Relationship between 45Ca movements, different calcium components and responses to acetylcholine and potassium in tracheal smooth muscle

Correlations between tension responses elicited with acetylcholine (ACh) and high K+ and corresponding alterations in Ca++ mobilization were obtained in rabbit and canine tracheal smooth muscle. Removal of Ca++ or preincubation with D-600 (50 microM) inhibited responses to K+ (50 or 80 mM) and low ACh (89 nM) and had only a small effect on responses to high ACh (8.9 microM). Conversely, solutions containing Sr++ instead of Ca++ inhibited responses to both concentrations of ACh to a greater degree than were those to K+. Washout of slow component 45Ca into a O-Ca solution was more rapid in rabbit trachea than reported previously for rabbit aorta. Washout of tracheal smooth muscle into an 80.8 mM La -substituted solution at 0.5 degrees C removed superficial (La -accessible) 45Ca and blocked both 45Ca uptake and most 45Ca efflux. D-600, which had no significant effect on control 45Ca uptake in rabbit aortic smooth muscle, decreased 45Ca uptake by 33% in rabbit tracheal smooth muscle. The uptake of 45Ca from the Ca++ binding sites with low affinity for Ca++ was increased by 80 mM K+, 50 mM K+ or 8.9 microM ACh, and the accumulation of Ca++ from the Ca++ binding sites with high affinity for 45Ca was inhibited by Sr++. The stronger effect of either Ca++ removal or D-600 on responses to K+ and the correspondingly greater effect of Sr++ on responses to ACh indicate that different Ca++ stores are present in tracheal smooth muscle. These Ca++ components appear to be qualitatively similar to those present in aortic smooth muscle but they differ quantitatively and are not as readily dissociated as are aortic Ca++ components.     

Specific actions of gallium on norepinephrine-induced tension and associated 45Ca movements in rabbit aortic smooth muscle

Gallium ion (Ga) dose-dependently (60-360 microM) inhibited contractions induced by norepinephrine (NE, 1 microM) in rabbit aortic (and media intimal) strips, but did not affect contractions elicited with high K+ (80 mM) solution. The initial phasic portion of the NE-induced response was either unaffected or only slightly (less than 10%) reduced, but the tonic portion of the response was inhibited completely by higher concentrations (greater than or equal to 300 microM) of Ga . In resting muscles, the equilibrated (90 min) 45Ca uptake was not altered by Ga (360 microM). Also, 45Ca efflux from either high- or low-affinity Ca++ binding sites was unaltered by Ga . The effects of Ga (360 microM) on 45Ca retained after a subsequent 60-min washout at 0.5 degrees C in an isosmotic (80.8 mM) La solution were also examined. High affinity La -resistant 45Ca released by NE (1 microM) was not altered by Ga . Under conditions favoring low affinity Ca++ uptake, 45Ca retention in control and K+-treated muscles was not changed by Ga , but the additional incremental 45Ca uptake associated with NE (in the presence of high K+) was blocked. Thus, Ga appears to have a selective inhibitory action on NE-associated 45Ca uptake without affecting either resting and high K+-induced 45Ca uptake or that 45Ca fraction released by NE. This action may result from a selective blockade by Ga of receptor-linked Ca++ channels in rabbit aortic smooth muscle.     

Block of 45Ca uptake into synaptosomes by methylmercury: Ca++- and Na+-dependence

