Light-mediated cyclic GMP hydrolysis controls important aspects of kinetics of retinal rod voltage response

Pulsatile injections of cyclic GMP into rod outer segments of the isolated toad retina cause transient depolarizations that are reduced in amplitude in proportion with the receptor potential by low Na+ Ringer's. This reduction in the amplitude of the cyclic GMP depolarization may be due to the direct effect of external Na+ concentration on dark current and an indirect effect resulting from the inactivation of a sodium-calcium exchange mechanism raising the intracellular Ca2+ concentration. By comparison the reduction in cyclic GMP response amplitude effected by illumination is accompanied by faster kinetics. This difference suggests that the reduced amplitude and speedier response reflect a light induced increase in phosphodiesterase (PDE) activity rather than the effects of Ca2+. Large doses of cyclic GMP can distort the kinetics of both the light response and the recovery from a depolarization caused by a pulse of cyclic GMP by similarly slowing both types of responses. This similarity in the kinetics of the cyclic GMP response and the initial hyperpolarizing phase of the receptor potential suggests that the kinetics of the initial phase of the receptor potential are controlled by light-mediated cyclic GMP hydrolysis.     

Calcium regulates some, but not all, aspects of light adaptation in rod photoreceptors

The role of calcium as a regulator of light adaptation in rod photoreceptors was examined by manipulation of the intracellular Ca2+ concentration through the use of the calcium ionophore A23187 and external Ca2+ buffers. These studies utilized suspensions of isolated and purified frog rod outer segments that retain their mitochondria-rich inner segments (OS-IS). Three criteria of the dark- and light-adapted flash response were characterized as a function of the Ca2+ concentration: (a) the time to peak, (b) the rate of recovery, and (c) the response amplitude or sensitivity. For all Ca2+ concentrations examined, the time to peak of the flash response was accelerated in the presence of background illumination, suggesting that mechanisms controlling this aspect of adaptation are independent of the Ca2+ concentration. The recovery kinetics of the flash response appeared to depend on the Ca2+ concentration. In 1 mM Ca2+-Ringer's and 300 nM Ca2+-Ringer's + A23187, background illumination enhanced the recovery rate of the response; however, in 10 and 100 nM Ca2+-Ringer's + A23187, the recovery rates were the same for dark- and light-adapted responses. This result implies that a critical level of Ca2+ may be necessary for background illumination to accelerate the recovery of the flash response. The sensitivity of the flash response in darkness (SDF) was dependent on the Ca2+ concentration. In 1 mM Ca2+-Ringer's SDF was 0.481 pA per bleached rhodopsin (Rh*); a background of four Rh*/s decreased SDF by half (Io). At 300 nM Ca2+ + A23187, SDF was reduced to 0.0307 pA/Rh* and Io increased to 60 Rh*/s. At 100 nM Ca2+ + A23187, SDF was reduced further to 0.0025 pA/Rh* and Io increased to 220 Rh*/s. In 10 nM Ca2+ + A23187, SDF was lowered to 0.00045 pA/Rh* and Io raised to 760 RhI/s. Using these values of SDF and Io for each respective Ca2+ concentration, the dependence of the flash sensitivity on background intensity could be described by the Weber-Fechner relation. Under low Ca2+ conditions + A23187, bright background illumination could desensitize the flash response. These results are consistent with the idea that the concentration of Ca2+ may set the absolute magnitude of response sensitivity in darkness, and that there exist mechanisms capable of adapting the photoresponse in the absence of significant changes in cytoplasmic Ca2+ concentration.     

Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors

Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities. At light intensities bleaching from 160 to 5.6 X 10(6) rhodopsin molecules/rod/s, decreases in the response latency for the cGMP kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 10(5) cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. It is unlikely, however, that light-induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors.     

