The role of cAMP and Ca on the modulation of junctional conductance: an integrated hypothesis

The role of cAMP and Ca in the modulation of junctional permeability is discussed. An integrated hypothesis is presented which proposes that cAMP modulates the junctional conductance through the activation of specific kinases and phosphorylation of gap junction proteins. A close-loop feed-back between cAMP and Ca is assumed to be relevant in the regulation of junctional conductance under physiological conditions. According to this hypothesis hormones modulate the junctional permeability through variations in the intracellular concentration of cAMP. It is known that in several tissues the cells are connected through low resistance intercellular junctions (Loewenstein, 1966; Bennett, 1973; De Mello, 1975, 1932a). Ions and small molecules can flow freely from cell-to-cell across narrow hydrophilic channels (De Mello, 1982a). This type of intercellular coupling is essential for the fast propagation of the impulse and the synchronization of electrical activity in excitable tissues (Bennett, 1973; De Mello, 1982a). It has been proposed that the exchange of chemical signals between cells is important for metabolic cooperation (Gilula et al. 1972) and growth control (Loewenstein, 1979). Therefore, the modulation of junctional conductance is a significant feature of cell biology. Evidence has been provided that the increase in free [Ca2+]i can produce cell decoupling in Chironomus salivary gland (Loewenstein et al., 1967) and in mammalian cardiac fibers (De Mello, 1972, 1975). The free [Ca2+]i required to suppress cell-to-cell coupling is difficult to determine because Ca ions are continuously taken up by mitochondria, sarcoplasmic reticulum or are extruded from the cell. In salivary gland a concentration of free [Ca2+]i of about 5-8 X 10(-5) M was found to be associated with cell decoupling (Loewenstein et al., 1967). The major difficulty here is that the concentration of the ion determined in the bulk of the cytosol is not necessarily the same near the gap junctions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.     

Effects of cAMP on intercellular coupling and osteoblast differentiation

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. Direct intercellular communication via gap junctions is an important component of bone homeostasis, coordinating cellular responses to external signals and promoting osteoblast differentiation. The cAMP pathway, a major intercellular signal transduction mechanism, regulates osteoblastic function and metabolism. We investigated the effects of this second messenger on junctional communication and on the expression of differentiation markers in human HOBIT osteoblastic cells. Increased levels of cAMP induce posttranslational modifications (i.e., phosphorylations) of connexin43 and enhancement of gap junction assembly, resulting in an increased junctional permeance to Lucifer yellow and to a positive modulation of intercellular Ca(2+) waves. Increased intercellular communication, however, was accompanied by a parallel decrease of alkaline phosphatase activity and by an increase of osteocalcin expression. cAMP-dependent stimulation of cell-to-cell coupling induces a complex modulation of bone differentiation markers.     

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium.

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.     

Methylxanthines stimulate calcium transport and inhibit cyclic nucleotide phosphodiesterases in abalone sperm

Experiments were designed to determine the mechanism by which methylxanthines elevate abalone sperm cAMP concentrations and induce the acrosome reaction (AR). Theophylline or, more effectively, 1-methyl-3-isobutylxanthine (MIX) inhibit the cyclic nucleotide phosphodiesterase activities of abalone sperm homogenates. 45Ca2+ uptake by sperm is also stimulated by theophylline, and more effectively by MIX, and this stimulatory effect is blocked by KCN. Verapamil, a compound known to antagonize Ca2+ conductance, has no effect on the Ca2+ or MIX-induced cAMP elevation at concentrations up to 200 microM. However, verapamil reduces the sperm cAMP elevation caused by the addition of Ca2+ plus MIX. This inhibition is not complete, even at 200 microM verapamil. The AR induced by Ca2+ plus MIX is completely inhibited by 200 microM verapamil. The data suggest that these methylxanthines elevate abalone sperm cyclic nucleotide concentrations by inhibiting cyclic nucleotide phosphodiesterase activities. Furthermore, since sperm cAMP metabolism is modulated by Ca2+ flux, methylxanthines also appear to elevate abalone sperm cAMP concentrations by their effects on Ca2+ transport. The Ca2+-induced cAMP elevation occurs through a verapamil-insensitive mechanism, whereas the potentiation by MIX of the Ca2+ effect to elevate cAMP occurs through both verapamil-insensitive and -sensitive mechanisms. The methylxanthine-induced AR is mediated by a primary effect on Ca2+ transport and occurs through a verapamil-sensitive mechanism. Cyclic AMP may play a role in the methylxanthine-induced AR, but does not appear to act as the primary mediator of this exocytotic event.     