Block of Ca++ influx into isolated nerve terminals by the neurotoxicant methylmercury (MeHg) was studied for its dependence on extracellular Ca++ and Na+. Depolarization-independent entry of 45Ca++ was determined in rat forebrain synaptosomes incubated in 5 mM K+ solution. 45Ca++ uptake was similarly measured after 1 ("fast" phase) or 10 sec ("total") of elevated K+ (41.25 mM)-induced depolarization or after 10 sec of elevated K+-induced depolarization after synaptosomes had been predepolarized for 10 sec in Ca++- and MeHg-free solutions ("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 microM and greater significantly reduced synaptosomal 45Ca++ uptake measured during 1 or 10 sec of incubation. In K+-depolarized synaptosomes, the estimated IC50 for block of total, fast and slow 45Ca++ uptake by MeHg is 75 microM; 250 microM MeHg reduced uptake by approximately 90%. The reversibility of block by extracellular Ca++ was tested by increasing the extracellular Ca++ concentration from 0.01 to 1.15 mM. When compared to control, 50 microM MeHg reduced total uptake of 45Ca++ by greater than or equal to 70% and reduced fast uptake by 20 to 60% at all concentrations of extracellular Ca++ tested. At Ca++ concentrations of 0.01 to 0.15 mM, MeHg (50 microM) reduced slow uptake by 75 to 90%, but did not affect slow uptake at higher Ca++ concentrations (greater than or equal to 0.30 mM). When the dependence of block of 45Ca++ uptake on extracellular Na+ was tested, equivalent levels of inhibition were caused by MeHg (25 microM) for fast uptake by synaptosomes in Na+-containing and Na+-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of K+ channel-dependent as well as -independent components of pinacidil-induced vasodilation

The mechanisms of pinacidil-induced direct vasodilation were studied in vitro in RMA and RAO. In RMA, pinacidil produced dose-dependent relaxations of norepinephrine (5 microM)-induced contractions with an IC50 of 0.2 microM. This component of pinacidil relaxation appeared to be dependent on K+ conductance because pretreatment with tetraethylammonium (10 mM), Ba++ (0.5 mM), glyburide (1 microM) and 20 mM K+ all caused a rightward shift of the pinacidil dose-response curve (DRC) and a corresponding increase in the pinacidil IC50. However, additional relaxation effects of pinacidil were still evident in the presence of various K+ channel blockers. Pinacidil also showed a relaxation DRC under the condition of 80 mM K+ contraction in both RMA and RAO with IC50 values of 27 and 50 microM, respectively. Pinacidil could also produce maximal relaxation in RMA and RAO remained unaffected in 145 mM K+ (zero Na+) depolarizing solution suggesting a lack of dependence on Na(+)-Ca++ exchange mechanism for this action of pinacidil. Studies using 1 or 3 min pulse labeling with 45Ca showed an absence of an inhibitory effect of pinacidil (at 50 and 100 microM) on unidirectional 45Ca influx stimulated by high-K+. Net 45Ca uptake studies showed that pinacidil inhibited high-K+ stimulated 45Ca uptake at 100 but not at 50 microM. Ryanodine (10-100 microM) was used as a tool to investigate the role of sarcoplasmic reticulum (SR) in this action of pinacidil. Under the condition in which ryanodine (10-100 microM) treatment was found to cause the SR to be nonfunctional, pinacidil relaxation DRC remained unaltered, suggesting a lack of a stimulatory effect of pinacidil on SR Ca++ accumulation. These data thus show that the K+ channel-independent effect of pinacidil does not involve to any significant degree an effect of pinacidil on plasmalemmal voltage-sensitive Ca++ channels, SR Ca++ stores, Na(+)-Ca++ exchange or membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between response to norepinephrine and removal of 45Ca from high-affinity binding sites by extracellular EDTA in rabbit aortic smooth muscle.

Divalent ion chelators (EDTA, EGTA) were found to remove 45Ca from high-affinity binding sites in isolated rabbit aortic smooth muscle in a concentration-dependent manner. Measurement of 14C-labeled EDTA uptake (10 to 60-min incubation periods) yielded tissue/medium ratios equivalent to the extracellular space [14C]-sucrose tissue/medium ratio). Addition of high EDTA concentrations (0.5--1.5 mM) elicits large but reversible increases in 45Ca efflux, rapidly removes virtually all 45Ca from muscles previously incubated with 45Ca for as short a time as 7 min (filling of extracellular and superficial sites) or as long as 180 min (more complete equilibration of 45Ca), and blocks the norepinephrine-induced contractile response (this response is only partially inhibited by 0.05 mM EDTA, a concentration too low to remove most of the 45Ca present). Furthermore, phosphatidyl serine, a compound known to decrease exchangeability of 45Ca in this tissue, inhibits the EDTA-induced increase in 45Ca loss. Thus, those slowly depleted Ca++ stores that are important for the contractile action of norepinephrine are removed by EDTA even though this chelator is confined to the extracellular space. Possibly, release or removal of high-affinity Ca++ is regulated by EDTA-accessible Ca++ bound at relatively superficial membrane sites.     

