Effect of forskolin on cytosolic Ca++ level and contraction in vascular smooth muscle

The effects of forskolin, an activator of adenylate cyclase, on cytoplasmic Ca++ level ([Ca++]cyt) measured simultaneously with muscle tension using fura-2-Ca++ fluorescence were examined in isolated smooth muscle of rat aorta. Forskolin decreased muscle tension and [Ca++]cyt in resting aorta whereas both norepinephrine and high K+ solution produced sustained increase in muscle tension and [Ca++]cyt. Addition of forskolin during the sustained contractions decreased muscle tension more strongly than [Ca++]cyt. Norepinephrine-induced contraction was more sensitive to forskolin than high K+-induced contraction. The inhibitory effect of forskolin was attenuated when the concentration of norepinephrine or K+ was increased. Cumulative addition of norepinephrine or K+ induced a concentration-dependent increase in both [Ca++]cyt and muscle tension and a positive [Ca++]cyt-tension correlation was observed. In the presence of 0.1 microM forskolin, the norepinephrine-induced increments in [Ca++]cyt and muscle tension were inhibited without changing the [Ca++]cyt-tension relationship. In the presence of a higher concentration (1 microM) of forskolin, muscle tension was inhibited more strongly with only a small additional decrease in [Ca++]cyt resulting in a shift of the [Ca++]cyt-tension relationship. Norepinephrine induced transient increments in [Ca++]cyt and muscle tension in Ca++-free solution and forskolin inhibited these changes. These results suggest that forskolin has concentration-dependent inhibitory effects on vascular contractility to decrease [Ca++]cyt at lower concentrations and to decrease the sensitivity of contractile elements to Ca++ at higher concentrations.     

Inhibitory effects of a synthetic atrial peptide on contractions and 45Ca fluxes in vascular smooth muscle

The mechanism of a synthetic atrial peptide (APII)-induced inhibition of smooth muscle contractility was investigated by studying its effects on tension development and 45Ca fluxes in isolated rabbit aorta. APII (10(-9) to 10(-7) M) produced a dose-dependent relaxation of contractions produced by alpha adrenoceptor activation with norepinephrine (NE; 10(-6) M). APII was a potent relaxant of NE contraction with an IC50 = 1.1 X 10(-8) M, with 10(-7) M APII causing a 97% relaxation. APII also produced a dose-dependent inhibition of NE contraction when added to the resting muscle before the exposure to NE. The relaxing effects of APII were found to be endothelium independent. In contrast, APII was only marginally effective in relaxing high-K+ contraction, with 10(-7) M APII causing only 17% relaxation. Furthermore, when a NE contraction was obtained on top of a high-K+ contraction, APII was still capable of relaxing the NE component. APII was similarly more effective in inhibiting NE-stimulated 45Ca influx than high-K+-stimulated 45Ca influx, indicating selective action of APII on the receptor-operated Ca++ channels. This was in contrast to D600, a well known Ca++ antagonist, which had a more selective inhibitory effect on the potential-operated Ca++ channels. The data presented indicate that APII is a potent relaxant of contractions produced by receptor-agonists involving 45Ca influx through receptor-operated Ca++ channels. APII may also prove to be a very useful tool to further distinguish and define receptor-operated Ca++ channels and potential-operated Ca++ channels in vascular smooth muscle.     

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)     

Cromakalim effects of acetylcholine-induced changes in cytosolic calcium and tension in swine trachealis.