Ca2+-dependent changes in cyclic GMP levels are not correlated with opening and closing of the light-dependent permeability of toad photoreceptors

We have measured the levels of 3',5'-guanosine monophosphate (cyclic GMP) in isolated retinas from toad to investigate their correlation to the opening and closing of the light-dependent permeability of photoreceptors. When Ca2+-induced changes in cyclic GMP concentration are compared with the Ca2+-induced changes in the permeability of photoreceptor light-dependent channel, four quantitative dissimilarities are noted. First, when extracellular Ca2+ ([Ca2+]o) is reduced from normal physiological levels to between 10(-6) and 10(-7) M, the light-dependent permeability is increased, but cyclic GMP levels are not significantly changed. Second, when [Ca2+]o is increased from 1.8 to 20 mM, the light-dependent permeability is suppressed, but cyclic GMP levels are decreased by only 10-15%, about one-quarter the decrease that can be obtained with bright illumination. Third, when [Ca2+]o is increased from 10(-8) M to 20 mM, the light-dependent permeability is closed rapidly, but the cyclic GMP decrease is slow. Fourth, when [Ca2+]o is lowered to 10(-8) M, the sensitivity of the light-dependent permeability to steady illumination is decreased by three to four orders of magnitude, but the sensitivity of the light-dependent decrease in cyclic GMP is not significantly affected. These observations indicate that there is no simple correlation between cyclic GMP levels and the permeability of the light-dependent channels and that Ca2+ can affect the conductance in the absence of changes in cyclic GMP content.     

Cyclic GMP directly regulates a cation conductance in membranes of bovine rods by a cooperative mechanism

Cyclic GMP causes the release of endogenous Ca2+ from rod outer segments, whose plasma membrane has been made permeable, or from isolated discs. Approximately 11,000 Ca2+ ions are released per disc at saturating concentrations of cyclic GMP. The velocity and the amplitude of the release of Ca2+ are dependent on the concentration of cyclic GMP. The maximal rate of the Ca2+ efflux is approximately 7 X 10(4) Ca2+ ions s-1 rod-1. The Ca2+ release by cyclic GMP is independent of light. The activation of the efflux occurred within a narrow range of the cyclic GMP concentration (30-80 microM) and does not obey a simple Michaelis-Menten scheme. Instead, the kinetic analysis of the Ca2+ efflux suggests that a minimum number of 2 molecules of cyclic GMP activates the ion conductance in a cooperative fashion. The release of Ca2+ by cyclic GMP requires a gradient of Ca2+ ions across the disc membrane. If the endogenous Ca2+ gradient is dissipated by means of the ionophore A23187, the release of Ca2+ by cyclic GMP is abolished. Ca2+ is released by analogues of cyclic GMP which are either modified at the 8-carbon position of the imidazole ring or by the deaza-analogue of cyclic GMP. Congeners of cyclic GMP which are modified at the ribose, phosphodiester, or pyrimidine portion of the molecule are ineffective. The hydrolysis of cyclic GMP by the light-regulated phosphodiesterase of rod outer segments is not a necessary condition for the Ca2+ release because 8-bromo-cyclic GMP, a congener resistant to hydrolysis, is a more powerful activator of the release than cyclic GMP itself. Ca2+ release by cyclic GMP is inhibited by organic and inorganic blockers of Ca2+ channels. The l-stereoisomer of cis-diltiazem blocks the release of Ca2+ at micromolar concentrations, whereas the d-form is much less effective. These results suggest that disc membranes contain a cationic conductance which is permeable to Ca2+ ions and which is regulated through the cooperative binding of at least 2 molecules of cyclic GMP to regulatory sites of the transport protein. By this mechanism, subtle changes in the concentration of cyclic GMP could promote large changes in the flux of Ca2+ ions across the disc membrane.     

The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum.