Displacement of the surface membrane bound calcium of the skeletal muscle fibers of the frog: effects of lanthanum and opioids

Three opioids, viz. methadone, morphine and meperidine, were tested separately for their effects on the rate of 45Ca++ efflux from frog toe muscles. The experimental procedures were designed to provide reliable measurements of the displacement of 45Ca++ from surface binding sites using the thin and light toe muscle. La (2 mM) was used to test the ability of the experimental procedure to detect the displacement of calcium bound superficially to the muscle fiber membrane. La not only enhances the rate of efflux of 45Ca++ from surface membrane superficial stores but also inhibits the efflux of intracellular calcium. Of the opioids tested, only methadone (10(-4) or 10(-3) M) caused a slight but significant increase in the rate of 45Ca++ efflux from the surface bound (i.e., the La -displaceable) store of calcium of the muscle. The "calcium-displacement" effect of methadone was much weaker than that seen with La . Morphine (10(-4) or 10(-3) M) or meperidine (10(-4) or 10(-3) M) did not show any such effect on the bound 45Ca++ desaturation rate even under experimental conditions modified to enable the detection of the displacement of even small amounts of 45Ca++ from superficial binding sites on the muscles.     

Alterations in 28Mg distribution and movements in rabbit aortic smooth muscle

The distribution and transmembrane fluxes of 28Mg were examined in the isolated media-intimal layer of rabbit aorta. Accumulation of 28Mg was slow and not complete after a 3-hr incubation. The major portion of the cellular Mg++ is not exchangeable. The 28Mg efflux rate was increased by 1.5 mM nonradioactive Mg++ after a time lag of 5 to 10 min; this increase was blocked reversibly by decreasing bathing solution temperature to 4 degrees C. A rapid and sustained increase in 28Mg efflux rate was elicited with added EDTA. Accumulation of 28Mg by rabbit aorta was increased more than 5-fold by substituting sucrose for NaCl in the bathing solution. Q10 values obtained for Mg++ accumulation in rabbit aorta incubated at different temperatures either in normal solution or low-Na+ solution ranged from 1.3 to 2.0. Uptake of 28Mg was inhibited substantially by 60 mM added K+, 1.5 or 15.0 mM La , 7 mM neomycin or 1 microgram/ml of antimycin A. These ions and drugs did not significantly increase 28Mg efflux when added during the slow component phase of the washout. Thus, the major portion of slowly accumulated Mg++ appears to be stored and exchanged intracellularly. The transmembrane movements of Mg++ depend upon simple diffusion, Mg++-Mg++ exchange and a transport process that is increased when Na+ is decreased. The EDTA-induced increase in 28Mg efflux rate may result from nonspecific membrane permeability increases, whereas ions and drugs decrease cellular Mg++ content by reducing uptake rather than increasing loss.     

Inositol 1,4,5-trisphosphate may regulate rat brain Cai++ by inhibiting membrane bound Na(+)-Ca++ exchanger.

The role of inositol 1,4,5-trisphosphate (1,4,5-IP3) in regulating cytosolic Ca++ by stimulating Ca++ release from intracellular organelles is well established. However, other modes of intracellular Ca++ regulation by 1,4,5-IP3 have not been determined. To determine if 1,4,5-IP3 may regulate cell cytosolic Ca++ by acting on plasma membrane bound Na(+)-Ca++ exchanger, we investigated Ca++ transport in synaptosomes using 45Ca++ as tracer. In the presence of either an inhibitor of voltage gated Na+ channels (tetrodotoxin) or the K+ ionophore (valinomycin), Ca++ uptake was significantly inhibited (P less than 0.05) by 1,4,5-IP3 in a concentration dependent manner, with half-maximal inhibition occurring at submicromolar concentrations between 10(-9) M and 10(-10) M 1,4,5-IP3. Similarly, Ca++ efflux by the exchanger was significantly inhibited 40% by 1,4,5-IP3. The inhibitory effect of 1,4,5-IP3 on the Na(+)-Ca++ exchanger was observed in the presence of Ca++ channel blockers, and in vesicles pretreated with caffeine to deplete the 1,4,5-IP3-sensitive stores of Ca++. These results suggest that during signal transduction in brain, 1,4,5-IP3 may increase cytosolic [Ca++] in part by inhibiting the Na(+)-Ca++ exchanger and thus, Ca++ efflux from cell.     