The effects of cromakalim, an ATP-sensitive K+ channel activator, on changes in cytosolic calcium concentration [( Ca++]i) and tension induced by acetylcholine (ACh; 0.1-10 microM) were examined in swine tracheal smooth muscle. Cromakalim (10 microM) hyperpolarized muscle cells by approximately 18 mV from -58 mV (resting membrane potential) to -76 mV. Cromakalim relaxed muscle contractions evoked by ACh at a concentration of 0.1 microM, but not at higher concentrations. Measurement of [Ca++]i using Fura-2 demonstrated that except at 0.1 microM ACh, cromakalim did not alter peak increases in [Ca++]i. At 0.1 microM ACh, the peak transient was decreased, but not eliminated. Cromakalim reduced steady-state increases in [Ca++]i at ACh less than or equal to 1 microM, but not 10 microM ACh. Tension was similarly affected. These data suggest that ACh-induced increases in steady-state [Ca++]i and tension are inhibited by cromakalim-induced hyperpolarization. The initial ACh-induced transient increase in [Ca++]i is not greatly altered. Cromakalim did not alter the transient peak tension and [Ca++]i relationship. The relationship between steady-state [Ca++]i/tension (EC50 = 321 nM) obtained for control, cromakalim inhibition and after glibenclamide reversal of cromakalim inhibition falls to the left of the peak transient [Ca++]i/tension relationship (EC50 = 587 nM). Thus, the Ca++ sensitivity of the contractile proteins during steady-state stimulation by ACh was increased from that at rest. We conclude that electromechanical coupling is important in ACh-induced contraction at concentrations less than 1 microM. Pharmacomechanical coupling with little or no sensitivity to changes in potential is important at higher ACh concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-independent activation of contractile apparatus in smooth muscle by calyculin-A

Calyculin-A (CL-A), a novel marine toxin isolated from Discodermia calyx, caused contraction in the smooth muscle of guinea pig taenia ceci and rat aorta in the presence or absence (with 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) of external Ca++ at concentrations ranging from 1 X 10(-8) to 1 X 10(-6) M. In the presence of external Ca++, the contraction induced by CL-A was accompanied by an increase in the cytosolic free Ca++ concentration [( Ca++]cyt) as measured by the fluorescence indicator fura-2. Verapamil (3 X 10(-6) M) inhibited the increase in [Ca++]cyt, but not tension development caused by CL-A. In the absence of external Ca++, CL-A still caused contraction without changing [Ca++]cyt. Thus, from studies with intact smooth muscle it was demonstrated that, in the absence of external Ca++, CL-A can induce a contraction that was not accompanied by an increase in [Ca++]cyt. In permeabilized taenia, CL-A caused contraction in the absence of Ca++ (with 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid) at concentrations similar to those required to contract intact tissue. This contraction was inhibited by the nonselective kinase inhibitors such as amiloride (1 X 10(-3) M) and K-252a (2 X 10(-5) M). Low concentrations of Ca++ (approximately 1 X 10(-6) M) augmented the CL-A-induced contraction in the permeabilized taenia. In native actomyosin prepared from chicken gizzard CL-A induced phosphorylation of the 20 kDa myosin light chain (MLC) in the absence of Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting rabbit mesenteric artery smooth muscle sensitivity to calcium antagonists

The sensitivity of rabbit isolated superior mesenteric artery to Ca++ antagonists was examined under various conditions. Relaxation dose-response curves for D600 or nifedipine were generated, and IC50 values were calculated. In the first series of experiments, D600 or nifedipine IC50 was found to be 20-25-fold greater for norepinephrine (NE, 5 microM) contraction than for 80 nM K+ contraction. Even when the tissues were depolarized with 80 mM K+ before NE contraction, D600 or nifedipine IC50 still remained significantly greater compared with 80 mM K+ alone and remained closer to that during NE alone. Also a protocol was designed to study NE-induced phasic contraction in EGTA-physiological salt solution (a functional indicator of intracellular Ca++ release) as well as NE-induced sustained contraction after readdition of Ca++. The effects of varying [K+]ex (0-80 nM range) on NE-induced [Ca++]i release as well as on the D600 IC50 for NE contraction was studied. Increasing [K+]ex was found to enhance NE-sensitive [Ca++]i release and lower the D600 IC50 for NE contraction. Thus, conditions causing an increase in the ability of NE to cause [Ca++]i release were associated with an increase in the sensitivity of NE contraction to D600. These data provide functional evidence that the receptor-agonist sensitive Ca++ influx process in vascular smooth muscle is not solely regulated by changes in membrane potential. Additional mechanisms, such as a modulatory role of [Ca++]i release, in this process are implicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of nicorandil on cytosolic calcium concentrations in quin2-loaded rat aortic vascular smooth muscle cells in primary culture