Effect of intracellular injection of cAMP on the electrical coupling of mammalian cardiac cells

The influence of cAMP on the electrical coupling of canine Purkinje fibers was investigated. It was found that the intracellular injection of the nucleotide enhances the cell-to-cell coupling appreciably. No change in the coupling coefficient (V2/V1) was found with the intracellular injection of 5-AMP. A slight decrease in input resistance (Vo/Io) was produced by cAMP injection and the time constant of the cell membrane (tau m) was also reduced. These findings indicate that the changes in intercellular coupling produced by cAMP were not related to an increase in resistance of the non-junctional membrane but to a decline in junctional resistance. The present results support the view that cAMP plays an important role in the modulation of junctional conductance in cardiac fibers.     

Interaction of cyclic AMP and Ca2+ in the control of electrical coupling in heart fibers

The influence of intracellular injection of cAMP on the electrical coupling of canine Purkinje cells was investigated. It was found that the nucleotide enhanced reversibly the cell-to-cell communication through an increase in junctional conductance. Dibutyryl cAMP (5 X 10(-4) M) plus theophylline (0.4 mM) decreased appreciably the intracellular longitudinal resistance (ri). The interactions of cAMP and Ca on the electrical coupling were also investigated. The nucleotide and Ca have opposite effects on the electrical coupling. In the presence of high [Ca2+]o solutions (6 mM), the intracellular injection of cAMP causes a transient increase in the coupling coefficient followed by an appreciable decrease in cell-to-cell coupling. This reduction in intracellular communication was reversed by injecting EGTA into the same cell. The results of this study support the view that cAMP is a modulator of junctional conductance in cardiac muscle and that the compound interacts with Ca in the control of intracellular communication.     

A method to determine the relative cAMP permeability of connexin channels

Here we present a method by which gap junction-mediated intercellular diffusion of adenosine 3',5'-cyclic monophosphate (cAMP) molecules can be monitored in "real-time" and the cAMP permeability of different gap junction channels can be compared. Intercellular cAMP diffusion was investigated throughout this study in human HeLa cells coexpressing murine connexin45 and cyclic nucleotide-gated (CNG) ion channels. The CNG channels were used as cAMP sensors, since CNG channel activation led to an increase of the cytosolic Ca2+ concentration, which was monitored by Ca2+ imaging. A cAMP gradient was generated between two contacting cells by restricting the photolysis of caged cAMP to only one cell. The intercellular diffusion of cAMP was measured by the increase in Ca2+ concentration in the neighboring cell. We developed a standardization procedure for the Ca2+ signal which allowed estimation of the amount of cAMP that diffused from cell to cell. The number of gap junction channels between each cell pair investigated was determined by double whole-cell patch-clamp measurements. On the basis of these data we calculated how many gap junction channels contributed to the diffusion of a certain amount of cAMP. The new method can be used to compare the selective permeabilities of different gap junction channels for cAMP and for cGMP which also activates the CNG channel.     

Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors.

Two properties were found to distinguish neuronal from muscle nicotinic acetylcholine receptors (nAChRs). First, neuronal nAChRs have a greater Ca2+ permeability. The high Ca2+ flux through neuronal nAChRs activates a Ca(2+)-dependent Cl- conductance, and the Ca2+ to Cs+ permeability ratio (PCa/PCs) is 7 times greater for neuronal than for muscle nAChRs. A second difference between the receptor types is that neuronal nAChRs are potently modulated by physiological levels of external Ca2+. Neuronal nAChR currents are enhanced by external Ca2+ in a dose-dependent manner. The results indicate that changes in extracellular Ca2+ modulate neuronal nAChRs and may modulate cholinergic synapses in the CNS. Also, activation of neuronal nAChRs produces a significant influx of Ca2+ that could be an important intracellular signal.     

