Functional importance of alpha adrenoceptor-mediated, D600-insensitive Ca++ entry in rabbit aorta

Specific receptor-linked Ca++ entry (RLCa++E) was studied separately from Ca++ release and potential-dependent Ca++ entry (PDCa++E) in rabbit aorta after incubation in a Ca++-free solution containing ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid to bind trace levels of Ca++ and including methoxyverapamil (D600) (10(-5) M) to inhibit PDCa++E. Adding norepinephrine (NE) under these conditions resulted in a transient response which was attributed to Ca++ release from a limited cellular store. Subsequent addition of Ca++ results in a sustained contraction that was dependent upon the concentration of agonist and Ca++. This maintained response which by definition was insensitive to D600 was attributed to RLCa++E, was extensively relaxed or inhibited by nitroprusside or nitroglycerin and was partially relaxed by KCl or tetraethylammonium. Contractions due to RLCa++E alone equaled or exceeded the peak tension attained solely as a result of Ca++ release. At higher NE concentrations (greater than 10(-7) M), RLCa++E provided sufficient Ca++ to attain and sustain maximal levels of developed tension without requiring any additional Ca++ from either PDCa++E or Ca++ release. This high flux capacity of these receptor-linked Ca++ channels may partially account for the relative insensitivity of contractile responses to these higher concentrations of NE to D600. The relative contribution of PDCa++E to the elevation of myoplasmic Ca++ and concurrent sensitivity to D600 was increased at lower concentrations of NE because the degree of Ca++ release and RLCa++E were relatively small. Additionally, it appears that RLCA++E may be attenuated by concomitant membrane depolarization.     

Effect of extracellular Ca++ omission on isolated hepatocytes. II. Loss of mitochondrial membrane potential and protection by inhibitors of uniport Ca++ transduction

Incubation of isolated rat hepatocytes in Ca++-free medium generates an oxidative stress which causes significant cell injury. Ruthenium red and La , which block Ca++ uptake through the mitochondrial uniport, totally prevented malondialdehyde formation, glutathione and protein thiol oxidation and vitamin E loss induced by Ca++ omission. Accordingly, these agents also prevented leakage of intracellular K+ and lactate dehydrogenase. Similar protective effects were provided by the Ca++ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. The absence of extracellular Ca++ resulted in a marked decline of the mitochondrial transmembrane potential which could be prevented by ruthenium red, ethylene glycol bis(beta-aminoethyl ether)-N,N'tetraacetic acid, the antioxidant vitamin E and the iron chelator, desferrioxamine. In contrast, oxidative stress induced by treatment with the redox active agent paraquat and 1,3-bis(2-chloroethyl)-1-nitrosourea had little effect on mitochondrial transmembrane potential and malondialdehyde formation and lactate dehydrogenase leakage were not affected by ruthenium red or La . These results indicate that the incubation of rat hepatocytes in the absence of extracellular Ca++ creates an unusual oxidative stress which markedly affects mitochondrial function. The ability of vitamin E and desferrioxamine to inhibit the loss of mitochondrial transmembrane potential indicates that oxidative damage is involved in producing mitochondrial dysfunction. Furthermore, the potent inhibitory effects of ruthenium red and La suggest that Ca++ movement through the uniport, perhaps indicative of mitochondrial Ca++ cycling, plays a major role in generating this oxidative stress and promoting cell injury.     

Angiotensin II amplifies arterial contractile response to norepinephrine without increasing Ca++ influx: role of protein kinase C.

We investigated whether the enhanced contractile response to norepinephrine caused by a subthreshold concentration of angiotensin II was associated with an increased 45Ca++ influx or net uptake. Rabbit facial artery segments were mounted isometrically to measure the 45Ca++ influx and net uptake in response to norepinephrine. The contractile response to norepinephrine (3 microM) in the presence of angiotensin II (0.1 nM) was 149.5 +/- 7.4% of control. This response amplification was not associated with changes in norepinephrine-induced 45Ca++ influx or net uptake. Angiotensin II also potentiated the contractile response to caffeine obtained in a Ca(++)-free buffer containing ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (2 mM) to 148.0 +/- 4.8% of control. In both cases, the amplification was prevented by pretreatment with either staurosporine (10 nM) or calphostin C (100 nM), two inhibitors of protein kinase C. We conclude that angiotensin II potentiation of norepinephrine-induced vascular tone occurs in the absence of changes in stimulated Ca++ entry. This potentiation may be due to an increase in intracellular sensitivity to Ca++, possibly mediated by protein kinase C.     

