Regulation of reactivated contraction in teleost retinal cone models by calcium and cyclic adenosine monophosphate

We have been using lysed cell models of teleost retinal cones to examine the mechanism of contraction in nonmuscle cells. We have previously reported that dark-adapted retinas can be lysed with the detergent Brij-58 to obtain cone motile models that undergo Ca++- and adenosine triphosphate (ATP)-dependent reactivated contraction. In this report we further dissect the roles of ATP and Ca++ in activation of contraction and force production by (a) characterizing the Ca++ and nucleotide requirements in more detail, (b) by analyzing the effects of inosine triphosphate (ITP) and the ATP analog ATP gamma S and (c) by testing effects of cyclic adenosine monophosphate (cAMP) on reactivated cone contraction. Exposing lysed cone models to differing free Ca++ concentrations produced reactivated contraction at rates proportional to the free Ca++ concentration between 3.16 X 10(-8) and 10(-6) M. A role for calmodulin (CaM) in this Ca++ regulation was suggested by the inhibition of reactivated contraction by the calmodulin inhibitors trifluoperazine and calmidazolium ( R24571 ). The results of analysis of nucleotide requirements in lysed cone models were consistent with those of smooth muscle studies suggesting a role for myosin phosphorylation in Ca++ regulation of contraction. ATP gamma S and ITP are particularly interesting in that ATP gamma S, on the one hand, can be used by kinases to phosphorylate proteins (e.g., myosin light chains) but resists cleavage by phosphatases or adenosine triphosphatases (ATPases), e.g., myosin ATPase. ITP, on the other hand, can be used by myosin ATPase but does not support Ca++/calmodulin mediated phosphorylation of myosin light chains by myosin light chain kinase. Thus, these nucleotides provide an opportunity to distinguish between the kinase and myosin ATPase requirements for ATP. When individual nucleotides were tested with cone motile models, the nucleotide requirement was highly specific for ATP; not only ITP and ATP gamma S, but also guanosine triphosphate, cytosine triphosphate, adenylyl-imidodiphosphate (AMPPNP) failed to support reactivated contraction when substituted for ATP throughout the incubation. However, if lysed cones were initially incubated with ATP gamma S and then subsequently incubated with ITP, the cones contracted to an extent that was comparable to that observed with ATP. As observed in skinned smooth muscle, adding cAMP to contraction medium strongly inhibited contraction in lysed cone models.     

Reactivation of contraction in detergent-lysed teleost retinal cones

Teleost retinal cones contract in the light and elongate in the dark. In the green sunfish, Lepomis cyanellus, the necklike myoid region of the cone contracts from as much as 120 micrometers (midnight dark- adapted) to 6 micrometers in fully light-adapted state. When dark- adapted fish are exposed to light (1.4 lux), cone myoids contract with a linear rate of 1.5 +/- 0.1 micrometers/min. We report here that detergent-lysed motile models of teleost retinal cones exhibit calcium- and ATP-dependent reactivated contraction, with morphology and rate comparable to that observed in vivo. For reactivation studies isolated dark-adapted retinas were lysed with nonionic detergent Brij-58 (0.1- 1.0%). In reactivation medium containing 10(-5) M free calcium and 4 mM ATP, the lysed cones contracted with normal morphology at in vivo rates (1.4 +/- 1 micrometer/min). Little contraction was observed if ATP or detergent was deleted from the medium or if free calcium levels were less than 10(-8) M. Ultrastructural examination of cone models lysed with 1% Brij-58 revealed that, in spite of extensive extraction of the cytoplasmic matrix, cytoskeletal components (thin filaments, intermediate filaments, microtubules) were still present. Thus we have produced extensively extracted motile models of teleost retinal cones which undergo calcium- and ATP-dependent reactivated contraction with normal morphology at physiological rate.     

