cyclic-gmp and 3--4--dichlorobenzamil

The oxidant species peroxynitrite, the reaction product of nitric oxide (NO.) and superoxide, has been implicated in several pathophysiological conditions of the heart. Here, we studied the mechanism of peroxynitrite-induced cardiac depression using specific drugs and simultaneous analyses of myocardial function and intracellular Ca(2+) ([Ca(2+)](i)).. Rat hearts were perfused retrogradely and left ventricular function (balloon method) and [Ca(2+)](i) transients were recorded on a beat-to-beat basis using the aequorin bioluminescence method. Peroxynitrite was infused at 10.8+/-0.93 microM via sideline for 10 min, followed by a 15-min recovery period to monitor irreversible effects. Test drugs were infused prior to and during peroxynitrite application.. Peroxynitrite depressed left ventricular developed pressure (LVDevP; -40%), yet increased systolic and diastolic [Ca(2+)](i) (1.3- and 2.3-fold, respectively; n=12). When Ca(2+) entry through Ca(2+) channels or Na(+)/Ca(2+) exchange transport was blocked using nicardipine (1 microM, n=3) or dichlorobenzamil (30 microM, n=5), respectively, cells showed lower [Ca(2+)](i) and accentuated negative inotropic action in response to peroxynitrite. Peroxynitrite slowed left ventricular relaxation and [Ca(2+)](i) transients, both of which were not affected by extracellular Ca(2+) restriction. Importantly, the oxidant greatly depressed myofilament responsiveness to Ca(2+), which was partly antagonized by Rp-8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate (Rp-cGMPS), an inhibitor of cGMP-dependent protein kinase. Also, the inhibitor partially restored left ventricular contractility (n=6). Peroxynitrite stimulated cardiac cGMP production and coronary cGMP efflux (n=6). Decomposed peroxynitrite had no effect in any of the tests.. Peroxynitrite depresses myocardial contractility by decreasing the ability of Ca(2+) to trigger contraction, and this effect is partly mediated by the cGMP/cGMP-dependent protein kinase pathway.

Topics: 1-Methyl-3-isobutylxanthine; Amiloride; Animals; Calcium; Calcium Channel Blockers; Cyclic GMP; Enzyme Inhibitors; Female; Homeostasis; Male; Myocardial Contraction; Myocardium; Nicardipine; Oxidants; Perfusion; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase

1. Late gestation fetal sheep were chronically catheterised in utero to allow measurement of the rate of production of lung liquid (Jv) from 132-143 days gestation (term, 147 days), and to test the hypothesis that cyclic nucleotide gated cation channels mediate a component of fetal lung liquid absorption. 2. In eight experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.005) reduction in Jv from +18. 12 +/- 3.52 to -10.27 +/- 5.26 ml h-1. Dichlorobenzamil (a blocker of cyclic nucleotide gated cation channels) at 1.5 x 10-5 M did not significantly inhibit the adrenaline-induced lung liquid absorption (Jv dichlorobenzamil, -5.77 +/- 2.78 ml h-1; P > 0.1) when the data were grouped, but did exert a significant gestational effect (r = 0. 90, P < 0.01). Subsequent addition of 10-4 M amiloride (a blocker of epithelial sodium channels) abolished the adrenaline-induced absorption of lung liquid (mean Jv amiloride, +6.45 +/- 1.59 ml h-1; P < 0.01 relative to Jv adrenaline and P < 0.005 relative to Jv dichlorobenzamil). 3. In seven experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.0005) reduction in Jv from +18.95 +/- 2. 98 to -10.08 +/- 3.75 ml h-1. Amiloride (10-4 M) inhibited the adrenaline response (Jv amiloride, +5.46 +/- 1.09 ml h-1; P < 0.005). However, subsequent addition of 1.5 x 10-5 M dichlorobenzamil had no additive effect to that of amiloride (Jv dichlorobenzamil, +4.58 +/- 0.93 ml h-1; P > 0.1). 4. In six experiments, the cGMP analogue 8-Br-cGMP at 10-4 M caused a significant (P < 0.05) reduction in Jv from +15.20 +/- 2.81 to +11.63 +/- 1.71 ml h-1. Amiloride (10-4 M) did not block the effect of 8-Br-cGMP (Jv amiloride, +14.00 +/- 2.49 ml h-1; not significantly different from 8-Br-cGMP). Subsequent addition of 1.5 x 10-5 M dichlorobenzamil also did not block the effect of 8-Br-cGMP (Jv dichlorobenzamil, +11.37 +/- 1.22 ml h-1; not significantly different from either Jv amiloride or Jv 8-Br-cGMP). 5. We conclude that, in fetal sheep, neither adrenaline nor cGMP stimulate lung liquid absorption by actions on cyclic nucleotide gated cation channels, and that the effect of cGMP on fetal lung liquid secretion is minor and does not involve epithelial sodium channels. The effect of dichlorobenzamil, when given before amiloride, was probably due to an action on amiloride sensitive epithelial sodium channels.