In the absence of cyclic nucleotides heart microsomes have two classes of calcium binding sites with binding constants of 0.69 and 0.071 micron-1 and capacities of 2.2 and 9.7 nmol/mg protein, respectively. Neither cyclic AMP nor monobutyryl cyclic AMP affect binding but cyclic GMP and monobutyryl cyclic GMP cause the complete loss of the high affinity calcium binding sites, Cyclic GMP (but not monobutyryl cyclic GMP) also causes a decrease in the binding constant of the low affinity binding sites. AMP, GMP and Tris-butyrate do not affect calcium binding. The effects of the cyclic nucleotides are direct and are not mediated by protein phosphorylation. Phosphorylation of microsomal proteins increases the binding constant but not the capacity of the high affinity calcium binding sites. The capacity and also, perhaps, binding constant of the low affinity sites is also increased by phosphorylation. In additon to their effects on calcium binding the cyclic nucleotides also affect the movements of calcium into and out of the microsomes. The effects are again direct and not mediated by protein phosphorylation. Cyclic GMP decreases the rate of Ca2+ efflux from preloaded cardiac microsomes and also appears to decrease the rate of uptake of Ca2+ by cardiac microsomes though this effect is less clear cut than the action on efflux. The cyclic nucleotide has a half maximal effect at a concentration of 100 microns. By contrast cyclic AMP increases the rate of influx of Ca2+ into heart microsomes and the rate of efflux of Ca2+ from preloaded preparations. The effect is, however, rather slight. It is suggested that the most obvious interpretation of these results is that cyclic GMP decreases the Ca2+ permeability of the cardiac microsomal membrane while cyclic AMP increases the permeability. In contrast to the results found with membrane preparations from certain other tissues phosphorylation of cardiac microsomal proteins does not appear to alter Ca2+ efflux or influx out of, or into, cardiac microsomal preparations. It is thus concluded that phosphorylation of cardiac microsomal proteins does not affect the Ca2+ permeability of the microsomal membrane.     

Transduction persists in rod photoreceptors after depletion of intracellular calcium

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.     

Blockade by NS-7, a neuroprotective compound, of both L-type and P/Q-type Ca2+ channels involving depolarization-stimulated nitric oxide synthase activity in primary neuronal culture

The effect of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), a neuroprotective compound, on Ca2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture. The NOS activity was estimated from the cyclic GMP formation. The KCl (25 mM)-stimulated cyclic GMP formation was totally abolished by a combined treatment with nifedipine and omega-agatoxin IVA (omega-Aga), whereas spontaneous cyclic GMP formation was partially but significantly reduced by nifedipine. In contrast to nifedipine, NS-7 blocked KCl-stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS-7 on L-type Ca2+ channels were compared. Nifedipine blocked equally the cyclic GMP formation stimulated by various concentrations of (+/-)-Bay K 8644, whereas NS-7 inhibited the maximal response without affecting the responses induced by low concentrations of (+/-)-Bay K 8644. The effects of NS-7 on L-type and P/Q-type Ca2+ channels involving KCl-stimulated cyclic GMP formation were subsequently examined. NS-7 suppressed the KCl-stimulated cyclic GMP formation measured in the presence of omega-Aga to almost the same extent as that determined in the presence of nifedipine. In contrast, NS-7 had no influence on ionomycin-induced enhancement of cyclic GMP formation. Finally, NS-7 reversed KCl-induced elevation of the intracellular free Ca2+ concentration. These findings suggest that NS-7 inhibits NOS activation in primary neuronal culture by reducing Ca2+ entry through L-type and P/Q-type Ca2+ channels, in which the inhibition is largely dependent on Ca2+ channel activity.     