Dynamics of cyclic GMP synthesis in retinal rods

In retinal rods, Ca(2+) exerts negative feedback control on cGMP synthesis by guanylate cyclase (GC). This feedback loop was disrupted in mouse rods lacking guanylate cyclase activating proteins GCAP1 and GCAP2 (GCAPs(-/-)). Comparison of the behavior of wild-type and GCAPs(-/-) rods allowed us to investigate the role of the feedback loop in normal rod function. We have found that regulation of GC is apparently the only Ca(2+) feedback loop operating during the single photon response. Analysis of the rods' light responses and cellular dark noise suggests that GC normally responds to light-driven changes in [Ca(2+)] rapidly and highly cooperatively. Rapid feedback to GC speeds the rod's temporal responsiveness and improves its signal-to-noise ratio by minimizing fluctuations in cGMP.     

Stress relaxation of fibroblasts activates a cyclic AMP signaling pathway

Mechanical force regulates gene expression and cell proliferation in a variety of cell types, but the mechanotransducers and signaling mechanisms involved are highly speculative. We studied the fibroblast signaling mechanism that is activated when cells are switched from mechanically stressed to mechanically relaxed conditions, i.e., stress relaxation. Within 10 min after initiation of stress relaxation, we observed a transient 10-20-fold increase in cytoplasmic cyclic AMP (cAMP) and a threefold increase in protein kinase A activity. The increase in cAMP depended on stimulation of adenylyl cyclase rather than inhibition of phosphodiesterase. Generation of cAMP was inhibited by indomethacin, and release of arachidonic acid was found to be an upstream step of the pathway. Activation of signaling also depended on influx of extracellular Ca2+ because addition of EGTA to the incubations at concentrations just sufficient to exceed Ca2+ in the medium inhibited the stress relaxation-dependent increase in free arachidonic acid and cAMP. This inhibition was overcome by adding CaCl2 to the medium. On the other hand, treating fibroblasts in mechanically stressed cultures with the calcium ionophore A23187-stimulated arachidonic acid and cAMP production even without stress relaxation. In summary, our results show that fibroblast stress relaxation results in activation of a Ca(2+)-dependent, adenylyl cyclase signaling pathway. Overall, the effect of stress relaxation on cAMP and PKA levels was equivalent to that observed after treatment of cells with forskolin.     

Actin-dependent cell elongation in teleost retinal rods: requirement for actin filament assembly

Teleost retinal rods elongate when exposed to light. Elongation is mediated by a narrow necklike region called the myoid. In the cichlid Sarotherodon mossambicus, the rod inner segment (composed of the myoid with adjacent ellipsoid) increases in length from 12 micrometers in the dark to 41 micrometers in the light. Long light-adapted myoids contain longitudinally oriented microtubules and bundles of parallel 60-A filaments that we have identified as actin by their ability to bind myosin subfragment 1. In short dark-adapted myoids, only microtubules are recognizable. Colchicine experiments reveal that light-induced rod elongation can occur in the absence of myoid microtubules. Intraocular injections of colchicine at concentrations that disrupt virtually all rod myoid microtubules do not block rod elongation. However, rod elongation is blocked by intraocular injections of cytochalasin B or cytochalasin D. The hierarchy of effectiveness of these drugs is consistent with their effectiveness in inhibiting actin assembly and in disrupting other actin-dependent motile processes. On the basis of ultrastructural observations and the results of these inhibitor studies, we propose that the forces responsible for rod elongation are dependent not on microtubules but on actin filament assembly.     

Dynamics of retinal waves are controlled by cyclic AMP

Waves of spontaneous activity sweep across the developing mammalian retina and influence the pattern of central connections made by ganglion cell axons. These waves are driven by synaptic input from amacrine cells. We show that cholinergic synaptic transmission during waves is not blocked by TTX, indicating that release from starburst amacrine cells is independent of sodium action potentials. The spatiotemporal properties of the waves are regulated by endogenous release of adenosine, which sets intracellular cAMP levels through activation of A2 receptors present on developing amacrine and ganglion cells. Increasing cAMP levels increase the size, speed, and frequency of the waves. Conversely, inhibiting adenylate cyclase or PKA prevents wave activity. Together, these results imply a novel mechanism in which levels of cAMP within an immature retinal circuit regulate the precise spatial and temporal patterns of spontaneous neural activity.     

