi and pyrogen

The effects of changes in ambient and central temperature, amines, PGEu and pyrogen were investigated with respect to the mechanism of Na+-Ca++ ratio in the posterior hypothalamus of the unrestrained cat. Guide tubes were implanted bilaterally above the posterior hypothalamic area of 23 cats so as to accommodate push-pull cannulae. After a Na+ or Ca++ sensitive site was identified by perfusion at 50 mul/min of an artificial CSF containing 10.4 mM excess Ca++ ions or 13.6 mM excess Na+ ions, several types of experiments were undertaken with the results summarized as follows: if the cat was exposed to a cold or warm environmental temperature as the posterior hypothalamus was perfused with excess cation, the typical hypothermia was produced by Ca++ and hyperthermia by Na+ ions. However, if the cat was exposed to peripheral cooling or warming 30 min prior to the perfusion, the fall or rise produced by Ca++ or Na+ was attenuated or prevented. In other experiments, 1.0 muCi 45Ca++ was injected in the ion sensitive site in the posterior hypothalamus to label stores of the cation. Raising of ambient temperature caused a retention of 45Ca++ in this hypothalmic area, whereas a cold environmental temperature enhanced the efflux of 45Ca++ at the same perfusion site. The magnitude of change in 45Ca++ efflux depended upon the intensity of the thermal challenge. Similarly, warming of the anterior hypothalmic, preoptic area by means of implanted thermodes caused an immediate diminution in 45Ca++ efflux in the posterior hypothalamus, whereas cooling of this anterior region augmented the extrusion of 45Ca++ ions from the posterior area. When substances which produce a temperature change were applied to the same thermosensitive zone, the direction of shift in 45Ca++ flux in the posterior area corresponded to the signal for heat production or heat loss. That is, the microinjection of 5-HT, PGE1 or Salmonella typhosa into the anterior hypothalamus enhanced the efflux of 45Ca++ in the posterior hypothalamus as hyperthermia developed, whereas a similar microinjection of norepinephrine reduced the 45Ca++ output from the same sites. Finally, locally anesthetizing the cells of the anterior hypothalamus by the nerve blocker, procaine, prevented the cold and heat-induced 45Ca++ eflux and retention, respectively. These results suggest that if the Na+-Ca++ ratio in the posterior hypothalamus establishes and maintains the set-point for body temperature of 37 degrees -38 degrees C, the mechanism of lability of Ca++ through changes in binding characteristics, transport, or metabolism of the cation serves two purposes: (1) the active defense of the set-point temperature through gradations in ion shifts; and (2) the upward or downward change in set-point value, pathological or normal, triggered by virtue of impulses relayed from the anterior hypothalamus.     

Role of endothelin receptors, calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1-8 Hz), under different conditions, at 37 degrees C and during cooling (30 degrees C). 2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30 degrees C, but not at 37 degrees C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43 +/- 7% to 190 +/- 25% for 8 Hz). 3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ETA receptors BQ-123 (10 microM) but not by the antagonist for endothelin ETB receptors BQ-788 (10 microM). The agonist for endothelin ETB receptors IRL-1620 (0.1 microM) did not modify the contraction to electrical stimulation. 4. The blocker of L-type Ca2+ channels verapamil (10 microM l-1) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca(2+)-channels NiCl2 (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30 degrees C. 5. Inhibition of nitric oxide synthesis with NG-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30 degrees C more than at 37 degrees C (for 8 Hz, this increment was 297 +/- 118% at 30 degrees C, and 66 +/- 15% at 37 degrees C). Endothelium removal increased the contraction to electrical stimulation at 30 degrees C (about 91% for 8 Hz) but not at 37 degrees C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30 degrees C. 6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca2+ entry after activation of endothelin ETA receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.     

The mechanism of contraction of rat aorta to various agonists

To elucidate the mechanism of contraction of the smooth muscle of the rat's aorta, its response to norepinephrine (NE), 5-hydroxytrypatamine (5-HT) and potassium chloride (KCI) was determined before and after pretreatment with reserpine and beta-diethylamionethyl 2-2-diphenylpropyl acetate (SKF 525-A). Unlike the rabbit's aorta, contraction of the rat's aorta induced by NE was inhibited by SKF 525-A. After the induction of maximal contraction, SKF 525-A induced a graded rapid relaxation after KCl, less so after 5-HT, and least after NE. Pretreatment with reserpine failed to induce supersensitivity to NE. After incubation in a Ca++-free or Na+ and Ca++-free Krebs solution, the rat's aorta failed to contract even on the addition of Ca++ or NE.     