Prolongation of actions of Ca2+ early in phototransduction by 9-demethylretinal

During adaptation Ca2+ acts on a step early in phototransduction, which is normally available for only a brief period after excitation. To investigate the identity of this step, we studied the effect of the light-induced decline in intracellular Ca2+ concentration on the response to a bright flash in normal rods, and in rods bleached and regenerated with 11-cis 9-demethylretinal, which forms a photopigment with a prolonged photoactivated lifetime. Changes in cytoplasmic Ca2+ were opposed by rapid superfusion of the outer segment with a 0Na+/0Ca2+ solution designed to minimize Ca2+ fluxes across the surface membrane. After regeneration of a bleached rod with 9-demethlyretinal, the response in Ringer's to a 440-nm bright flash was prolonged in comparison with the unbleached control, and the response remained in saturation for 10-15s. If the dynamic fall in Ca2+i induced by the flash was delayed by stepping the outer segment to 0Na+/0Ca2+ solution just before the flash and returning it to Ringer's shortly before recovery, then the response saturation was prolonged further, increasing linearly by 0.41 +/- 0.01 of the time spent in this solution. In contrast, even long exposures to 0Na+/0Ca2+ solution of rods containing native photopigment evoked only a modest response prolongation on the return to Ringer's. Furthermore, if the rod was preexposed to steady subsaturating light, thereby reducing the cytoplasmic calcium concentration, then the prolongation of the bright flash response evoked by 0Na+/0Ca2+ solution was reduced in a graded manner with increasing background intensity. These results indicate that altering the chromophore of rhodopsin prolongs the time course of the Ca2+-dependent step early in the transduction cascade so that it dominates response recovery, and suggest that it is associated with photopigment quenching by phosphorylation.     

Physiological evidence that light-mediated decrease in cyclic GMP is an intermediary process in retinal rod transduction

Brief, intracellularly injected pulses of cyclic GMP transiently depolarized toad retinal rod outer segments (ROS). The depolarization is antagonized by light, perhaps by the activation of phosphodiesterase (PDE), as shown in the biochemical studies of others. As measured by the antagonism of cyclic GMP pulses by light, PDE activity peaks after the peak of the receptor potential and has approximately the same recovery time as the membrane voltage after weak illumination, but recovers more slowly than the membrane potential after strong illumination, as sensitivity does in other preparations. A cyclic GMP pulse delivered just after the hyperpolarizing phase of the receptor potential tends to turn off the light response. The kinetics of recovery from this turnoff are similar to those of the initial phase of the receptor potential. This similarity suggests that the initial phase of the receptor potential is controlled by light-activated PDE. Both EGTA and saturating doses of cyclic GMP block the light response, but only cyclic GMP increases response latency, which suggests that if calcium is involved in transduction, it is controlled by the hydrolysis of cyclic GMP. After brief pulses of cyclic AMP, a new steady state of increased depolarization occasionally develops. The effects described above also occur under these conditions. The results are consistent with the hypothesis that light-activated hydrolysis of cGMP is an intermediary process in transduction.     

Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell

The present studies were performed to determine the role of cyclic GMP in regulating agonist mediated calcium entry in the pancreatic acinar cell. In guinea pig-dispersed pancreatic acini the findings demonstrated that carbachol stimulated a transient 20-40-fold rise in cellular cyclic GMP followed by a sustained 3-4-fold rise in cellular cyclic GMP. The guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), caused a dose-dependent inhibition of carbachol-stimulated increases in cellular cyclic GMP both during the initial transient large increase in cyclic GMP and the sustained increase in cyclic GMP. LY83583 also inhibited cellular Ca2+ influx during carbachol stimulation and reloading of the agonist-sensitive pool of Ca2+ at the termination of carbachol stimulation with atropine. The effect of the inhibition on reloading of the agonist-sensitive pool was secondary to its effects on the plasma membrane C2+ entry. The addition of dibutyryl cyclic GMP to LY83583-treated acini restored Ca2+ influx across the plasma membrane. Nitroprusside increased both cellular cyclic GMP and the rate of Ca2+ influx. During periods when plasma membrane Ca2+ entry was activated, cellular cyclic GMP levels were increased. These results suggest that agonist-induced increases in cellular cyclic GMP are necessary and sufficient to mediate the effects of the agonist on the plasma membrane Ca2+ entry mechanism.     