Dependency of cyclic AMP-induced insulin release on intra- and extracellular calcium in rat islets of Langerhans.

Calcium and cyclic AMP are important in the stimulation of insulin release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) raises islet cAMP levels and causes insulin release at nonstimulatory glucose concentrations. In isolated rat pancreatic islets maintained for 2 d in tissue culture, the effects of IBMX on insulin release and 45Ca++ fluxes were compared with those of glucose. During perifusion at 1 mM Ca++, 16.7 mM glucose elicited a biphasic insulin release, whereas 1 mM IBMX in the presence of 2.8 mM glucose caused a monophasic release. Decreasing extracellular Ca++ a monophasic release. Decreasing extracellular Ca++ to 0.1 mM during stimulation reduced the glucose effect by 80% but did not alter IBMX-induced release. Both glucose and IBMX stimulated 45Ca++ uptake (5 min). 45Ca++ efflux from islets loaded to isotopic equilibrium (46 h) was increased by both substances. IBMX stimulation of insulin release, of 45Ca++ uptake, and of efflux were not inhibited by blockade of Ca++ uptake with verapamil, whereas glucose-induced changes are known to be inhibited. Because IBMX-induced insulin release remained unaltered at 0.1 mM calcium, it appears that cAMP-stimulated insulin release is controlled by intracellular calcium. This is supported by perifusion experiments at 0 Ca++ when IBMX stimulated net Ca++ efflux. In addition, glucose-stimulated insulin release was potentiated by IBMX. These results suggest that cAMP induced insulin release is mediated by increases in cytosolic Ca++ and that cAMP causes dislocation of Ca++ from intracellular stores.     

Inhibition of fast- and slow-phase depolarization-dependent synaptosomal calcium uptake by ethanol

Uptake of 45Ca++ by synaptosomes isolated from cerebral cortex, cerebellum, midbrain and brain stem of male, Sprague-Dawley rats was measured at 1-, 3-, 5-, 15-, 30- and 60-sec time periods. At 1 sec, the Ca++ uptake rate by cerebrocortical synaptosomes was 1.45 mumol/sec/g of protein, whereas the 60-sec rate was 0.03 mumol/sec/g of protein. In vitro addition of ethanol, 80 mM, inhibited depolarization-dependent (65 mM KCl) 45Ca++ uptake by synaptosomes but the time-response relationships varied depending upon the brain region studied. In cerebrocortical synaptosomes, ethanol significantly inhibited only the fast-phase component of 45Ca++ uptake (1 and 3 sec). Ethanol inhibited 45Ca++ uptake by midbrain synaptosomes at all measurement times studied (1, 3, 5 and 15 sec), whereas in cerebellum and brain stem ethanol inhibited 45Ca++ uptake at 3- and 5-sec time periods. Ethanol at concentrations of 25, 50, 100 and 150 mM inhibited 45Ca++ uptake by 9.0, 15.9, 24.8 and 30.7%, respectively, in cerebrocortical synaptosomes. In vitro ethanol, 80 mM, added to cerebrocortical synaptosomes isolated from rats fed a nutritionally adequate liquid ethanol diet did not significantly inhibit depolarization-dependent 45Ca++ uptake. The results of this study show that pharmacologically relevant ethanol concentrations inhibit voltage-dependent 45Ca++ uptake into synaptosomes. This inhibitory action may, at least in part, underlie some of the intoxicating effects of ethanol. In addition, chronic administration of ethanol resulted in an apparent adaptive response such that addition of ethanol no longer blocked 45Ca++ uptake. This adaptive response involving the calcium channel may represent a cellular mechanism for functional tolerance development.     