Protein phosphorylation and compartments of cyclic AMP in the control of cardiac contraction

The roles of cyclic AMP, cyclic AMP-dependent protein kinase and the phosphorylation of specific proteins in the regulation of cardiac contractility are briefly reviewed. Criteria for determining whether changes in cyclic AMP and protein phosphorylation are involved in a physiological response are discussed. Although cyclic AMP-dependent phosphorylation of the voltage-operated Ca^2– channel, phospholamban, troponin-I and C-protein have all been implicated in the response of the heart to inotropic agents which elevate cyclic AMP, none of these phosphorylations satisfy all of the criteria completely. Evidence is presented that there are compartments of cyclic AMP in heart which are coupled to different functional responses.Abbreviations cAMP 3,5 cyclic adenosine monophosphate - PDE cyclic nucleotide phosphodiesterase - cA-PrK cAMP-dependent protein kinase - SR sarcoplasmic reticulum - PGE₁ prostaglandin E₁ - Tn-I troponin I     

Modulation by internal protons of native cyclic nucleotide-gated channels from retinal rods

Ion channels directly activated by cyclic nucleotides are present in the plasma membrane of retinal rod outer segments. These channels can be modulated by several factors including internal pH (pH(i)). Native cyclic nucleotide-gated channels were studied in excised membrane patches from the outer segment of retinal rods of the salamander. Channels were activated by cGMP or cAMP and currents as a function of voltage and cyclic nucleotide concentrations were measured as pH(i) was varied between 7.6 and 5.0. Increasing internal proton concentrations reduced the current activated by cGMP without modifying the concentration (K(1/2)) of cGMP necessary for half-activation of the maximal current. This effect could be well described as a reduction of single-channel current by protonation of a single acidic residue with a pK(1) of 5.1. When channels were activated by cAMP a more complex phenomenon was observed. K(1/2) for cAMP decreased by increasing internal proton concentration whereas maximal currents activated by cAMP increased by lowering pH(i) from 7.6 to 5.7-5.5 and then decreased from pH(i) 5.5 to 5.0. This behavior was attributed both to a reduction in single-channel current as measured with cGMP and to an increase in channel open probability induced by the binding of three protons to sites with a pK(2) of 6.     

Interrelations between cyclic AMP, cyclic GMP and contraction in guinea pig gallbladder stimulated by cholecystokinin.

The changes in isometric tension and in concentrations of cyclic AMP and cyclic GMP in guinea pig gallbladder muscle induced by the C-terminal octapeptide of cholecystokinin (C8-CCK) were studied before and after the addition of indomethacin 3 × 10^?6 g/ml. The contractile response to the hormone was not affected by indomethacin, nor was the associated decrease in cyclic AMP concentration. However, indomethacin completely prevented the increase in cyclic GMP following addition of C8-CCK. The results suggest that in isolated guinea pig gallbladder, cyclic GMP is not essential for the C8-CCK-induced decrease in cyclic AMP concentration, and that the contractile response induced by the hormone is independent of this nucleotide.     

Ovarian contraction in various teleost families

Fishes from 12 species (a minimum of three fish of each species), representing nine families were examined for ovarian contraction. An ovary from each fish was removed, placed in a muscle bath and assessed for contractions using a force-displacement transducer. After at least 30 min of observation for spontaneous contractions, acetylcholine (ACh) was added to the muscle bath to assess the ovary's capacity for contraction. At least one third of the ovaries from each species contracted spontaneously or in response to 10⁻⁵ M ACh. Mean ± s . e . frequencies of spontaneous contractions ranged from 7·5 ± 2·0 h⁻¹ in the Atlantic silverside Menidia menidia to 380·3 ± 101·5 h⁻¹ in the sheepshead minnow Cyprinodon variegatus . Simultaneously tested ovaries from a single white perch Morone americana contracted at rates of 31·4 and 81·9 h⁻¹. This is consistent with independent generation of the contractile rhythm in the two ovaries. In three species (goldfish Carassius auratus , mummichog Fundulus heteroclitus and white perch), the tunica albuginea was removed from the ovary and found to be capable of independent contraction. This suggests that this membrane contributes to the observed contractions. The presence of ovarian contractions in this varied group of fish species indicates that the contractions play an important role in reproduction.     