An extended Ca2+-hypothesis of visual transduction with a role for cyclic GMP

A model is described having the following features: Light induces Ca2+ release from vertebrate rod outer segments discs via pores composed of multimeric rhodopsin. Cytoplasmic Ca2+ reversibly blocks Na+ channels of the surface membrane, with the time course of development and amplitude of the response to light being influenced by restrictions on intradiscal Ca2+ diffusion. The falling phase of response reflects a decline in cytoplasmic [Ca2+] due to a Ca2+-binding protein controlled by cyclic GMP so that its binding capacity is increased by the reduction in cytoplasmic [cyclic GMP] which follows rhodopsin bleaching.     

The heterogeneity of bovine growth hormone. Extraction from the pituitary of components with different biological and immunological properties.

Evidence is available to suggest that Ca2+-calmodulin and cyclic nucleotides are involved in the regulation of ion transport in rabbit ileum. Since both Ca2+-calmodulin and cyclic nucleotides exert many of their effects by phosphorylation, the effects of Ca2+-calmodulin and cyclic nucleotides on phosphorylation of purified microvillus membrane from rabbit ileal mucosa were evaluated. Ca2+-calmodulin increased phosphorylation of five microvillus-membrane peptides, with Mr values of 137000, 77000, 58000, 53000 and 50000. The increases in phosphorylation caused by Ca2+-calmodulin were: Mr-137000 peptide, 111 +/- 26%; Mr-77000 peptide, 71 +/- 17%; Mr-58000 peptide, 51 +/- 8%; Mr-53000 peptide, 113 +/- 20%. These increases were maximal at 1 microM-calmodulin and 0.3-0.9 microM free Ca2+; concentrations of Ca2+ causing half-maximal effects on phosphorylation for the different peptides were 0.06-0.12 microM. Cyclic AMP and cyclic GMP increased phosphorylation of two peptides, of Mr 137000 and 85000. The concentrations of cyclic nucleotides giving half-maximal phosphorylation of the Mr-137000 peptide were 0.3 microM-cyclic AMP and 4.6 microM-cyclic GMP, and for the Mr-85000 peptide, 3.9 microM-cyclic AMP and 0.05 microM-cyclic GMP. The maximal increase in phosphorylation of the Mr-137000 peptide was 200% for cyclic AMP and 95% for cyclic GMP, and that of the Mr-85000 peptide was 220% for cyclic AMP and 120% for cyclic GMP. These studies demonstrate the existence of Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-dependent protein kinases and substrate proteins in purified rabbit ileal microvillus membranes and that Ca2+ can regulate phosphorylation of these proteins over the presumed physiological concentration range of cytosol free Ca2+.     

Guanosine 3'',5''-cyclic monophosphate and the in vitro physiology of frog photoreceptor membranes

Frog rod outer segments freshly detached from dark-adapted retinas contain approximately 1-2 molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) for every 100 molecules of visual pigment present. This cyclic GMP decays to 5'-GMP, and the conversion is accelerated upon illumination of the outer segments. Bleaching one rhodopsin molecule can lead to the hydrolysis of 1,000-2,000 molecules of cyclic GMP within 100-300 ms. The decline in cyclic GMP concentration becomes larger as illumination increases, and varies with the logarithm of light intensity at levels which bleach between 5 X 10(2) and 5 X 10(5) rhodopsin molecules per outer segment-second. Light suppression of plasma membrane permeability, assayed in vitro as light suppression of outer segment swelling in a modified Ringer's solution, occurs over this same range of light intensity. The correlation between cyclic GMP and permeability or swelling is maintained in the presence of two pharmacological perturbations: papaverine, a phosphodiesterase inhibitor, increases both cyclic GMP levels and the dark permeability of the plasma membrane; and beta,gamma-methylene ATP increases the effectiveness of light in suppressing both permeability and cyclic GMP levels.     

