Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.     

Involvement of calmodulin and myosin light chain kinase in activation of mTRPC5 expressed in HEK cells

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CaM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 microM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 microg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPgammaS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPgammaS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.     

Inhibition of mitosis in PtK2 cells by CAPP1-calmodulin

Various indirect evidence has indicated that calcium ions and the calcium-binding regulator protein, calmodulin, may regulate mitosis in higher eukaryotes. We have used the competitive antagonist, CAPP1-calmodulin, to antagonize intracellular calmodulin and test the hypothesis that calmodulin serves as a regulator of mitosis. We find that CAPP1-calmodulin inhibits the transit of cells through metaphase at estimated intracellular concentrations up to that of native calmodulin; beyond that level, the inhibition of mitosis vanishes. The membrane-permeant anticalmodulin agents, W7 and calmidazolium, also inhibit the progress of cells through metaphase. The similarity of the inhibitory curves for CAPP1-calmodulin, W7, and calmidazolium suggests that all these agents inhibit mitosis by antagonizing intracellular calmodulin. In order to test whether this inhibition of metaphase transit is due to an effect of the agents on intracellular free calcium, we used the calcium indicator Fura-2 to measure intracellular calcium levels after CAPP1-calmodulin injection or during calmidazolium treatment. We found that, while intracellular calcium levels are modestly elevated during calmidazolium treatment, they were unaffected by CAPP1-calmodulin, a result suggesting that mitosis inhibition was not due to an effect on intracellular free calcium. The reasons for the anomalous dose-response behavior of these drugs are not known; however, the behavior of cells at drug levels below the point of anomaly supports the hypothesis that calmodulin acts as a regulator of mitosis in these cells.     

The lens as a nonuniform spherical syncytium.

The effective intracellular resistivity Ri of the ocular lens is a measure of the coupling between cells. Since degradation of coupling may accompany cataracts, measurements of Ri are of considerable interest. Experimental results show that the lens is a nonuniform syncytium in which Ri is much higher in the nuclear region than in the cortex. A theory describing the lens as a radially nonuniform spherical syncytium is proposed, solved, and described as a simple equivalent circuit. The impedance of the lens is measured with new circuitry which permits the accurate application and measurement of current and voltage over a wide bandwidth without arbitrary compensation of unstable capacitances. The fit of the nonuniform theory to experimental data is satisfactory and the parameters determined are consistent with theoretical assumptions. In the outer region (cortex) of the lens Ri = 2.4 k omega-cm, probably as a consequence of differences in coupling and cytoplasmic resistivity. The radial resistivity of the cortex is some five times the circumferential resistivity, demonstrating a marked anisotropy in the preparation, probably reflecting the anisotropy in the orientation of lens fibers and distribution of gap junctions. Current can flow in the circumferential direction without crossing from fiber to fiber; current can flow in the radial direction only by crossing from fiber to fiber.     

Phase-shifts of the Bulla ocular circadian pacemaker in the presence of calmodulin antagonists

Previous work has shown that light-induced phase shifts of the Bulla ocular circadian pacemaker require extracellular calcium, suggesting the possibility that the action of calcium as a second messenger via calmodulin is an element in the phase shifting mechanism. The calmodulin antagonists calmidazolium, trifluoperazine (TFP) and W7 were applied with phase shifting light pulses. Light phase shifts were not blocked by calmidazolium or TFP, suggesting that calmodulin does not mediate light-induced phase shifts. Period changes were observed with treatments of both TFP and W7, but not with calmidazolium and are probably not calmodulin-mediated.     

A new peptide (1150Da) selectively activates the calcium-calmodulin sensitive isoform of cyclic nucleotide phosphodiesterase from human myometrium.

The cyclic nucleotide phosphodiesterase enzymatic system is examined in extracts of human myometrium and four individual phosphodiesterase isoforms have been isolated and characterized. A new thermostable peptide, recently purified in rat and calf myometrium, is able to stimulate up to 55-fold, the calcium-calmodulin dependent phosphodiesterase isoform. Activation of cAMP hydrolysis is by far the most marked with a 55-fold maximal stimulation at a concentration of 0.1 microM peptide and a IC50 value estimated at 30nM. For cGMP hydrolysis, the maximal effect (x25) obtained at 40nM peptide is lesser and the IC50 value is in the 10nM range. Furthermore, we verified that classical calmodulin antagonists such as calmidazolium or trifluoroperazine did not change stimulation of the calcium-calmodulin phosphodiesterase by the peptide, indicating that the myometrial peptide is different from calmodulin. To our knowledge, this is the first evidence for such a strong and selective stimulation of one isoform of the phosphodiesterase enzymatic system by a natural peptide.     

Effect of calmidazolium and phorbol ester on catecholamine secretion from adrenal chromaffin cells

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 μM calmidazolium. Catecholamine release due to 55 mM K⁺ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca²⁺ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.     