Biophysical properties of gap junctions between freshly dispersed pairs of mouse pancreatic beta cells.

Coupling between beta cells through gap junctions has been postulated as a principal mechanism of electrical synchronization of glucose-induced activity throughout the islet of Langerhans. We characterized junctional conductance between isolated pairs of mouse pancreatic beta cells by whole-cell recording with two independent patch-clamp circuits. Most pairs were coupled (67%, n = 155), although the mean junctional conductance (gj) (215 +/- 110 pS) was lower than reported in other tissues. Coupling could be recorded for long periods, up to 40 min. Voltage imposed across the junctional or nonjunctional membranes had no effect on gj. Up to several hours of treatment to increase intracellular cAMP levels did not affect gj. Electrically coupled pairs did not show transfer of the dye Lucifer yellow. Octanol (2 mM) reversibly decreased gj. Lower concentrations of octanol (0.5 mM) and heptanol (0.5 mM) than required to uncouple beta cells decreased voltage-dependent K+ and Ca2+ currents in nonjunctional membranes. Although gj recorded in these experiments would be expected to be provided by current flowing through only a few channels of the unitary conductance previously reported for other gap junctions, no unitary junctional currents were observed even during reversible suppression of gj by octanol. This result suggests either that the single channel conductance of gap junction channels between beta cells is smaller than in other tissues (less than 20 pS) or that the small mean conductance is due to transitions between open and closed states that are too rapid or too slow to be resolved.     

The mechanisms in NO-dependent regulation of electrical and mechanical activity in smooth muscles

The role of nitric oxide (NO) and its implication in intracellular and intercellular signaling pathways attract an attention of many research teams up to now. Away of its signaling functions. NO is considered as one of the key molecules in maintenance of balance between the physiological and pathological processes due to cytoprotective and cytotoxic functions of this molecule. In this regard, elucidation of the NO-dependent mechanisms, involved into the physiological processes and pathophysiological reactions, remains an urgent problem of conntemporary biology and medicine. Analysis of obtained results establishes a relative contribution of electro- and pharmaco-mechanical coupling mechanisms in NO-dependent regulation of smooth muscle cels (SMC) functions. The authors show that elevation of intracellular Ca2+ concentration by biologically active substances promotes relaxing effect of NO through both voltage-dependent and -independent intracellular mechanisms of calcium redistribution. Namely the peculiarities of considered mechanisms in each certain type of SMCs cause the final direction of alterations in contractility and membrane potential. It has been shown that voltage-dependent effects of NO are mediated by suppression of calcium and/or sodium components and modulation of Ca2+ -dependent and ATP-seisitive potassium components of SMC membrane permeability, Voltage-independent NO control of mechanical smooth muscles activity mainly is mediated by 1) modulation of protein kinase C (PK-C) branch of calcium signaling system, 2) ratio of cyclic nucleotides intracellular concentrations (cGMP/cAMP), and 3) directional mode of electrosilent Na+, K+, 2Cl- -cotransport. Our results show that the features of the myogenic effects of NO are caused by the peculiarities of PK-C operation in SMC.     

Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation.

The Ca2+ permeability of a nicotinic acetylcholine receptor (nAChR) in the rat CNS was determined using both current and fluorescence measurements on medial habenula neurons. The elementary slope conductance of the nAChR channel was 11 pS in pure external Ca2+ (100 mM) and 42 pS in standard solution. Ca2+ influx through nAChRs resulted in the rise of cytosolic Ca2+ concentration ([Ca2+]i) to the micromolar range. This increase was maximal under voltage conditions (below -50 mV) in which Ca2+ influx through voltage-activated channels was minimal. Ca2+ influx through nAChRs directly activated a Ca(2+)-dependent Cl- conductance. In addition, it caused a decrease in the GABAA response that outlasted the rise in [Ca2+]i. These results underscore the physiological significance of Ca2+ influx through nAChR channel in the CNS.     