Release of endogenous norepinephrine from rat hypothalamus by stimulation of N-methyl-D-aspartic acid receptors.

Release of endogenous dopamine and norepinephrine (NE) from rat hypothalamic slices superfused with Mg(++)-free medium in the presence of nomifensine and tyrosine was measured by high-performance liquid chromatography coupled to an electrochemical detector. Superfusion with L-glutamic acid or N-methyl-D-aspartic acid elicited a concentration-dependent release of NE but not of dopamine. The release of NE was transient, returning toward basal values despite the continued presence of the amino acid. Superfusion with 20 mM K+ caused a release of NE that declined at a slower rate. Mg++, DL-2-amino-5-phosphonopentanoic acid and MK-801 (D-5-methyl-10,11,dihydro-5H-dibenzo[a,d] cyclohepten-5-10-imine maleate), but not 6-cyano-7-nitroquinoxaline-2,3-dione, inhibited the L-glutamic acid-evoked release of NE. The release of NE by L-glutamic acid was virtually abolished by tetrodotoxin and by elimination of Ca++ from and inclusion of 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid in the superfusion medium. Repeated L-glutamic acid applications displayed a decreased response, whereas repeated exposure to 20 mM K+ did not. Exposure to L-glutamic acid in the absence of Ca++ (plus 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) or in the presence of DL-2-amino-5-phosphonopentanoic acid did not reduce the effects seen on subsequent exposure to L-glutamic acid. Exposure to L-glutamic acid in the absence of Mg++ reduced the effect of a subsequent exposure to L-glutamic acid. These observations provide evidence for an indirect modulation of rat hypothalamic endogenous NE by the N-methyl-D-aspartate receptor.     

Contribution of Ca++ and calmodulin to the action of norepinephrine on renal prostaglandin synthesis and vascular tone

We have investigated the contribution of Ca++ and calmodulin to the action of norepinephrine (NE) on prostaglandin (PG) synthesis and vascular tone in the Tyrode's perfused rat kidney. Lowering the Ca++ concentration (0.6 mM) reduced and raising the Ca++ concentration (5.4 mM) enhanced the renal vasoconstriction and PG output elicited by NE. Calcium channel blockers diltiazem or nimodipine inhibited the vasoconstriction and PG output caused by NE. Ca++-free Tyrode's solution containing ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid abolished NE-induced vasoconstriction and reduced PG output by 25 to 38%. Addition of intracellular Ca++ antagonists 8-(diethylamino) octyl 3,4,5 trimethoxybenzoate, dantrolene or ryanodine to Ca++-free Tyrode's solution inhibited NE-induced PG output. Calmodulin inhibitors trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide or calmidazolium diminished PG output and the renal vasoconstriction elicited by NE in the presence and absence of Ca++. Mepacrine and indomethacin inhibited NE-induced renal vasoconstriction and PG output. Arachidonic acid-induced PG output was abolished by indomethacin but was unaltered by mepacrine, Ca++ antagonists or calmodulin inhibitors. We conclude that NE produces renal vasoconstriction by a mechanism that depends primarily on extracellular Ca++ and calmodulin, whereas NE-induced PG output depends on both extra- and intracellular Ca++ and calmodulin.     