Influence of calcium on guanosine 3'',5''-cyclic monophosphate levels in frog rod outer segments

In the presence of 10(-9) M calcium, rod outer segments freshly detached from dark-adapted frog retinas contain between 0.01 and 0.02 moles of guanosine 3',5'-cyclic monophosphate (cyclic GMP) per mole of rhodopsin. The dark level of cyclic GMP is reduced approximately 50% by illumination that bleaches 5 x 10(5) rhodopsin molecules/outer segments. The dark levels of cyclic GMP also can be suppressed to approximately 0.007 mol/mol of rhodopsin by increasing the concentration of calcium from 10(-9) M to 2 x 10(-9) M, and they remain at this level as calcium concentration is raised to 10(-3) M. The final level to which illumination reduces cyclic GMP in unaffected by the calcium concentration between 10(-9) and 10(-3) M. The maximal light-induced decrease in cyclic GMP occurs within 1 s from the onset of illumination at all calcium concentrations. The magnitude and time-course of the light-induced decrease in cyclic GMP measured in these experiments are comparable to values obtained previously (Woodruff et al. 1977. J. Gen. Physiol. 69:677-679; Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). The data are consistent with a role for cyclic GMP in visual transduction irrespective of the calcium concentration.     

The role of calcium and guanosine 3':5'-monophosphate in the action of acetylcholine on thyroid metabolism

The role of calcium and guanosine 3':5'-monophosphate (cyclic GMP) in the regulation of thyroid metabolism has been investigated in dog thyroid slices. Carbamoylcholine enhanced glucose carbon-1 oxidation, protein iodination, cyclic GMP accumulation and decreased thyrotropin-induced adenosine 3':5'-monophosphate (cyclic AMP) accumulation and iodine secretion; it did not affect protein synthesis. The effects of carbamoylcholine were reproduced under various experimental conditions by supplementary calcium in the medium, ouabain, and in media in which Na+ had been replaced by choline chloride. They were inhibited by lanthanum. These results further support the hypothesis that free intracellular Ca2+ is the intracellular signal for carbamoylcholine effects and suggest that a Na+ -gradient-driven Ca2+ extrusion mechanism operates in the thyroid cell. Mn2+ reproduced the effect of Ca2+ on glucose oxidation, protein iodination and cyclic GMP accumulation in Ca2+ -depleted slices and medium, and thus mimicked some intracellular effects of Ca2+. On the other hand Mn2+ inhibited the carbamoylcholine effect on thyrotropin-induced thyroid secretion and cyclic AMP accumulation, and Ca2+ inhibited the Mn2+-induced cyclic GMP accumulation. This suggests that the two ions compete for the same channel. Similarly Mn2+ inhibited calcium effects in the presence of ionophore A23187. Procaine inhibited protein iodination under all conditions suggesting a primary effect; it also inhibited all carbamoylcholine and ouabain actions. However the drug did not inhibit the effects of choline chloride and its action was reversed by raising carbamoylcholine but not Ca2+ concentration; it is therefore doubtful that procaine acts by blocking Ca2+ channels. In media without added Ca2+, Mn2+ increased cyclic GMP accumulation but did not decrease thyrotropin-induced cyclic AMP accumulation or iodine secretion, which suggests that cyclic GMP cannot be the sole mediator of the latter two effects of carbamoylcholine.     

Calcium induces cyclic GMP formation in Dictyostelium

Cyclic GMP is rapidly formed a few seconds after binding of chemotactic signalling molecules to specific receptors on the cell surface of Dictyostelium amoebae. This phenomenon could be mimicked by addition of a pulse of Ca2+ to permeabilised amoebae. The concentration of Ca2+ for half-maximal response was 60 microM. Other ions (K+, Na+, Mg+ or Mn+) had no effect. A pulse of 5 microM IP3 produced a cyclic GMP response of similar magnitude but IP2 elicited no response. The data provide strong support for the hypothesis that cell surface receptor binding induces cyclic GMP formation by liberating Ca2+ from internal stores.     