We made use of quin2 microfluorometry to observe the effects of nicorandil (2-nicotinamidoethyl nitrate) on cytosolic Ca++ concentrations [( Ca++]i) in rat aortic vascular smooth muscle cells in primary culture. Regardless of whether cells were at rest, in a state of Ca++-depletion or at K+-depolarization, nicorandil rapidly and dose-dependently decreased [Ca++]i to the lower steady-state level. Nicorandil dose-dependently inhibited norepinephrine-induced Ca++ transients in physiological salt solution containing 1 mM Ca++. Nicorandil accelerated the reduction of [Ca++]i observed when the cells were exposed to Ca++-free solution. When the cells were treated with nicorandil in Ca++-free solution, Ca++ transients induced by the first application of caffeine were little affected, but those induced by subsequent repetitive caffeine applications were reduced strongly and progressively. In contrast, pretreatment with nicorandil markedly inhibited Ca++ transients induced by the first application of norepinephrine, in Ca++-free solution. These effects of nicorandil on [Ca++]i and Ca++ transients were similar to those seen with nitroglycerin. The denitrated compound of nicorandil, N-(2-hydroxyethyl)nicotinamide, had no such effect. Thus, it is apparently the nitrate moiety of the chemical structure by which nicorandil actively and strongly reduces [Ca++]i in vascular smooth muscle cells. The reduction of [Ca++]i by nicorandil may result in a decrease in Ca++ in the norepinephrine-sensitive store; hence, the reduction of [Ca++]i elevation by norepinephrine.     

Feedback inhibition of cyclic adenosine monophosphate-stimulated Na+ transport in the rabbit cortical collecting duct via Na(+)-dependent basolateral Ca++ entry.

Arginine vasopressin (AVP) transiently stimulates Na+ transport in the rabbit cortical collecting duct (CCD). However, the sustained effect of both AVP and its putative second messenger, cyclic adenosine monophosphate (cAMP), on Na+ transport in the rabbit CCD is inhibitory. Because maneuvers that increase [Ca++]i inhibit Na+ transport, the effects of AVP and cell-permeable cAMP analogues, on [Ca++]i were investigated in fura-2-loaded in vitro microperfused rabbit CCDs. Low-dose AVP (23-230 pM) selectively stimulated Ca++ influx, whereas 23 nM AVP additionally released calcium from intracellular stores. 8-chlorophenylthio-cAMP (8CPTcAMP) and 8-bromo-cAMP (8-Br-cAMP) also increased CCD [Ca++]i. The 8CPTcAMP-stimulated [Ca++]i increase was totally dependent on basolateral [Ca++]. In the absence of cAMP, peritubular Na+ removal produced a marked increase in [Ca++]i, which was also dependent on bath [Ca++], suggesting the existence of basolateral Na+/Ca++ exchange. Luminal Na+ removal in the absence of cAMP did not alter CCD [Ca++]i, but it completely blocked the cAMP-stimulated [Ca++]i increase. Thus the cAMP-dependent Ca++ increase is totally dependent on both luminal Na+ and basolateral Ca++, suggesting the [Ca++]i increase is secondary to cAMP effects on luminal Na+ entry and its coupling to basolateral Na+/Ca++ exchange. 8CPTcAMP inhibits lumen-to-bath 22Na flux [JNa(l-b)] in CCDs bathed in a normal Ca++ bath (2.4 mM). However, when bath Ca++ was lowered to 100 nM, a maneuver that also blocks the 8CPTcAMP [Ca++]i increase, 8CPTcAMP stimulated, rather than inhibited JNa(l-b). These results suggest that cAMP formation initially stimulates CCD Na+ transport, and that increased apical Na+ entry secondarily activates basolateral Ca++ entry. The cAMP-dependent [Ca++]i increase leads to inhibition Na+ transport in the rabbit CCD.     

Myosin phosphorylation-independent contraction induced by phorbol ester in vascular smooth muscle.