Calcium channel blockers: correlation among receptor binding, calcium uptake and contractility in vitro

Ten known calcium channel blockers were studied for inhibition of K+-induced 45Ca++ uptake into rabbit aortic smooth muscle cells in culture, and for displacement of [3H]nitrendipine [2,6-dimethyl-3-carbomethoxy-5-carbomethoxy-4-(3-nitro)phenyl-1, 4-dihydroxypyridine] binding to rat ventricular membrane preparations, in order to relate their effects on receptor binding with their inhibitory activities on 45Ca++ uptake and on contractile responses of vascular smooth muscle. Steady-state 45Ca++ uptake increased with K+ concentration in a dose-dependent manner. With 25 to 50 mM K+, Ca++ uptake was 0.6 nmol of Ca++ per one million cells. All calcium channel blockers inhibited K+-induced 45Ca++ uptake and [3H]nitrendipine binding in a dose-dependent fashion. The enatiomeric dihydropyridines 202-791 [isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-nitro-3-pyridinecarboxylate] exhibited marked stereoselectivity in both studies, the agonist (+)-202-791 significantly enhancing 45Ca++ uptake at 15 to 50 mM K+. The similarity between the order of potency in inhibiting 45Ca++ uptake and displacing [3H]nitrendipine resulted in a highly significant linear (1:1) correlation. An equally significant correlation was also established for the 10 blockers between their inhibitory potencies on 45Ca++ uptake and the contractile response of rabbit aortic strips as cited in the literature. These findings support the hypothesis that calcium channel blockers block contraction of vascular muscle by inhibiting cellular calcium uptake through voltage-dependent calcium channels as a result of binding to receptors associated with these channels. The aortic cells possess channels that are functionally similar to those found in intact vascular tissue.     

Effects of antibiotics on uptake of calcium into isolated nerve terminals

The goal of the present study was to determine whether several antibiotics which are known to block neuromuscular transmission would impair depolarization-dependent and/or -independent uptake of calcium into isolated nerve terminals prepared from forebrain synaptosomes of rats by conventional methods. Antibiotics tested for potential block of Ca++ uptake included the aminoglycosides neomycin and streptomycin, the lincosamide clindamycin, oxytetracycline and polymyxin B. Drugs were applied in concentrations ranging from 1 to 1000 microM. Uptake of 45Ca was determined during depolarization induced by an elevated K+ concentration (77.5 mM). Influxes of 45Ca during 1 and 10 sec of depolarization were used to assess Ca++ uptake via a "fast, inactivating path" and total uptake, respectively. Uptake of 45Ca during 10 sec of depolarization into synaptosomes which were previously depolarized for 10 sec in the presence of 77.5 mM K+ but in the absence of external Ca++ was used to measure uptake during a "slow, noninactivating path." Total depolarization-dependent uptake of 45Ca was depressed significantly by all antibiotics tested except oxytetracycline; however, the various agents differed with respect to their efficacy and potency as blockers of Ca influx. The fast component of uptake, which is thought to be associated with neurotransmitter release, was decreased significantly by all antibiotics. Neomycin and polymyxin were the most potent and most effective at lowering fast phase 45Ca influx; streptomycin, was intermediate in effectiveness whereas clindamycin and oxytetracycline were only effective at concentrations greater than or equal to 100 microM. Only clindamycin, streptomycin and polymyxin B caused significant reductions in the "slow" phase of 45Ca uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antinociceptive effects of lanthanum and cerium in nontolerant and morphine tolerant-dependent animals.

Intracerebroventricularly (i.vt.) administered lanthanum chloride was found to produce antinociceptive effects as measured by the mouse tail-flick and hot plate tests and the rat tail-flick test. These antinociceptive effects were reduced by peripheral administration of the narcotic antagonist naloxone or by i.vt. administration of calcium chloride. In addition, animals made tolerant to morphine were also tolerant to the effects of La+++. In morphine-dependent mice, the incidence of both abrupt and naloxone-precipitated withdrawal jumping was reduced by La+++ administration. In view of the known inhibitory effects of La+++ on Ca++ binding and movement, these findings suggest that alterations in Ca++ localization may be involved in the analgetic action of narcotic drugs as well as the development of narcotic tolerance and dependence.     