Light-induced extracellular changes of calcium and sodium concentrations in the Planarian ocellus

Ion-selective double-barreled microelectrodes inserted into a planarian ocellus were used to monitor the ocellus potential and the changes in extracellular concentrations of Ca2+ (Ca(o)) and Na+ (Na(o)) caused by a 0.5-sec light flash or sustained (120s) illumination. Ca(o) and Na(o) were slightly decreased following a flash. Sustained illumination caused a biphasic change in Ca(o) (a rapid decrease followed by a slow increase) and a tonic decrease in Na(o). When Na+ in the planarian saline was replaced by Li+ or choline+, the increase in Ca(o) was prevented: sustained illumination induced only a decrease in Ca(o). These results suggest that illumination induces influxes of both Ca+ and Na+ into planarian photoreceptors, and that the Ca2+ influx is rapidly followed by a Na-dependent Ca2+ efflux due to Na-Ca exchange.     

Cyclic 3',5'-AMP phosphodiesterase of rabbit aorta.

Cyclic AMP and cyclic GMP phosphodiesterase activities (3' : 5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca2+ in the presence of the activator. However, the activity of the cyclic AMP phosphodiesterase was not significantly stimulated by Ca2+ up to 100 muM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60-70%) of the media enzyme was found in 105 000 times g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 times g supernatant the main kinetic parameters were specified as follows: 1) The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg2+ was achieved at 4mM of MgC12. 2) High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3) Activity-substrate concentration relationship revealed a high affinity (Km equals 1.0 muM) and low affinity (Km equals 45 muM) for cyclic AMP. The homogenate and 105 000 times g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent Km values for cyclic AMP hydrolysis were 1.2 muM and 36-40 muM and an enzyme extracted by sonication from 105 000 times g precipitate also exhibited non-linear kinetics (Km equals 5.1 muM and 70 muM). 4) Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I5 o at 0.62 muM, I5 o of the media at 0.62 muM and I5 o of the adventitia at 1.0 muM) than on the brain (I5 o at 8.5 muM) and serum (I5 o at 20 muM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.     

Nicorandil reduces the basal level of cytosolic free calcium in single guinea pig ventricular myocytes.

Using fluorescent Ca2+ indicator fura-2 and whole-cell patch-clamp techniques, we examined the effect of 2-nicotinamidoethyl nitrate (nicorandil) on the intracellular free Ca2+ concentration ([Ca2+]i) and electrical properties in single guinea pig ventricular myocytes. Nicorandil (10 nM approximately 1 mM) reduced the resting level [Ca2+]i monitored as fura-2 fluorescence ratio in a concentration-dependent manner. Dibutyryl guanosine 3':5'-cyclic monophosphate (cyclic GMP), a membrane permeable cyclic GMP analogue, mimicked the nicorandil action. Neither application of caffeine (10 mM) nor deprivation of extracellular Na+ ions could prevent the nicorandil action on [Ca2+]i. In contrast, the nicorandil effect was virtually blocked by sodium orthovanadate (40 microM), a Ca2+ pumping ATPase inhibitor. During electrophysiological experiments, nicorandil shortened action potential durations (205 +/- 80 ms to 153 +/- 76 ms) by increasing a glibenclamide-sensitive outward K+ conductance. However, the drug produced little hyperpolarization (approximately 2 mV) because the resting potential of ventricular myocytes was close to the K+ equilibrium potential. The involvement of voltage-dependent Ca-channel current and Na-Ca exchanger was considered to be minimal under physiological conditions. It is thus concluded that nicorandil decreases basal [Ca2+]i via cyclic GMP-mediated activation of the plasma membrane Ca2+ pump in guinea pig ventricular myocytes.