In isolated rat aorta, carotid artery, tail artery, rabbit aorta and mesenteric artery, but not in ear artery, 1 microM 12-deoxyphorbol 13-isobutyrate (DPB) induced a sustained contraction. However, DPB increased cytosolic Ca++ concentration ([Ca++]i) only in rat aorta and carotid artery. Similar results were obtained with phorbol 12,13-dibutyrate, although the inactive phorbol ester, 4-alpha-phorbol 12,13-dibutyrate, was ineffective. In rat aorta, DPB-induced contraction was followed by an increase in 20 kDa myosin light chain (MLC) phosphorylation. Both contraction and MLC phosphorylation stimulated by DPB were greater than those due to high K+ for a given increase in [Ca++]i. A Ca++ channel blocker, verapamil, decreased the DPB-induced increments in [Ca++]i and MLC phosphorylation to their respective resting levels, although contraction was inhibited only slightly. In the absence of external Ca++ (with 0.5 mM ethyleneglycol bis(beta-aminoethyl-ether)tetraacetic acid), DPB induced sustained contraction without increasing [Ca++]i or MLC phosphorylation. This contraction was followed by an increase in stiffness and force recovery after a shortening step. These results suggest that the contraction induced by DPB in rat aorta is due to increase in [Ca++]i followed by MLC phosphorylation and Ca++ sensitization of MLC phosphorylation. In the presence of verapamil or in the absence of external Ca++, DPB may increase cross-bridge cycling by activating an unknown mechanism that is not dependent on an increase in MLC phosphorylation.     

Concentration-dependence of acetylcholine-induced changes in calcium and tension in swine trachealis

Acetylcholine (ACh)-induced increases in intracellular calcium concentration ([Ca++]i) and tension were measured simultaneously in swine tracheal smooth muscle strips loaded with the calcium-sensitive fluorescent dye Fura-2. ACh at concentrations greater than or equal to 3 x 10(-8) M induced concentration-dependent increases in tension which reached a maximum at 10(-4) M. Increases in [Ca++]i occurred at all [ACh] tested (10(-8) to 10(-4) M). After addition of ACh at concentrations greater than 3 x 10(-7) M, [Ca++]i increased rapidly to a concentration dependent-peak then declined to a concentration-independent steady state approximately 250 nM above the resting [Ca++]i of 257 +/- 12 nM. There was a steep relationship (slope factor greater than 3) between the peak tension and the peak [Ca++]i reached at each [ACh]. The rate of decline of [Ca++]i to the steady state at [ACh] greater than 73 x 10(-7) M was well correlated with the peak [Ca++]i reached. We conclude that the peak increase in calcium induced by ACh sets the level of tension to be attained and the rate of decline of the transient increase in [Ca++]i. The steady-state [Ca++]i is sufficient for maintenance of tension.     

Diastolic dysfunction in hypertrophic cardiomyopathy. Effect on active force generation during systole.

We tested the hypothesis that intracellular Ca++ [( Ca++]i) overload underlies the diastolic dysfunction of patients with hypertrophic cardiomyopathy. Myocardial tissue was obtained at the time of surgery or transplantation from patients with hypertrophic cardiomyopathy and was compared with control myocardium obtained from patients without heart disease. The isometric contractions and electrophysiologic properties of all myocardial specimens were recorded by standard techniques and [Ca++]i was measured with the bioluminescent calcium indicator aequorin. In contrast to the controls, action potentials, Ca++ transients, and isometric contraction and relaxation were markedly prolonged in the hypertrophic myocardium, and the Ca++ transients consisted of two distinct components. At 38 degrees C and 1 Hz pacing frequency, a state of relative Ca++ overload appeared develop, which produced a rise in end-diastolic [Ca++]i, incomplete relaxation, and fusion of twitches with a resultant decrease in active tension development. We also found that drugs with increase [Ca++]i, such as digitalis, exacerbated these abnormalities, whereas drugs that lower [Ca++]i, such as verapamil, or agents that increase cyclic AMP, such as forskolin, prevented them. These results may explain why patients with hypertrophic cardiomyopathy tolerate tachycardia poorly, and may have important implications with regard to the pharmacologic treatment of patients with hypertrophic cardiomyopathy.     

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.     

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.     

Effects of nitroglycerin on ATP-induced Ca(++)-mobilization, Ca(++)-activated K channels and contraction of cultured smooth muscle cells of porcine coronary artery.

Extracellular application of ATP transiently increases the cytosolic-free Ca++ concentration ([Ca++]i) in cultured smooth muscle cells of porcine coronary artery, and this activates large conductance Ca(++)-activated K (Kca) channels. In the present study effects of nitroglycerin (NG) and 4-aminopyridine (4-AP) on [Ca++]; and contraction were studied. 4-AP blocked Kca channels and enhanced the rise of [Ca++]i with oscillation, which led to contraction of the cells. NG activated the Kca channels of 300 picosiemens and inhibited 4-AP-induced contraction and oscillation of [Ca++]i. These results suggest that the vasorelaxant effect of NG involves hyperpolarization of the cell membrane by activating the Kca channels. NG also cause rapid decrease of [Ca++]i during the Ca(++)-mobilization by ATP in Ca-free solution. Similar effects were observed with cyclic GMP, suggesting that the effects of NG on the Kca channels and [Ca++]i were mediated by cyclic GMP.     