Modulation of the Ca++-evoked release of [3H]dopamine from striatal synaptosomes by dopamine (D2) agonists and antagonists

Release of [3H]dopamine ([3H]DA) from striatal synaptosomes is evoked most commonly by elevating potassium levels in the presence of calcium. However, it has been difficult to show that DA agonists or antagonists can modify K+-evoked release of [3H]DA. DA. In this study [3H]DA release evoked by exposure of synaptosomes (isolated and superfused previously with 0.0 mM Ca++ and 0.1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) to 1.25 mM Ca++ can be modulated by the DA (D2) agonists apomorphine, pergolide and quinpirole and antagonists l-sulpiride and domperidone. The release was evoked under low potassium (6 mM or less) concentrations and the potassium concentration in the superfusion medium was not elevated before or during Ca++ exposure. Analysis of the superfusates obtained during Ca++ exposure revealed that approximately 80% of the tritium released was [3H]DA. The ability of DA (D2) agonists to inhibit the Ca++-evoked release from synaptosomes superfused with 9 mM K+ was greatly reduced. Therefore, prolonged depolarization may block DA (D2) regulation of [3H]DA release from synaptosomes. The Ca++-evoked release of [3H]DA was reduced greatly when 1 microM tetrodotoxin was present indicating sodium channels play a role in triggering the processes involved in Ca++-evoked [3H]DA release.     

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)     

Effects of 2-nicotinamidoethyl nitrate (nicorandil; SG-75) and its derivative on smooth muscle cells of the canine mesenteric artery

To clarify the mechanism of vasodilating actions of nicotinamidoethyl nitrate (nicorandil; SG-75) in relation to the chemical structure, we studied the effects of SG-75 and its derivatives [nitrate containing structure; 3,5-bis([2'- nitroxyethyl ] aminocarbonyl )pyridine (SG-114); nicotinamide derivatives: N-(2-hydroxyethyl)nicotinamide (SG-86) and N-(2- nicotinyloxyethyl )-nicotinamide; (SG-103)] on the electrical and mechanical properties of smooth muscle cells of the canine mesenteric artery. SG-75 significantly and SG-114 slightly hyperpolarized the membrane but SG-86 or SG-103 did not. The excitatory junction potential and spike potential evoked by perivascular nerve or direct muscle stimulation were markedly inhibited by SG-75 due to hyperpolarization of the membrane. SG-114 slightly inhibited but SG-86 or SG-103 did not inhibit the excitatory junction potential or spike potential. The K-induced contraction was inhibited by SG-75 (below 39.2 mM) or without hyperpolarization (over 39.2 mM) of the membrane, but SG-114 inhibited the contraction with no hyperpolarization. In concentrations over 39.2 mM K0, SG-114 inhibited the contraction more potently than did SG-75. On the other hand, the norepinephrine-induced contraction was inhibited by SG-75 or SG-114 to the same extent, due to additional hyperpolarization of the membrane, in the case of SG-75. Both agents inhibited but SG-86 or SG-103 did not inhibit the norepinephrine-induced contraction in the Ca-free 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid containing solution. After the complete depletion of the stored Ca, application of Ca in the presence of SG-75 or SG-114 enabled estimation of the reduction in the amount of Ca stored in the cell, determined by the amplitude of the subsequently produced caffeine-induced contraction in Ca-free ethylene glycol bis(beta-aminoethyl ether)N,N'- tetraacetic acid containing solution. The effects of SG-75 or SG-114 on the norepinephrine-induced contraction in Ca-free solution also indicated a reduction in the Ca stored in the cell. It would appear that SG-75 hyperpolarizes the membrane due to the SG-75 moiety and not to the nitrate residue alone. The relaxation of the tissue induced by SG-75 or SG-114 is due to nitrate action, as observed in the case of nitroglycerin. SG-114 possesses a stronger potency with regard to relaxation of the tissue; however, in vivo, SG-75 may have a more potent vasodilating action than SG-114, as the former inhibits neuromuscular transmission mechanisms.     