Calcium transport abnormality in uremic rat brain synaptosomes.

Brain calcium is elevated in patients and laboratory animals with uremia. The significance of this finding is unclear. We evaluated calcium transport in brain of both normal and acutely uremic rats (blood urea nitrogen = 250 mg/dl) by performing studies in synaptosomes from rat brain cerebral cortex. Synaptosomes are vesicular presynaptic nerve endings from brain that contain mitochondria and are metabolically active. Two mechanisms of calcium transport were evaluated using radioactive 45Ca++ as a tracer. Both mechanisms were evaluated in the absence of exogenously administered parathyroid hormone (PTH). We first evaluated Na+-Ca++ exchange in vesicles that were loaded with NaCl in an external media containing 10 microM CaCl2. Both initial rates of calcium transport and equilibrium levels of calcium accumulation in synaptosomes prepared from uremic rats were significantly greater (P less than 0.005) than in normal. To assess calcium efflux, ATP-dependent calcium uptake (1 mM ATP) was studied in inverted plasma membrane vesicles loaded with KCl. In the uremic synaptosomes, a significant increase (P less than 0.005) in ATP-dependent calcium uptake was observed as compared with the normal. These studies show that (a) Calcium accumulation via the Na+-Ca++ exchanger is increased in synaptosomes prepared from uremic rat brain. (b) Calcium influx into inverted plasma membrane vesicles from uremic rats via the ATP-dependent calcium transport mechanism is increased when compared with normal. (c) The increased calcium accumulation in uremia by both Na+-Ca++ exchange and ATP-dependent calcium transport mechanism appears to be a result of increased synaptosomal membrane permeability to calcium. Both these abnormalities of calcium transport in uremia would tend to increase brain extracellular calcium in vivo. The defects observed in uremia do not appear to be readily reversible, and the relationship to PTH is presently unclear. These abnormalities may affect neurotransmission in the uremic state.     

Mechanism of action of cobra cardiotoxin in the skeletal muscle.

Cobra cardiotoxin (CTX) is a potent polypeptide in inducing irreversible contracture of the chick biventer cervicis muscle. this polypeptide is about 2000 times more potent than caffeine in inducing contracture of this muscle preparation but the rate of CTX contracture is slower. Studies on the interaction on CTX with divalent cations showed that low Ca0++ (10(-31-2 mM)-Krebs' enhanced markedly while ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid-Krebs' or high Ca++ (12 mM) inhibited completely CTX contracture; 10 mM Mg++ enhanced but 20 mM Mg++ or 10 mM Zn++ inhibited CTX contracture. Neither Na+ nor tetrodotoxin affected CTX contracture. Comparison of CTX and caffeine contracture showed that ethylenediamine tetraacetic acid (1 mM) and Mn++ (5 mM) inhibited CTX contracture completely but inhibited caffeine contracture only partially; procaine did not inhibit CTX contracture but inhibited caffeine contracture competitively; by contrast, N-ethylmaleimide inhibited CTX but not caffeine contracture. Neither caffeine nor K+ contracture was inhibited by 12 mM Ca++. CTX could induce contracture in the depolarized muscle and the muscle with T-tubule destroyed or closed, whereas K+ failed to induce contracture in the latter. Caffeine contracture was inhibited in the muscle with a previous elicitation of CTX contracture. This antagonistic effect of CTX could be prevented by calcium. Moreover, CTX increased both 45Ca++ efflux and 45Ca++ uptake. Both Zn++ (0.6 mM) and Mg++ (10 mM) but not protamine and polylysine mimicked CTX to increase 45 Ca++ uptake. Zn++ (0.6mM) was also found to be effective in replacing Ca0++ to induce CTX contracture in 10(-6) M Ca0++. CTX increased Ca and Na but decreased K contents of the muscle. The binding of radioactive iodinated CTX was inhibited not only by unlabeled CTX but also by 10mM Ca++. All of these findings suggest that CTX may affect a membrane calcium binding site and may induce contracture by releasing the membrane calcium rather than by increasing Na+ permeability of the muscle membrane.     