Receptor-coupled G proteins mediate contraction and Ca++ mobilization in isolated intestinal muscle cells.

Guanosine 5'-O-(gamma-thio)triphosphate (GTP[S]), NaF and cholecystokinin-octapeptide (CCK-8) were used to examine the participation of G proteins in agonist-induced contraction of smooth muscle cells isolated separately from circular and longitudinal muscle layer of guinea pig intestine. All three agents stimulated inositol 1,4,5-triphosphate (InsP3) production and protein kinase C activity to the same extent in permeabilized (GTP[S] and CCK-8) and nonpermeabilized (NaF and CCK-8) muscle cells. InsP3 production was 9 to 13 times higher in circular muscle cells consistent with preferential hydrolysis of phosphatidylinositol 4,5-biphosphate in this cell type. InsP3 production and protein kinase C activation in permeabilized muscle cells were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM). Maximal concentrations of GTP[S] (100 microM), CCK-8 (1 nM) and InsP3 (1 microM) elicited similar increases in [Ca++]i, net 45Ca++ efflux and contraction in permeabilized circular, but not longitudinal, muscle cells [( Ca++]i: 224 +/- 35 nM, 279 +/- 29 nM and 288 +/- 45 nM increase above basal level; 45Ca++ efflux: 35 +/- 2%, 34 +/- 3% and 37 +/- 3% decrease in cell Ca++ content; contraction: 26 +/- 2%, 24 +/- 2% and 25 +/- 2% decrease in cell length). The responses to GTP[S] and CCK-8 were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM) and heparin (10 micrograms/ml), whereas the response to InsP3 was abolished by heparin only. Maximal concentrations of NaF and CCK-8 elicited similar increases in [Ca++]i and contraction in nonpermeabilized circular and longitudinal muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Interaction of hydralazine and hydrazone derivatives with contractile mechanisms in rabbit aortic smooth muscle

The mechanism of action and relative potency of hydralazine (H) and tow hydrazone derivatives were investigated using isolated rabbit aortic strips. H, hydralazine acetone hydrazone (HA) and hydralazine butanone hydrazone (HBH) relaxed established K+ and norepinephrine (NE) contractures, and inhibited the development of contractures to these two agents on preincubation. H, HA and HBH increased the threshold to Ca++ and decreased the maximum tension responses during K+-Ca++-contractures (HA greater than H, P less than .05; HBH greater than H P less than .01). The Ca++-dependent and Ca++-independent components of NE contractures were both inhibited by H, HA and HBH. NE contractures were more sensitive to the effects of H than K+ contractures. These results are consistent with the conclusion that H and hydrazone derivatives produce effects on vascular muscle both by interactions with the fluxes of Ca++ from the extracellular space and effects on release from cell stores. However, other possibilities need to be assessed experimentally.     

Histamine-induced intracellular free Ca++, inositol phosphates and electrical changes in murine N1E-115 neuroblastoma cells