Regulation of Ca++ influx into striatal neurons by kainic acid

We investigated the mechanisms by which kainic acid (KA) produces increases in [Ca++]i in single striatal neurons in vitro using fura-2-based microfluorimetry. When neurons were depolarized by perfusion with high K+ or veratridine containing solutions, [Ca++]i rose rapidly to a peak and then declined to a lower sustained plateau that persisted as long as the depolarizing stimulus. The peak high K+-induced rise in [Ca++]i occurred at [K+]o greater than 50 mM and the plateau was largest at 30 mM K+. [K+]o that was greater than 70 mM caused the magnitude of the plateau to decrease. Responses to high K+ stimulation were completely dependent on [Ca++]o and presumably represented Ca++ influx. Nitrendipine partially blocked the peak of the high K+-induced response and completely blocked the sustained plateau Ca++ influx. The nitrendipine-resistant portion of the high K+ response could be completely blocked by predepolarization of the cell in Ca++-free solution. KA also produced large increases in [Ca++]i that were abolished on removal of external Ca++. Predepolarization/nitrendipine greatly reduced the effect of lower [KA] (100 microM). However, KA-induced increases in [Ca++]i became increasingly resistant to block of voltage-sensitive Ca++ channels as [KA] rose above 100 microM, indicating a second route of Ca++ entry that may be the KA receptor-gated ionophore. About one-half the responses to KA (100 microM) also displayed a large oscillation. [Ca++]i rose to a peak, fell and then rose again before finally declining to a plateau level. This oscillation was abolished when all external Na+ was replaced by Li+ and may result from alterations in the buffering of [Ca++]i as a result of KA-induced Na+ influx.     

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.     

Membrane events and ionic processes involved in dopamine release from tuberoinfundibular neurons. II. Effect of the inhibition of the Na+-Ca++ exchange by amiloride

In the present study we investigated the effect of amiloride, a rather specific inhibitor of the membrane Na+-Ca++ exchange system, on the release of endogenous dopamine (DA) and "previously taken-up" [3H]DA from tuberoinfundibular dopaminergic neurons. Amiloride (300 microM) stimulated either endogenous DA or [3H]DA release. Amiloride-induced stimulation of [3H]DA release was prevented in a Ca++-free plus ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid medium. Amiloride, at the same concentration, reinforced both high K+- and electrically-induced stimulation of [3H]DA release. These results are explained on the basis of the ability of amiloride in blocking the Na+-Ca++ exchange system, therefore causing an elevation of intracellular Ca++ levels in resting conditions, and a further accumulation of Ca++ ions after high K+- or electrically elicited opening of voltage-operated channels specific for Ca++ ions. The enhanced intracellular Ca++ availability may trigger the stimulation of neurotransmitter release. In addition, amiloride was able to block in a dose-dependent manner (70-300 microM) the ouabain-induced [3H]DA release, suggesting that, when intracellular concentrations of Na+ are increased by the blockade of Na+,K+-adenosine triphosphatase the Na+-Ca+;+ exchange carrier reverses its resting mode of operation, mediating the influx of extracellular Ca++ ions. Amiloride, by blocking the Na+-Ca++ exchange mechanism, prevents the ouabain-elicited entrance of extracellular Ca++ ions, therefore inhibiting [3H]DA release stimulated by the cardioactive glycoside. Collectively, the results of the present study seem to be compatible with the idea that the Na+-Ca++ exchange mechanism is involved in the regulation of [3H]DA release from tuberoinfundibular dopaminergic neurons, through the regulation of Ca++ movements across the plasma membrane.     

Energetic aspects of the regulation of Ca++ sensitivity of permeabilized rabbit mesenteric artery: possible involvement of a second Ca++ regulatory system in smooth muscle contraction