Time-dependent inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by calmidazolium chloride in rat GH3 cells.

The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P₃-5-phosphatase from GH₃ cells, with an ₅₀ value of 100 μM following a 10-min preincubation with enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.     

Calmodulin antagonists differentiate between Ni(2+)- and Mn(2+)-stimulated phosphatase activity of calcineurin.

The interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazolium, a potent calmodulin antagonist, inhibited the Ni(2+)-stimulated calmodulin-independent phosphatase activity to much the same extent as it did the Ca2+/calmodulin-stimulated activity. Other calmodulin antagonists, such as trifluoperazine, thioridazine, and W-7, also inhibited the Ni(2+)-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn(2+)-stimulated phosphatase activity and the other calmodulin antagonists examined increased the Mn(2+)-stimulated activity, in the absence of calmodulin. With the addition of an equimolar amount, as to the inhibited holoenzyme, of the purified B subunit of calcineurin, the Ni(2+)-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5 microM calmidazolium. When the amount of additional B subunit was increased, the phosphatase activity recovered to 94% of the control level, thereby implying that calmidazolium inhibits the Ni(2+)-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn(2+)-stimulated phosphatase activity also recovered from the inhibition by calmidazolium, but a much larger amount of the B subunit was necessary for the recovery. These results indicate that the Ni(2+)- and Mn(2+)-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni(2+)-stimulated phosphatase activity.     

Developmental changes in the effects of carbachol and morphine on cGMP contents of plasma, heart, and lung of mice

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor--guanylate cyclase system and opioid receptors.     

A proposed mechanism for the potentiation of cAMP-mediated acid secretion by carbachol

Acid secretion in isolated rabbit gastric glands was monitored by the accumulation of [(14)C]aminopyrine. Stimulation of the glands with carbachol synergistically augmented the response to dibutyryl cAMP. The augmentation persisted even after carbachol was washed out and was resistant to chelated extracellular Ca(2+) and to inhibitors of either protein kinase C or calmodulin kinase II. Cytochalasin D at 10 microM preferentially blocked the secretory effect of carbachol and its synergism with cAMP, whereas it had no effect on histamine- or cAMP-stimulated acid secretion within 15 min. Cytochalasin D inhibited the carbachol-stimulated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase due to release from the Ca(2+) store. Treatment of the glands with cytochalasin D redistributed type 3 inositol 1,4,5-trisphosphate receptor (the major subtype in the parietal cell) from the fraction containing membranes of large size to the microsomal fraction, suggesting a dissociation of the store from the plasma membrane. These findings suggest that intracellular Ca(2+) release by cholinergic stimulation is critical for determining synergism with cAMP in parietal cell activation and that functional coupling between the Ca(2+) store and the receptor is maintained by actin microfilaments.     

A proton nuclear magnetic resonance and molecular modeling study of calmidazolium (R24571) binding to calmodulin and skeletal muscle troponin C

1H NMR spectroscopy at 360 MHz has been used to study the interactions between the calmodulin function inhibitor calmidazolium (R24571) and (i) calmodulin (CaM) and (ii) skeletal muscle troponin C (sTnC). One equivalent of racemic calmidazolium binds tightly to CaM and perturbs a number of protein signals, corresponding to residues in both dicalcium-binding domains, in a manner characteristic of slow exchange. Calmidazolium binds with lower affinity to sTnC but still induces widespread perturbations in both domains. Extensive spectral overlap precludes definite assignment of intermolecular nuclear Overhauser effect (NOEs) although intraprotein NOEs do indicate the nature of some drug-induced conformational changes. Relaxation enhancements induced by two spin-labeled calmidazolium analogues demonstrate that several methionine residues of CaM, significantly immobilized by calmidazolium binding, are in fact located at or near its binding sites. These and other residue-specific broadening effects have enabled low resolution models to be constructed of the predominantly hydrophobic drug-binding sites on each domain of CaM. The hydrophobic portions of calmidazolium itself, and its analogues, contact side chains of Ala-15, Leu-18, Phe-19, Val-35, Met-36, Leu-37, Leu-39, Met-51, Met-71, Met-72, and Met-76 in the N-terminal domain of calmodulin, and Ala-88, Val-91, Phe-92, Val-108, Met-109, Leu-112, Phe-141, and Met-145 in its C-terminal domain. The model, and an analogous one of sTnC, can be used to rationalize drug-induced changes in intraprotein NOEs. Issues pertaining to the possible simultaneous binding of calmidazolium to both globular domains of the proteins are discussed in terms of the experimental results and the overall structures of each protein.     

Time-dependent inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by calmidazolium chloride in rat GH3 cells

The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P₃-5-phosphatase from GH₃ cells, with an IC₅₀ value of 100 μM following a 10-min preincubation with enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.     

Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin

We evaluated the effect of several classes of calmodulin inhibitors on the activity of calmodulin prepared from C6 astrocytoma cells and studied the activity of these drugs as inhibitors of the growth of C6 cells in tissue culture. There was a good correlation between the activity of the drugs as inhibitors of calmodulin and their activity as inhibitors of cell growth. The most potent compounds were calmidazolium and melittin as compared to the phenothiazines, trifluoperazine, chlorpromazine, chlorpromazine-sulfoxide or the diphenylbutylpiperidine, pimozide. The mechanism by which the inhibition of calmodulin leads to the death of cells could not be attributed entirely to inhibition of the calmodulin-sensitive cyclic nucleotide phosphodiesterase. Calmodulin is a heat stable, calcium-binding protein involved in numerous biological processes. Recent evidence indicates that calcium and calmodulin may be important for cellular proliferation. For example, this protein changes in concentration during the cell cycle; is involved in the disassembly of the mitotic apparatus; is increased in concentration in rapidly growing hepatomas and in transformed fibroblasts. Weiss and co-workers demonstrated that phenothiazines and structurally similar drugs are capable of binding to and inhibiting the activity of calmodulin. It has been recently observed that certain drugs that inhibit the activity of calmodulin also inhibit the growth of malignant cells in vitro and in vivo. In these studies, however, there was no direct correlation of the effect of the drugs on the calmodulin from the cell type under investigation with cytotoxicity. To learn more about the relationship between a drug's ability to inhibit calmodulin and its antiproliferative activity, we correlated the effect of drugs on the activity of calmodulin prepared from the C6 astrocytoma cell line with their effect on cellular proliferation. Since many inhibitors of calmodulin readily cross the blood-brain barrier and since no acceptable treatment for malignancies of the central nervous system exist, we chose this cell line as a model for elucidating the potential antineoplastic effects of calmodulin inhibitors.     

Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin.

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.     

Extracellular Calmodulin Stimulates the Transplasma Membrane Redox Reaction of Root Protoplasts in Zea mays

Using ferricyanide as the membrane impermeable electron acceptor, the effects of extracellular calmodulin on transplasma membrane redox reaction of the root protoplasts in Zea mays L. were studied. The calmodulin antagonists (calmidazolium, W7-agarose) and anti-calmodulin serum had inhibitory effect on the extracellular reduction of ferricyanide with their concentration that yielded 50% inhibition were 1.5 μmoL/L, 10 μmol/L and 10 mg/L respectively. Inhibition of calmidazolimn could be restored by calmodulin completely. And the reduction of ferricyanide could be specifically stimulated by the exogerous purified calmodulin. These results suggested that transplasma membrane redox system of root protoplasts in Zea mays L. could be modulated by calmodulin outside the plasma membrane.     

Effects of calcium ionophore, A23187 and calmodulin inhibitors on intercellular communication in the rat myometrium

We have studied the effect of a calcium ionophore, A23187, and the purported calmodulin inhibitors, calmidazolium and chlorpromazine, on direct intercellular communication between smooth muscle cells in the myometrium of delivering rats. The extent of cell-to-cell coupling was determined by exposing one portion of small strips of longitudinal myometrium to 2-[3H] deoxy-D-glucose (2-DG) and determining the distribution and apparent diffusion coefficient (Da) for this tracer after a 5-h period for diffusion. The distribution and Da for 2-DG were significantly (p less than 0.05) reduced by exposure to A23187 in Krebs-Ringer solution with 2.5 mM Ca++, partially reduced in Krebs solution with A23187 and low Ca++ (1-10 microM), but the drug had no effect when used with Ca++-free solutions with [ethylenebis (oxyethylene-nitrilo)] tetraacetic acid (EGTA). The calmodulin inhibitors blocked the effects of A23187 in a dose-dependent fashion, and at higher concentrations, the extent of 2-DG diffusion was not different from that in control tissues. Surprisingly, however, a dose-dependent reduction in coupling was also observed in tissues exposed to the calmodulin inhibitors alone. Structural studies failed to reveal any change in the area of gap junctions between the myometrial cells following the above treatments, suggesting that the reduced exchange of 2-DG resulted from a decrease in the permeability of gap junctions between the muscle fibers.     