Effects of sevoflurane or halothane on contractile responses of isolated canine basilar artery

Although volatile anesthetic is known as a cerebral vasodilator, its mechanism is not clear. The purpose of this study was to investigate effects of sevoflurane or halothane on contractions induced by high K+ and serotonin in the isolated canine basilar artery. Cylindrical segments of canine basilar artery were placed in Krebs solution oxygenated with 95% O2 and 5% CO2 at 37 degrees C. They were then constricted with cumulative administration of 10 to 60 mM KCl, or with 10(-9) to 10(-6) M serotonin and exposed to either sevoflurane or halothane at concentration of 1.0 and 2.0 MAC. Halothane and sevoflurane at concentration of 1.0 and 2.0 MAC decreased contractile responses evoked by KCl to a similar degree. The attenuation by either of the two anesthetics at concentration of 2.0 MAC were equivalent to the inhibitions by diltiazem 2 x 10(-7) M. Contractile responses to serotonin above 3 x 10(-7) M were depressed by halothane 1.0 MAC, but not by sevoflurane 1.0 MAC. Sevoflurane and halothane at concentration of 2.0 MAC decreased contractile responses evoked by serotonin at concentrations above 3 x 10(-8) M and 10(-8) M. Removal of the endothelium did not alter the response of the basilar artery contracted by serotonin to either anesthetic. These findings suggest that sevoflurane and halothane depress the voltage-dependent Ca2+ channels due to decreases of contractile responses to high K+. Our results also demonstrate that sevoflurane is a less potent vasodilator of the basilar artery contracted by serotonin than halothane.     

Influence of lubeluzole on voltage-sensitive Ca++ channels in isolated rat neurons

Lubeluzole is neuroprotective in a photochemical stroke model, whereas the (R)-enantiomer of the same molecule is not [De Ryck M, Keersmaekers R, Duytschaever H, Claes C, Clincke G, Janssen M and Van Reet G (1996) J Pharmacol Exp Ther 279:748-758]. We investigated the effects of lubeluzole and the (R)-enantiomer on voltage-sensitive Ca++ channels of isolated rat dorsal root ganglion cells, using whole-cell voltage-clamp, with Ba++ as the charge carrier. Both compounds blocked the low-voltage-activated Ba++ current (iLVA or T current) with an IC50 value of 1.2 microM. Lubeluzole and the (R)-enantiomer also blocked the high-voltage-activated calcium channel current (iHVA), with IC50 values of 2.6 and 3.5 microM, respectively, and accelerated the apparent inactivation of iHVA. This acceleration was more pronounced with lubeluzole than with the (R)-enantiomer at 3 and 10 microM. Both compounds produced a clear tonic block of iLVA and iHVA, even in the absence of previous stimulation. Lubeluzole and the (R)-enantiomer induced a negative shift of the inactivation curve of iLVA and showed down the recovery from inactivation. This resulted in a stronger inhibition of iLVA at more depolarized conditioning potentials and higher stimulation frequencies. The block of iHVA was voltage and frequency dependent. Lubeluzole and the (R)-enantiomer also blocked iHVA in isolated rat superior cervical ganglion cells and cerebellar Purkinje cells. The Ca++ channel-blocking properties of lubeluzole may contribute to its neuroprotective effect. However, the small difference between the two enantiomers in inhibition of Ca++ channel currents does not explain the stereospecificity of the neuroprotective properties of lubeluzole in vitro and in vivo.     

Influences of Mg++ and reserpine on calcium fluxes and sensitivity of the rat aorta

Rat aorta became supersensitive and subsensitive to noradrenaline (NA), respectively, in Mg++-free and 3.6 mM Mg++ media reserpine treatment reduced the sensitivity in normal or high (3.6 mM) Mg++ media but had no effect in Mg++-free medium. Incubation of aortae in Mg++-free medium enchanced 45Ca++ uptake and efflux in rat aorta, whereas, it reduced 45Ca++ efflux without a change in 45Ca++ uptake by rabbit aorta. Reserpine pretreatment enhanced 45Ca++ efflux from rat aorta without a changed in 45Ca++ uptake in normal Mg++-free medium. Unlike in rat aorta, reserpine enhanced 45Ca++ uptake by and reduced efflux from rabbit aorta in Mg++-free medium but not in normal medium. These results suggest that the failure of reserpine to induce supersensitivity in rat aorta to NA may be due to poor capacity of the muscle to retain Ca++ and probably also due to an enhanced antagonism of Mg++ on Ca++ movements.     