The effects of phosphagen concentrations and adenosine-5'-O-(2-thiodiphosphate)(ADP beta S), a nonhydrolyzable ADP analog, on the pCa++ tension relationships were investigated, using alpha-toxin permeabilized rabbit mesenteric artery. The removal of creatine phosphate (CP) greatly affected the Ca++ sensitivity and induced a leftward shift of the pCa++ tension curve. Addition of ADP beta S (10-300 microM) also caused a leftward shift of the pCa++ tension curve. Ca++ solutions (0.3-10 microM) containing 0.1 mM ATP did not induce contraction. However, the addition of CP in the presence of 0.1 mM ATP dose-dependently increased force development which reached a maximum around 3 mM CP. A 10 microM Ca++ solution containing 0.1 mM ATP and 1 mM CP was much more effective in inducing contraction than a 10 microM Ca++ solution containing 1.1 mM ATP alone, although the total concentration of phosphagen (ATP + CP) was the same. Application of 0.1 mM ATP solution containing various concentrations of Ca++ after the maximal Ca+(+)-induced contraction relaxed the tissue, with the higher Ca++ concentrations inducing the faster relaxation. The same pattern of the relaxation was seen when the tissue was pretreated with adenosine-5'-O-(3-thiotriphosphate) beforehand. The contractile state observed in the Ca+(+)-free solution containing 0.1 mM ATP and 0.1 mM CP was completely relaxed by 1 mM vanadate, consistent with the idea that the sustained contraction was due to accumulation of the actomyosin-ADP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ca++ utilization in the constriction of rat aorta to full and partial alpha-1 adrenoceptor agonists

l-Norepinephrine and l-phenylephrine were full agonists and cirazoline, SKF d-89748, Sgd 101/75 and SKF l-89748 were partial agonists in contracting rat isolated aortic rings. Clonidine, l-amidephrine and St 587 were found ineffective. Nifedipine (10(-8)-10(-6) M) abolished the contractions to Sgd 101/75 and to high K+ with similar potency but only partially inhibited the contractions to the other alpha adrenoceptor agonists. Norepinephrine, phenylephrine, cirazoline and Sgd 101/75 were full agonists in stimulating 45Ca++ influx, which amounted to 50% of the maximal influx produced by high K+. SKF d- and l-89748 behaved as partial agonists, whereas St 587, amidephrine and clonidine were virtually inactive. Nifedipine was equally effective in blocking the influx of 45Ca++ produced by K+ and the alpha adrenoceptor agonists. Norepinephrine stimulated 45Ca++ efflux to an extent similar to that for high K+. In the following order of decreasing efficacy, phenylephrine, cirazoline and SKF d- and l-89748 caused significant stimulation of 45Ca++ efflux. Sgd 101/75, amidephrine, clonidine and St 587 were without effect. However, Sgd 101/75 (10(-5) M) antagonized the 45Ca++ efflux of norepinephrine. Nifedipine (3 X 10(-7) M) completely suppressed the K+-induced 45Ca++ efflux but only partly affected the 45Ca++ efflux caused by the alpha adrenoceptor stimulants. A highly significant (r = 0.975) linear relationship was found between the nifedipine-resistant contractile response and the 45Ca++ efflux obtained in the presence of nifedipine. The data suggest that the stimulation of alpha-1 adrenoceptors in rat aorta can activate two distinct processes of Ca++ utilization for contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction: mode of action

Neuropeptide Y (NPY), which co-exists with noradrenaline (NA) in postganglionic sympathetic nerves, was able to potentiate NA-evoked constriction in certain isolated rabbit blood vessels. The phenomenon was observed in the femoral, the gastroepiploic and the pulmonary arteries but not in the femoral or the gastroepiploic veins or in the aorta. Thus, NPY potentiated NA-evoked vasoconstriction predominantly in muscular arteries with alpha-1 adrenoceptors. NPY-related peptides, such as peptide YY and to some extent pancreatic polypeptide shared this ability, whereas calcitonin gene-related peptide or LPLRFamide did not. The mode of action by which NPY potentiates NA-evoked vasoconstriction was analyzed using the femoral artery. Pretreatment of the vessel with cocaine, a blocker of amine re-uptake, or rolipram, an inhibitor of phosphodiesterase, left the potentiation unaffected, whereas Na+ deficiency or ouabain, an inhibitor of Na+/K+-adenosine triphosphatase, abolished this effect of NPY. Nifedipine, a blocker of Ca++ entry, or removal of extracellular Ca++ shortly before the application of NPY had little effect. After prolonged exposure to a Ca++-free medium (with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) the maximum response to NA was greatly reduced and the potentiating effect of NPY was abolished. Thus, the potentiation of NA-evoked vasoconstriction by NPY seems to depend upon the presence of Na+ but not upon a Ca++ influx. An intracellular sequestered Ca++ pool appears to play a critical role.     