Apparent intracellular free Ca++ concentration [(Ca++]i) was measured in differentiated N1E-115 neuroblastoma by microinjecting cells with aequorin (estimated intracellular concentration, 4 microM) and measuring light emission. Histamine produced a transient, dose-dependent increase in [Ca++]i. Pyrilamine blocked completely the response to histamine whereas cimetidine had no effect. Omitting Ca++ from the external medium reversibly blocked the response. As well as a rise in [Ca++]i, histamine caused a concomitant cell hyperpolarization that was not blocked by ouabain, low Cl-, tetraethylammonium chloride/tetradotoxin or metiamide but was blocked by apamin and pyrilamine. A secondary small depolarization caused by histamine was also blocked by apamin but not by ouabain, low Cl- or tetraethylammonium chloride/tetrodotoxin. Direct iontophoretic injection of Ca++ into cells caused only hyperpolarization. Injection of inositol 1,4,5-trisphosphate [IP3(1,4,5)] caused an increase in [Ca++]i and rapid hyperpolarization. Inositol 1,3,4-trisphosphate [IP3(1,3,4)] caused an increase in [Ca++]i, rapid hyperpolarization and a slower depolarization. Repeated injections of IP3(1,3,4) led to a diminished [Ca++]i response and decreased hyperpolarization but had no effect on depolarization. Inositol 1,3,4,5-tetrakisphosphate was without effect on [Ca++]i or on cellular membrane potential. The results suggest that histamine causes an H1 receptor-dependent increase in [Ca++]i, probably by the increased entry of extracellular Ca++, although there may be a contribution from intracellular Ca++ released by IP3(1,4,5). The increase in [Ca++]i activates K+ channels leading to cell hyperpolarization. IP3(1,3,4) formed from inositol 1,3,4,5-tetrakisphosphate, which is itself a product of IP3(1,4,5), causes a slower depolarization by a mechanism that does not involve Na+ channels or an increase in [Ca++]i.     

The Na(+)-dependent release of endogenous dopamine and noradrenaline from rat brain synaptosomes

The involvement of intrasynaptosomal-free Ca++ concentration [( Ca++]i) in Na(+)-dependent release of endogenous dopamine and noradrenaline from rat brain synaptosomes was studied. The release of endogenous dopamine and noradrenaline from rat whole brain synaptosomes were measured using high-performance liquid chromatography with electrochemical detector. The change of [Ca++]i was measured fluorometrically using a Ca++ indicator, Quin-2. Whether extracellular Ca++ was present or not, 30 microM veratridine, a Na(+)-ionophore, increased the release of endogenous dopamine and noradrenaline. In the presence of 1.25 mM Ca++, 30 microM veratridine increased [Ca++]i. In contrast, in the absence of extracellular Ca++, veratridine did not affect [Ca++]i. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) increased the release of dopamine and noradrenaline in Ca-Mg-free medium. This stimulatory effect of EGTA seemed to be the result of an increase in the influx of Na+ through Ca++ channels in the absence of divalent cation. In Ca-Mg-free medium, EGTA caused a slight decrease in [Ca++]i. The EGTA-stimulated release of dopamine and noradrenaline was blocked by La which also significantly blocked the decrease in [Ca++]i observed after the addition of EGTA. These results suggest that the Na(+)-dependent release of dopamine and noradrenaline may not depend on a change in [Ca++]i. Veratridine (30 microM)-induced release of dopamine and noradrenaline were detected simultaneously. However, the time needed to induce the maximal stimulatory effect of veratridine on the release of dopamine was apparently shorter than that of noradrenaline. This delay might suggest that the Na(+)-dependent release process of dopamine is not similar to that of noradrenaline.     

Effect of ouabain on the concentration of free cytosolic Ca++ and on contractility in cultured rat cardiac myocytes

Effects of ouabain on [Ca++]i and on contractility was measured in quin2 and fura2 loaded cultured neonatal rat cardiac myocytes. Addition of ouabain (5 x 10(-8) to 5 x 10(-6) M) to cultured myocytes exposed to balanced buffered salt solution (BSS) caused a transient increase in [Ca++]i, followed by slow oscillations for about 10 min, and by an elevated steady state level of [Ca++]i thereafter. Concentrations of ouabain between 10(-7) and 5 x 10(-7) M caused an increase in the amplitude of systolic motion (ASM) whereas concentrations above 10(-6) caused a decrease in the ASM, an increase in the beating frequency and an upward shift of the base line, indicating impaired relaxation. When ouabain was added to cardiac myocytes exposed to Ca++-free BSS the increase in [Ca++]i was not observed, but only a transient decrease. To investigate the effect of [K+]o on the ouabain-induced changes in [Ca++]i, ouabain was added to cells exposed to BSS containing low K+ concentration (1 mM instead of 5 mM in balanced BSS). In this medium the increase in ASM by ouabain was similar to that in balanced BSS. Addition of ouabain caused a transient decrease in [Ca++]i. There was no initial increase in [Ca++]i and the steady state level of [Ca++]i was not elevated as compared with the same cells before the addition of ouabain. Similar results were observed in cells loaded with quin2 or with fura2. In view of these results the mechanism of action of ouabain on cardiac myocytes is discussed.