Temperature-sensitive effects of potassium channel openers on isolated guinea pig myocardium and aorta

The effects of K+ channel openers, NIP-121, cromakalim, and pinacidil, on isolated myocardium and aorta were investigated at two different temperatures, 23 degrees C and 37 degrees C. In right ventricular myocardium, NIP-121 shortened the action-potential duration with little influence on other action-potential parameters at 37 degrees C, but not at 23 degrees C. In whole-cell clamped ventricular myocytes, NIP-121 induced a glibenclamide-sensitive outward current at 37 degrees C but not at 23 degrees C. No difference in tissue adenosine triphosphate (ATP) concentration was detected between ventricular myocardia incubated at 37 degrees C and at 23 degrees C. In aortic preparations precontracted with norepinephrine, NIP-121, cromakalim, and pinacidil produced endothelium-independent relaxation at 37 degrees C, which was antagonized by glibenclamide. The vasorelaxant effects were greatly reduced at 23 degrees C. Thus we demonstrated that the effects of K+ channel openers on the myocardium and vascular smooth muscle are temperature sensitive.     

Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America

We studied the role of Ca++ and protein kinase C (PKC) in alpha-1A adrenergic receptor (AR)-mediated activation of mitogen-activated protein kinase pathways in PC12 cells. In PC12 cells stably transfected with the human alpha-1A AR, norepinephrine (NE) strongly activated both extracellular signal regulated kinases (ERKs) and c-jun-NH2-terminal kinases (JNK). Ten nanomolar thapsigargin (TG) increased cytoplasmic Ca++ at least as much as NE but did not activate ERKs or JNK. Higher concentrations of TG caused a small activation of ERKs but not JNK. Emptying [Ca++]i stores by pretreatment with TG prevented the NE-stimulated increase in [Ca++]i but not ERK or JNK activation. The Ca++ chelator bis(2-aminophenoxy)ethane-N-N-N'-N'-tetraacetate (BAPTA) dose dependently abolished NE-stimulated Ca++ responses but not ERK or JNK activation. NE increased tyrosine phosphorylation of Pyk2, and this response was neither blocked by BAPTA nor mimicked by TG. The phorbol ester tumor promoting agent (TPA) caused a dose-dependent activation of ERKs that was potentiated by 10 nM TG. TPA caused only a small activation of JNK relative to that caused by NE, which was not affected by TG. The potent PKC inhibitor bisindolylmaleimide I dose dependently inhibited ERK and JNK activation by TPA, but not NE. ATP and UTP activated similar mitogen-activated protein kinase responses through endogenous P2Y2 receptors, and these responses were not blocked by BAPTA or bisindolylmaleimide I, suggesting that these results may be generalizable to other Gq/11-coupled receptors. The results suggest that Ca++ release and PKC activation are neither necessary nor sufficient for alpha-1A AR-mediated activation of mitogenic responses in PC12 cells.     

Cooling effects on nitric oxide production by rabbit ear and femoral arteries during cholinergic stimulation

1. Ear (cutaneous) and femoral (deep) arteries from rabbit were perfused at 37 degrees C and 24 degrees C (cooling) and the production of nitrite, as an index of nitric oxide production, was measured under basal conditions and cholinergic stimulation. 2. In both types of arteries under control conditions, the basal production of nitrite was similar at 24 degrees C and 37 degrees C. Compared with the control conditions, the basal production of nitrite was significantly lower in ear and femoral arteries without endothelium or treated with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) but it was similar in those treated with atropine (10(-6) M). 3. At 37 degrees C, methacholine (10(-7)-10(-5) M) increased the production of nitrite in ear and femoral arteries; this increase persisted during 30-60 min and was practically abolished by L-NAME (10(-4) M), atropine (10(-6) M), or removal of the endothelium. In ear arteries the total nitrite production to activation with methacholine was higher at 24 degrees C than at 37 degrees C due to this production persisted increased for a longer period (> 150 min), whereas in femoral arteries it was lower at 24 degrees C than at 37 degrees C. 4. It is suggested that: (a) the endothelium of rabbit ear and femoral arteries produce nitric oxide under basal conditions, which is increased by cholinergic stimulation, and (b) cooling potentiates endothelial nitric oxide production to cholinergic stimulation in cutaneous arteries, whereas it inhibits this production in deep arteries.     

Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43 degrees C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca2+ concentration ([Ca2+]i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca2+]i from 52.5 +/- 9.5 nM (mean +/- S.D., n = 18) to 171.0 +/- 15.6 nM in cells between 15 and 25 microns in diameter. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. The Na(+)-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Effects of bis(2-chloroethyl)sulfide on ATP receptor-mediated responses of the rat vas deferens: possible relationship to cytotoxicity