Abnormally high rate of cyclic AMP excretion from an Escherichia coli mutant deficient in cyclic AMP receptor protein

By labeling adenosine 3′, 5′-cyclic monophosphate (cyclic AMP) with [³²P] phosphate and chromatographing it on a thin-layer alumina plate, we have determined the extra- and intracellular amounts of cyclic AMP in an $\text{Escherichia coli}$ CRP^? mutant (deficient in a cyclic AMP receptor protein) and its isogenic CRP⁺ cell. The CRP^? cell was found to excrete cyclic AMP at an abnormally high rate as compared to the CRP⁺ cell when growing on glucose or glycerol, which can be correlated with the abnormally high intracellular levels of cyclic AMP in the CRP^? cell.     

Insulin activates the plasma-membrane and dense-vesicle cyclic AMP phosphodiesterase in hepatocytes by distinct routes.

Insulin elicits the activation of two distinct membrane-bound cyclic AMP phosphodiesterases when incubated at 37 degrees C for 5 min with intact hepatocytes: the 'dense-vesicle' enzyme and the peripheral-plasma-membrane enzyme. In hepatocytes the lysosomotropic agents chloroquine, methylamine and NH4Cl, as well as intracellular ATP depletion elicited by fructose or incubation with insulin at 22 degrees C, blocks selectively the activation of the 'dense-vesicle' enzyme. Incubation of hepatocytes with bacitracin, leupeptin and a variety of proteinase inhibitors failed to affect insulin's activation of these two cyclic AMP phosphodiesterases by distinct routes. It is suggested that activation of the 'dense-vesicle' enzyme occurs through a pathway triggered by the endocytosis, processing and recycling of the insulin receptor. This might involve the delivery, with subsequent activation, of a latent phosphodiesterase into this fraction.     

Elevation of intracellular cyclic AMP inhibits NF-κB-mediated thymosin β4 expression in melanoma cells

Thymosin β4 (Tβ4) is a major actin-sequestering protein that has been implicated in the growth, survival, motility, and metastasis of certain tumors and is considered an indicator for malignant progression. Therefore, identifying compounds that can downregulate Tβ4 expression is very important for the development of anti-cancer chemotherapies. In this study, we investigated the effects of elevated cAMP on Tβ4 expression and the metastatic potential of murine B16 melanoma cells. In addition, we also dissected the mechanism underlying cAMP-mediated Tβ4 suppression. We found that treatment with the cAMP-inducing compounds α-MSH (α-melanocyte stimulating hormone) and IBMX (3-isobutyl-1-methylxanthine) significantly suppressed Tβ4 expression and regulated EMT-associated genes through the suppression of NF-κB activation in B16F10 cells. Along with decreased Tβ4 expression, the in vitro invasiveness and anchorage-independent growth in a semi-solid agar of these cells were also inhibited. In animal experiments, the metastatic potential of the α-MSH- or IBMX-treated B16F10 melanoma cells was decreased compared to untreated control cells. Collectively, our data demonstrate that elevated intracellular cAMP significantly suppresses Tβ4 expression and reduces MMP-9 activity, which leads to decreased metastatic potential. Moreover, suppression of NF-κB activation by α-MSH or IBMX is critical for inhibiting Tβ4 expression.     

Metformin activates AMP kinase through inhibition of AMP deaminase

The mechanism for how metformin activates AMPK (AMP-activated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action.     

Interdomain interaction of cyclic AMP receptor protein in the absence of cyclic AMP

Interdomain interaction of apo-cyclic AMP receptor protein (apo-CRP) was qualified using its isolated domains. The cAMP-binding domain was prepared by a limited proteolysis, while the DNA-binding domain was constructed as a recombinant protein. Three different regions making interdomain contacts in apo-CRP were identified by a sequence-specific comparison of the HSQC spectra. The results indicated that apo-CRP possesses characteristic modules of interdomain interaction that are properly organized to suppress activity and to sense and transfer the cAMP binding signals. Particularly, the inertness of the DNA-binding motif in apo-CRP was attributable to the participation of F-helices in the interdomain contacts.     

Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods

The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.