Irreversible suppression of calcium entry into nerve terminals by methylmercury

Measurements of uptake of 45Ca into rat forebrain synaptosomes depolarized with high K+ and EPP amplitudes at the rat neuromuscular junction were used to assess the effects of methylmercury (MeHg) on voltage-dependent Ca++ uptake and subsequent transmitter release at model central and peripheral synapses. The objectives were to: determine whether MeHg altered uptake of 45Ca into purified synaptosomes depolarized by high K+; compare its effects with those produced by HgCl2; ascertain whether the "fast" or "slow" components of Ca++ uptake were affected preferentially by MeHg; and determine whether a functional correlate to the effects on 45Ca uptake could be observed electrophysiologically at the mammalian neuromuscular junction. HgCl2 (10-500 microM) produced a concentration-dependent decrease of total depolarization-induced 45Ca uptake. Peak inhibition occurred at 200 microM Hg++ which suppressed nerve terminal Ca++ uptake to approximately 5% of Hg-free control values, a result similar to that obtained previously by others. Similarly, MeHg also suppressed total 45Ca uptake although the maximal inhibition produced (70% at 200 microM MeHg) was less than that produced by HgCl2. The effect of MeHg was apparent both in nonpreviously depolarized synaptosomes after a 1-sec depolarization ("fast uptake") and after 10-sec incubation in synaptosomes predepolarized with 41 mM K+ in Ca-free solutions before addition of MeHg and 45Ca ("slow uptake"). A significant decrease in the slow phase of 45Ca uptake occurred with 200 and 500 microM MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of extracellular calcium in contractions produced by activation of postsynaptic alpha-2 adrenoceptors in the canine saphenous vein

Canine saphenous vein (CSV) has been shown to contain both postsynaptic alpha-1 and alpha-2 adrenoceptors. Our previous studies have shown that activation of postsynaptic alpha-1 adrenoceptors in this tissue utilizes both extracellular and intracellular Ca++ to produce contractions. In the present study, the source of calcium mobilized by activation of postsynaptic alpha-2 adrenoceptors in CSV was elucidated. Contractions of tissue rings to the supramaximal concentrations of three selective alpha-2 agonists, B-HT 920, M-7 and clonidine, were determined in the absence and presence of 5 mM La . In the presence of La , clonidine and M-7 produced small but statistically significant contractions (8-14% of control) which were abolished when the alpha-1 adrenoceptors were inactivated by phenoxybenzamine (10(-7) M, 30 min). In contrast, contractions to B-HT 920 were abolished completely in the presence of La . All the three alpha-2 agonists stimulated 45Ca++ uptake into CSV (0.3-0.4 mmol/kg wet weight, 10 min). 45Ca++ efflux studies demonstrated that the selective alpha-2 agonist, B-HT 920 (10(-5) M plus 10(-7) M phenoxybenzamine), did not induce an increase in the rate of 45Ca++ efflux. In contrast, an augmented 45Ca++ efflux rate was observed with the alpha-1 agonist, phenylephrine (10(-4) M plus 10(-7) M rauwolscine). These results suggest that activation of postsynaptic alpha-2 adrenoceptors in CSV utilizes primarily extracellular Ca++ to produce contractions.     

Modulation of levels of free calcium within synaptosomes by organochlorine insecticides

Effects of the organochlorine insecticides chlordecone, mirex, 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane on free intrasynaptosomal Ca2+ [( Ca++]i), synaptosomal 45Ca uptake and synaptosomal plasma and mitochondrial membrane potentials in vitro were studied. Chlordecone (10-50 microM) increased [Ca++]i from the resting level of 370 nM in a dose- and time-dependent manner to above 1.5 microM. This took place in the presence of 1 mM extrasynaptosomal Ca++ but not in nominally Ca++-free medium. Verapamil, a voltage sensitive Ca++ channel blocker, inhibited the initial increase of [Ca++]i caused by chlordecone, by 40%. Chlordecone also elevated [Ca++]i in synaptosomes in which mitochondrial Ca++ uptake had been abolished by valinomycin. Chlordecone depolarized partially the synaptosomal plasma membrane and, to a lesser extent, the potential of mitochondria within synaptosomes. However, chlordecone appeared to inhibit synaptosomal K+-stimulated and unstimulated 45Ca++ uptake by 20 to 30%. Inasmuch as chlordecone also stimulated release of 45Ca++ and the fluorescent dye fura-2 from preloaded synaptosomes, the apparent inhibition of uptake might be due to lysis of some synaptosomes by chlordecone. The effect of chlordecone on [Ca++]i decreased when the total amount of tissue in incubations was increased. [Ca++]i was only elevated marginally by mirex at the same concentration range. The results suggest that chlordecone increases free intrasynaptosomal Ca++ mainly by increasing influx of extrasynaptosomal Ca++. The principal mechanism appears to be a nonspecific leakage of Ca++ through the plasma membrane but some Ca++ may pass through voltage-sensitive Ca++ channels due to chlordecone-induced membrane depolarization.     