Neurotoxic action of veratridine in rat brain neuronal cultures: mechanism of neuroprotection by Ca++ antagonists nonselective for slow Ca++ channels

The effect of various Ca++ antagonists and local anesthetics on neuronal cell degeneration induced by veratridine was studied in primary rat brain neuronal cultures. Cell death was quantified by measuring lactate dehydrogenase (LDH) released in the culture medium. The neuronal cell degeneration was Ca+(+)-dependent because, in the absence of extracellular Ca++, 16 hr of exposure to 30 microM veratridine failed to produce release of LDH. Ca++ antagonists, nonselective for slow Ca++ channels (flunarizine, cinnarizine, lidoflazine, prenylamine and bepridil) inhibited veratridine-induced release of LDH with IC50 values between 0.11 and 0.47 microM. Ca++ antagonists selective for slow Ca++ channels were less potent and inhibited veratridine-induced release of LDH at concentrations in the following order of potency: nicardipine greater than gallopamil and verapamil greater than niludipine greater than nitrendipine greater than nifedipine greater than nimodipine greater than diltiazem. Tested local anesthetics were incomplete inhibitors of veratridine-induced release of LDH. A good correlation was found between the potency of the drugs to inhibit released LDH induced by 30 microM veratridine in neuronal cultures and their binding affinity for the batrachotoxin binding site of Na+ channels in rat cortex synaptosomal preparation. It is concluded that protection against veratridine-induced neurotoxicity can be mediated by blocking a veratridine-sensitive Na+ channel. It is a property of certain nonselective Ca++ antagonists. There is apparently no direct relationship with Ca++ antagonistic activity. The effect is unrelated to local anesthetic activity.     

Block of neuronal Ca++ influx by the anti-ischemic agent TA3090

We examined the effect of the novel anti-ischemic drug, TA3090 on Ca++ entry into peripheral and central neurons. TA3090 inhibited voltage-dependent Ca++ entry into rat dorsal root ganglion (DRG) and sympathetic neurons. The degree of inhibition produced by the drug was voltage-dependent. TA3090 also inhibited Ca++ entry into DRG cells elicited by trains of action potentials. TA3090 inhibited Ca++ influx into neurons from the hippocampus. As in peripheral neurons, TA3090 blocked Ca++ influx associated with a Ca++ current in voltage-clamped cells or elicited by trains of action potentials. Furthermore, TA3090 blocked Ca++ influx stimulated by glutamate or N-methyl-D-aspartate. In addition, TA3090 blocked excitatory glutamate-mediated synaptic transmission between hippocampal pyramidal neurons. These data indicate that the protective effects of TA3090 in the brain under ischemic conditions may be partly or completely due to its ability to inhibit neuronal Ca++ influx.     

Contraction mediated by Ca++ release in circular and Ca++ influx in longitudinal intestinal muscle cells

The source of Ca++ responsible for contraction was examined in muscle cells isolated separately from the circular and longitudinal muscle layers of guinea pig and human intestine. Contraction was measured by scanning micrometry and cytosolic-free Ca++ ([Ca++]i) with the fluorescent indicator, quin2. In both species, contraction induced in circular muscle cells by cholecystokinin-8 (CCK-8) and acetylcholine was not affected by withdrawal of Ca++ from the medium or addition of the Ca++ channel blocker, methoxyverapamil, whereas contraction induced by both agonists in longitudinal muscle cells and by depolarizing concentrations of K+ in both cell types was abolished. Depletion of intracellular Ca++ stores with caffeine in Ca++-free medium abolished the response in circular muscle cells. Readdition of Ca++ to the medium for 30 sec restored the response in longitudinal but not circular muscle cells. [Ca++]i, measured in guinea pig muscle cells, increased 3- to 4-fold above resting levels (circular, 70.8 +/- 8.1 nM; longitudinal, 77.4 +/- 9.7 nM) in response to all three contractile agents. The increase in [Ca++]i induced by CCK-8 and acetylcholine in circular muscle cells was not affected by withdrawal of Ca++ from the medium or addition of methoxyverapamil, whereas the response to both agonists in longitudinal muscle cells and to 20 mM K+ in both cell types was abolished. It was concluded that cells from adjacent muscle layers of the intestine mobilize Ca++ differently during agonist-induced contraction, i.e., by Ca++ release in circular and Ca++ influx in longitudinal muscle cells.     

Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle

The Ca dependence of contraction and myosin phosphorylation was investigated in canine tracheal smooth muscle stimulated with carbachol, K or serotonin. Previous studies of tracheal muscle showed carbachol concentration-response curves for contraction and myosin phosphorylation were superposable. In contrast, there was a striking difference in the Ca++ sensitivities of tension and myosin phosphorylation when Ca++ concentration-response curves were constructed in the presence of 10(-7) M carbachol. Significant phosphorylation (greater than 0.3 moles phosphate/mole 20,000 dalton myosin light chain) was observed in the absence of active tension. In the present study, carbachol (10(-7) and 10(-6) M) and serotonin (10(-5) M) also induced significant myosin phosphorylation in low Ca++ solutions (0-0.025 mM CaCl2) without proportional increases in tension. K+ depolarization in Ca++-free physiological salt solution (60 mM KCl, 10(-6) M atropine) yielded phosphorylation not significantly different from basal levels. All stimulants induced active stress after readmission of Ca. The Ca++ dependence curve for myosin phosphorylation in muscles stimulated with carbachol was shifted up and to the left of the force curve. Atropine (10(-6) M) significantly reduced phosphorylation induced by carbachol in Ca++-free solutions, as did 3 X 10(-6) M nifedipine and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Phorbol 12-myristate, 13-acetate or phorbol 12,13-dibutyrate did not increase basal phosphorylation or phosphorylation in low Ca++ solutions, suggesting that protein kinase C did not phosphorylate myosin in this case. Myosin phosphorylation under these conditions is not sufficient to support contraction, and is reduced by treatments that decrease Ca++ entry.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor agonists induce myosin phosphorylation-dependent and phosphorylation-independent contractions in vascular smooth muscle.

In isolated rat aorta, 72.7 mM KCI, 10 microM prostaglandin F2 alpha, 30 nM endothelin-1 and 1 microM norepinephrine increased muscle tension, cytosolic Ca++ concentration ([Ca++]i) and 20 kDa myosin light chain (MLC) phosphorylation. The levels of contractile tension and MLC phosphorylation at a given [Ca++]i were greatest in the presence of endothelin-1 followed by prostaglandin F2 alpha greater than norepinephrine greater than high K+. Verapamil inhibited the high K(+)-induced increments to their respective resting levels. Verapamil also almost completely inhibited the receptor agonist-induced increments in [Ca++]i and MLC phosphorylation, although a part of the contraction was not inhibited. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid further decreased [Ca++]i and muscle tension, suggesting that a part of the contraction is regulated by [Ca++]i below a resting level. Receptor agonists induced sustained contraction in the absence of external Ca++ which was not followed by the increase in [Ca++]i or MLC phosphorylation. This contraction was followed by the increments in shortening velocity and stiffness. In the rabbit mesenteric artery permeabilized with Staphylococcus aureus, alpha-toxin, norepinephrine and endothelin-1 shifted the Ca(++)-tension curve to the left in the presence of GTP. From these results, it is suggested that high K(+)-induced sustained contraction of vascular smooth muscle is attributable to an increase in [Ca++]i followed by an increase in MLC phosphorylation. In addition to this fundamental mechanism, receptor agonists increase Ca+ sensitivity of MLC phosphorylation when [Ca++]i is higher than resting level resulting in a greater contraction than that induced by high K+ for a given increase in [Ca++]i.(ABSTRACT TRUNCATED AT 250 WORDS)