Extracellular ATP is a broad-spectrum cytotoxic agent that produces effects via cell surface P2 purinoceptors. The ligand-gated P2X purinoceptor subtype has very high sequence homology with the RP-2 gene, which encodes for apoptosis. The P2X RNA found in rat vas deferens is expressed preferentially by apoptotic thymocytes. P2X purinoceptor-mediated phasic (twitch) motor responses of the isolated rat vas deferens to neurogenic or exogenous ATP were rapidly, specifically and irreversibly potentiated by bis(2-chloroethyl)sulfide (HD 10-100 microM). Both untreated and HD-potentiated neurogenic responses were Ca++ dependent, blocked in the absence of Ca++ plus 0.1 mM EGTA, by the neuronal Ca++ channel blocker omega-conotoxin-MVIIC (3 microM), by the P2 purinoceptor antagonist suramin (100 microM) and by tetrodotoxin (100 nM). HD also potentiated the effects of ATP on isolated guinea pig taenia caecum, where the nucleotide acts at G protein-coupled P2Y purinoceptor subtypes to cause relaxation. HD failed to inhibit the metabolism of ATP by ecto-ATPase in vas deferens or to cause the release of endogenous ATP. Potentiation of the twitch response to electric field stimulation by HD was attenuated or eliminated in tissues excised from rats previously challenged with topically applied HD, suggesting that HD absorbed into the systemic circulation had already effected maximal potentiation of ATP responses before in vitro testing. The physiological consequences of HD-induced potentiation of the extracellular actions of ATP are discussed in relation to apoptosis and necrosis.     

Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells. A comparative study

We have measured the [Ca2+] in the endoplasmic reticulum ([Ca2+]er) of intact HeLa cells at both 22 degrees C and 37 degrees C using endoplamsic reticulum-targeted, low Ca2+ affinity aequorin reconstituted with coelenterazine n. Aequorin consumption was much slower at 22 degrees C, and this allowed performing a much longer study of the dynamics of [Ca2+]er. The steady-state [Ca2+]er (500-600 microM) was not modified by the temperature, although both the rates of pumping and leak were decreased at 22 degrees C. The behavior of both [Ca2+]er and cytoplasmic [Ca2+] ([Ca2+]c) after the addition of increasing concentrations of agonists and/or Ca2+-ATPase inhibitors, or following incubation in Ca2+-free medium were compared. We show that agonists induce a fast but relatively small decrease in [Ca2+]er, which is enough to produce a sharp increase in [Ca2+]c. Termination of Ca2+ release is controlled by feedback inhibition of the inositol 1,4,5-trisphosphate receptors by [Ca2+]c, a mechanism that appears to be designed to release the minimum amount of Ca2+ necessary to produced the required [Ca2+]c signal. We also show that Ca2+ release is inhibited progressively when [Ca2+]er decreases below a threshold of about 150 microM, even in the absence of Ca2+ pumping or -Ca2+-c increase. This effect is consistent with a regulation of the inositol 1,4,5-trisphosphate-gated channels by [Ca2+]er.     

Attenuated mutants of Staphylococcus aureus with two temperature-sensitive lesions: isolation and characterization

The feasibility of constructing attenuated mutants of Staphylococcus aureus with two temperature-sensitive (ts) lesions for ultimate development of a live-attenuated strain was investigated. Temperature-sensitive S. aureus strain G/1/2, which grows well at 31 degrees C but does not replicate at 37 degrees C, was subjected to chemical mutagenesis. After two enrichment cycles, fifteen mutants able to grow at 25 degrees C but unable to grow at 31 degrees C, were identified. Growth curves with temperature shifts from 25 to 31 degrees C, and from 31 to 37 degrees C confirmed that these were mutants with two lesions (dts), each with a different cut-off temperature. The reversion frequency of mutant G/1/2 at 37 degrees C was 2 x 10(-6) whereas those of several dts mutants were much lower (dts7: 7 x 10(-9) and dts12: 1 x 10(-9)). There was no increase in ts mutation reversion rate in response to prolonged incubation at 37 degrees C. The data support the further development of these mutants for use as a stable attenuated vaccine.     

Effects of temperature on norepinephrine-induced sinus acceleration and overdrive suppression in the isolated dog atrium

Using an isolated, blood-perfused atrium preparation, the effects of temperature on SA nodal pacemaker activity were investigated in 9 preparations. The PP interval decreased as temperature was raised. Regular sinus rhythm and atrial contraction were maintained above approximately 26 degrees C. Below 26 degrees C, sinus depolarization still showed a regular rate, although atrial contractions had ceased. At about 24 degrees C, atrial rhythm became irregular. Below 20 degrees C, atrial depolarization disappeared. Chronotropic responses to norepinephrine were suppressed at decreased temperatures, not only with respect to maximum PP shortening but also to the threshold dose for inducing sinus acceleration. Overdrive suppression was not influenced significantly by decreasing temperature. These results indicate that a temperature decrease causes suppression of SA nodal pacemaker activity, although the SA node continues to function regularly until about 25 degrees C.