Gentamicin blockade of slow Ca++ channels in atrial myocardium of guinea pigs.

Cardiac dysfunction is occasionally detected in patients undergoing treatment with amino-glycoside antibiotics, however, the mechanism responsible for the negative inotropic effect of these agents has not been identified. In the present investigation electrically driven left atria of guinea pigs were used to study the effects of gentamicin on calcium ion (Ca++)-dependent contractile events in heart muscle isolated from in vivo influences. When atria were first inactivated by excess potassium ion (K+; 22mM) and contractions were then restored by isoproterenol (an experimental model that accentuates the contractile dependence of myocardial fibers on influx of Ca++ through specific "slow channels" of the sarcolemma), the cardiac depressant activity of gentamicin (0.1 mM) was profoundly augmented. Conversely, the negative inotropic effect of tetrodotoxin (23.5 micron) was abolished by the same experimental conditions. Also, gentamicin (1 mM) and La+++ (0.5 mM) markedly decreased the positive inotropic response to increased frequency of stimulation; whereas, D600 (1.05 micron) converted the positive frequency-force relationship to a negative relationship. Present data indicate a direct cardiac depressant action of gentamicin, and suggest that this antibiotic adversely affects either the transport system responsible for Ca++ movement through slow channels of the sarcolemma, the availability of Ca++ for translocation to these sites, or both.     

Effects of 2-nicotinamidoethyl nitrate on agonist-sensitive Ca++ release and Ca++ entry in rabbit aorta

The effects of 2-nicotinamidoethyl nitrate (SG-75) on norepinephrine (NE)- and KCI-induced responses in rabbit aorta were quantitated, correlated with 45Ca studies and compared with the effects of nifedipine (NIF) on similar parameters. NE- and KCI-induced dose-response relationships were differentially depressed by SG-75 (NE much greater than KCI) and NIF (KCI much greater than NE). Responses to KCI were relatively insensitive to prior SG-75, yet moderately relaxed by subsequent SG-75. Conversely, NIF markedly inhibited and completely relaxed similar responses. Responses to NE were relaxed and inhibited with SG-75, but unaffected by NIF. Responses to NE in La or O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 (with and without KCI) solutions were phasic, reduced by SG-75 and insensitive to NIF. NE-dependent, Ca++-induced responses in a O-Ca++ + ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid plus D600 solution (with and without KCI) were attenuated by SG-75. Equilibrated (60 min) La -resistant (residual), high apparent affinity Ca++ binding was increased 26% with SG-75 and decreased 34% with NIF, yet neither altered the rate of exchange (10 min). Rate of exchange at low apparent affinity, residual sites was increased 21% by SG-75 without altering equilibrated values, whereas NIF reduced equilibrated values 11%, without affecting rate. NE reduced, SG-75 + NE augmented and NIF + NE decreased, in an additive fashion, high apparent affinity, residual bound Ca++. Residual Ca++ binding at low apparent affinity sites was increased with 160 mM substituted KCI (380%). This increase was only partially inhibited with SG-75, and eliminated by NIF. Net Ca++ efflux was persistently slowed by SG-75 and unaltered by NIF. The primary effects of SG-75 appear to be depression of Ca++ release and inhibition of receptor-operated (potential-independent) Ca++ entry, with limited attenuation of voltage-dependent Ca++ entry. NIF primarily inhibits voltage-dependent Ca++ entry.