Topics: Amiloride; Animals; Biological Transport; Catheterization; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Diuretics; Epinephrine; Extravascular Lung Water; Fetal Organ Maturity; Ion Channels; Lung; Sheep; Sodium Channel Blockers

Speract, a decapeptide from Strongylocentrotus purpuratus sea urchin eggs, transiently stimulates a membrane guanylyl cyclase and activates a K(+)-selective channel that hyperpolarizes sperm. However, previous studies of sperm and of sperm membrane vesicles reached conflicting conclusions about the mechanisms that open these channels. We find that speract hyperpolarizes and increases the cGMP content of flagellar vesicles. We confirm previous findings that intravesicular GTPgammaS and GTP enhance this hyperpolarization, but not GDPbetaS. The G protein activators AlF(-)(4) and mastoparan also are ineffective. Thus, it is unlikely that a G protein participates in the speract response. In contrast, hyperpolarization responses to speract are increased by 3-isobutyl-1-methylxanthine, which preferentially inhibits cGMP-selective phosphodiesterases of sperm, and the 8Br-cGMP derivative hyperpolarizes vesicles in the absence of speract. The responses to speract and to 8Br-cGMP have similar ionic selectivities (K(+) > Rb(+) > > Li(+) > Na(+)) and sensitivities to the channel blockers 4-aminopiridine and 3, 4-dichlorobenzamil, indicating that they likely result from opening of the same K(+) channel. Inhibitors that preferentially inhibit cAMP-selective phosphodiesterases do not alter responses to speract, and permeant cAMP analogs do not hyperpolarize vesicles. In addition, inhibitors of protein kinases and phosphatases fail to alter vesicle hyperpolarization by speract. The increase in vesicular cGMP content produced by speract therefore may directly mediate opening of the channel that hyperpolarizes sperm membrane vesicles. Similar mechanisms presumably operate in intact sperm.

Topics: 4-Aminopyridine; Amiloride; Animals; Cations, Monovalent; Cell Membrane; Cyclic GMP; Enzyme Inhibitors; GTP-Binding Proteins; Guanosine Diphosphate; Male; Membrane Potentials; Oligopeptides; Phosphoprotein Phosphatases; Potassium Channel Blockers; Potassium Channels; Protein Kinase Inhibitors; Sea Urchins; Signal Transduction; Sperm Tail; Spermatozoa; Thionucleotides

1. Hyperpolarizing scallop photoreceptors, like vertebrate rods, use cGMP as an internal messenger and their light-sensing structure is also of ciliary origin. To ascertain possible functional similarities between the light-sensitive conductances in the two classes of visual cells, we examined in scallop photoreceptors the effects of several antagonists of the photocurrent of rods. 2. Extracellular application of L-cis-diltiazem rapidly and reversibly suppressed the photocurrent. The effect was stereospecific and dose dependent, with a K1/2 of approximately 400 microM. Intracellular dialysis at lower doses (100-200 microM) also induced a substantial inhibition. 3. L-cis-Diltiazem reduced the light-activated conductance without shifting the intensity-response curve. Furthermore, the drug also blocked the current directly evoked by application of cGMP. These observations indicate that the inhibitory effects result from blockage of the conductance, rather than from impairment of the activating cascade. 4. The fractional blockage increased e-fold per approximately 55 mV depolarization, regardless of the side of drug application, as if the charged form of L-cis-diltiazem can only access the blocking site from the intracellular compartment. 5. The amiloride derivative 3',4'-dichlorobenzamil potently suppressed the photocurrent (K1/2 approximately 5 microM), without affecting its kinetics or operating range. Amiloride itself was also effective at higher concentrations. 6. The pharmacological resemblance of these light-dependent channels to those of rods and cones indicates that significant aspects of the transduction cascade are conserved across disparate sensory cells of ciliary origin.