Decreased endothelial nitric-oxide synthase (eNOS) activity resulting from abnormal interaction between eNOS and its regulatory proteins in hypoxia-induced pulmonary hypertension

In the pulmonary artery isolated from 1-week hypoxia-induced pulmonary hypertensive rats, endothelial NO production stimulated by carbachol was decreased significantly in in situ visualization using diaminofluorescein-2 diacetate and also in cGMP content. This change was followed by the decrease in carbachol-induced endothelium-dependent relaxation. Protein expression of endothelial NO synthase (eNOS) and its regulatory proteins, caveolin-1 and heat shock protein 90, did not change in the hypoxic pulmonary artery, indicating that chronic hypoxia impairs eNOS activity at posttranslational level. In the hypoxic pulmonary artery, the increase in intracellular Ca(2+) level stimulated by carbachol but not by ionomycin was reduced. We next focused on changes in Ca(2+) sensitivity of the eNOS activation system. A morphological study revealed atrophy of endothelial cells and a peripheral condensation of eNOS in hypoxic endothelial cells preserving co-localization between eNOS and Golgi or plasma membranes. However, eNOS was tightly coupled with caveolin-1, and was dissociated from heat shock protein 90 or calmodulin in the hypoxic pulmonary artery in either the presence or absence of carbachol. Furthermore, eNOS Ser(1177) phosphorylation in both conditions significantly decreased without affecting Akt phosphorylation in the hypoxic artery. In conclusion, chronic hypoxia impairs endothelial Ca(2+) metabolism and normal coupling between eNOS and caveolin-1 resulted in eNOS inactivity.     

Identification of calmodulin-like activity in term human amnion: effect of calmodulin inhibitors on prostaglandin biosynthesis

Human amnion prostaglandin E2 (PGE2) synthesis increases with the onset of labour, and this synthesis is Ca2+-dependent. To understand better the mechanism of Ca2+-stimulated PGE2 biosynthesis, studies were performed to identify the presence of the intracellular Ca2+-mediator, calmodulin, in human amnion and to examine its role in PGE2 synthesis. Calmodulin-like activity was identified by the ability of the microsomal and cytosolic fractions of the 105,000g centrifugation of amnion homogenate to stimulate cyclic AMP-dependent phosphodiesterase activity. Cytosolic fractions consistently stimulated phosphodiesterase activity more than microsomal fractions (P less than 0.001) in paired samples from term human amnions. This activity was calcium-dependent. The cytosolic and microsomal factors increased the Vmax but not the Km of phosphodiesterase. There were no differences in these parameters with the onset of labour. The distribution of calmodulin-like activity between microsomes and cytosol was similar to the distribution of calmodulin mass as determined by radioimmunoassay. Three structurally different inhibitors of calmodulin activity, calmidazolium, trifluoperazine and W7, were tested for their ability to inhibit cytosolic factor-stimulated phosphodiesterase activity and to inhibit PGE2 output from dispersed amnion cells obtained before the onset of labour at term (cesarean section cells) or after spontaneous labour and vaginal delivery (spontaneous labour cells). The 50% inhibitory concentrations of the calmodulin antagonists in the phosphodiesterase assay were: trifluoperazine (6.7 microM), calmidazolium (0.11 microM), and W7 (24 microM). Trifluoperazine inhibited both basal and calcium ionophore (A23187)-stimulated PGE2 output from cesarean section cells and spontaneous labour amnion cells. Calmidazolium inhibited basal PGE2 output in cesarean section cells and spontaneous labour cells, but had no effect on A23187-stimulated output. W7 inhibited only the ionophore-stimulated PGE2 output in cesarean section amnion cells. The rank order of inhibition of both phosphodiesterase activation and basal PGE2 output was: calmidazolium greater than trifluoperazine greater than W7. These results suggest that human amnion contains calmodulin and that its distribution, concentration and activity remain unchanged with the onset of labour. The data suggest, although not conclusively, that calmodulin may, in part, play a role in amnion cell PGE2 production. Further investigation of calmodulin effects upon specific enzymes in the PGE2 synthetic pathway will be necessary to elucidate a role for calmodulin in PGE2 production.     

Regulation of a potassium-selective current in rabbit corneal epithelium by cyclic GMP,carbachol and diltiazem

Summary The effects of cyclic GMP (cGMP), carbachol and diltiazem on a potassium-selective, delayed-rectifier current in freshly dissociated rabbit corneal epithelial cells were studied using a modified perforated-patch-clamp technique. The current was stimulated by both 500 m cGMP (2.3–4.5-fold, mean = 2.9) and 250 nm carbachol, a muscarinic agonist (1.12–7.04-fold, mean = 3.8), and the stimulated current was totally blocked by diltiazem (10 m). The effects of cGMP appeared to be, at least in part, different from those of carbachol as they required the presence of external calcium. Single-channel data suggest that cGMP and carbachol activate the potassium current by increasing the open probability of the channel via a second-messenger system and that the action of diltiazem is probably through a direct blocking effect on the open channel.We are grateful to Erika Wohlfiel for secretarial help, Helen Hendrickson for cell preparation, and Joan Rae for software development. The work was supported by NIH grants EY06005 and EY03282 and an unrestricted award from Research to Prevent Blindness.