Topics: Amiloride; Animals; Calcium Channel Blockers; Cyclic GMP; Diltiazem; Dose-Response Relationship, Drug; Electric Conductivity; Ion Channel Gating; Mollusca; Patch-Clamp Techniques; Photoreceptor Cells, Invertebrate; Retinal Rod Photoreceptor Cells

We have previously demonstrated that activation of the Na(+)-Ca2+ exchanger in the reverse mode causes Ca2+ influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na(+)-Ca2+ exchanger is involved in agonist-induced Ca2+ signaling in cultured rat astrocytes. The astrocytic intracellular Ca2+ concentration ([Ca2+]i) was increased by L-glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin-induced increase in [Ca2+]i. The NA-induced Ca2+ signal was also attenuated by S-nitroso-L-cysteine and 8-bromo cyclic GMP, whereas it was enhanced by 3,4-dichlorobenzamil, an inhibitor of the Na(+)-Ca2+ exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na(+)-Ca2+ exchanger enhanced the ionomycin-induced increase in [Ca2+]i and blocked the effects of SNP and 8-bromo cyclic GMP in reducing the NA-induced Ca2+ signal. Furthermore, the ionomycin-induced Ca2+ signal was enhanced by removal of extracellular Na+ and pretreatment with ascorbic acid. These findings indicate that the Na(+)-Ca2+ exchanger is a target for NO modulation of elevated [Ca2+]i and that the exchanger plays a role in Ca2+ efflux when [Ca2+]i is raised above basal levels in astrocytes.

Topics: Adenosine Triphosphate; Amiloride; Animals; Animals, Newborn; Astrocytes; Base Sequence; Bucladesine; Calcium; Carrier Proteins; Cells, Cultured; Cerebral Cortex; Cyclic GMP; Cysteine; Glutamic Acid; Ionomycin; Kinetics; Nitric Oxide; Nitroprusside; Norepinephrine; Oligonucleotides, Antisense; Phosphatidylinositols; Rats; Rats, Sprague-Dawley; S-Nitrosothiols; Signal Transduction; Sodium-Calcium Exchanger

Vertebrate rod photoreceptors in the dark maintain an inward current across the outer segment membrane. The photoresponse results from a light-induced suppression of this dark current. The light-regulated current is not sensitive to either tetrodotoxin or amiloride, potent blockers of Na+ channels. Here, we report that a derivative of amiloride, 3',4'-dichlorobenzamil (DCPA), completely suppresses the dark current and light response recorded from rod photoreceptors. DCPA also blocks a cyclic GMP-activated current in excised patches of rod plasma membrane and a cGMP-induced Ca++ flux from rod disk membranes. These results are consistent with the notion that the Ca++ flux mechanism in the disk membrane and the light-regulated conductance in the plasma membrane are identical. DCPA also inhibits the Na/Ca exchange mechanism in intact rods, but at a 5-10-fold-higher concentration than is required to block the cGMP-activated flux and current. The blocking action of DCPA in 10 nM Ca++ is different from that in 1 mM Ca++, which suggests either that the conductance state of the light-regulated channel may be modified in high and low concentrations of Ca++, or that there may be two ionic channels in the rod outer segment membrane.

Topics: Amiloride; Animals; Calcium; Cattle; Cyclic GMP; In Vitro Techniques; Light; Neural Conduction; Photoreceptor Cells; Rana catesbeiana; Rod Cell Outer Segment