Ca2+-dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC)

Cell function depends on the distribution of cytosolic and mitochondrial factors across the outer mitochondrial membrane (OMM). Passage of metabolites through the OMM has been attributed to the voltage-dependent anion-selective channel (VDAC), which can form a large conductance and permanently open a channel in lipid bilayers. However, recent data indicate that the transport of metabolites through the OMM is controlled in the cells. Recognizing that the bilayer studies had been commonly conducted at supraphysiological [Ca2+] and [K+], we determined the effect of Ca2+ on VDAC activity. In liposomes, the purified VDAC displays Ca2+-dependent control of the molecular cut-off size and shows Ca2+-regulated Ca2+ permeability in the physiological [Ca2+] range. In bilayer experiments, at submicromolar [Ca2+], the purified VDAC or isolated OMM does not show sustained large conductance but rather exhibits gating between a nonconducting state and various subconductance states. Ca2+ addition causes a reversible increase in the conductance and may evoke channel opening to full conductance. Furthermore, single cell imaging data indicate that Ca2+ may facilitate the cation and ATP transport across the OMM. Thus, the VDAC gating is dependent on the physiological concentrations of cations, allowing the OMM to control the passage of ions and some small molecules. The OMM barrier is likely to decrease during the calcium signal.     

The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: evidence for a Ca2+-dependent mechanism

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, while in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of interleukin-1 (IL-1) in the synovial fluid. We have examined the effects of IL-1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL-1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose-dependent increase in the expression of connexin 43 and by an enhanced connexin 43 immunostaining at sites of cell-to-cell contact. IL-1 stimulation induced a dose-dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL-1-dependent up-regulation of connexin 43 could be prevented by intracellular Ca2+ chelation, but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of connexin 43. IL-1 is one of the most potent cytokines that promotes cartilage catabolism: its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.     

Unitary permeability of gap junction channels to second messengers measured by FRET microscopy

Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP(3)) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single gap junction channels to second messengers. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP(3). The unitary permeabilities to cAMP (47 x 10(-3) +/- 15 x 10(-3) microm(3)/s) and InsP(3) (60 x 10(-3) +/- 12 x 10(-3) microm(3)/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 +/- 3 x 10(-3) microm(3)/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways.     

Mobilization of the hormone-sensitive calcium pool increases hepatocyte tight junctional permeability in the perfused rat liver

Hepatocyte tight junctional permeability has been shown to be regulated by hormones that exert their effects via phospholipase C activation. However, the precise transduction pathway involved in this effect is not known. The present study has employed the selective inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), to examine the effect of the mobilization of the endoplasmic reticular Ca2+ pool on tight junctional permeability in the perfused rat liver. Infusion of tBuBHQ followed by a bolus infusion of horseradish peroxidase (HRP) resulted in a significant increase in the first peak of biliary HRP, a measure of junctional permeability, whereas transcellular (vesicular) transport of HRP was not affected. Therefore, we conclude that the effect of hormones on tight junctional permeability is mediated, at least in part, by the mobilization of intracellular Ca2+.     

Modulation of cytosolic calcium signaling by protein kinase A-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors

Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.     

Hormonal regulation of cell junction permeability: Upregulation by catecholamine and prostaglandin E1

Summary By cellular activation with hormones, we test the proposition (Loewenstein, W.R.,Physiol. Rev. 61:829, 1981) that the permeability of cell junction is upregulated through elevation of the level of cyclic AMP. Cultured rat glioma C-6 cells, with -adrenergic receptors, and human lung WI-38 cells, with prostaglandin receptors, were exposed to catecholamine (isoproterenol) and prostaglandin E₁, respectively, while their junctions were probed with microinjected fluorescent-labelled mono-, di-, and triglutamate. Junctional permeability, as indexed by the proportion of cell interfaces transferring the probes, rose after the hormone treatments. The increase in permeability took several hours to develop and was associated with an increase in the number of gap-junctional membrane particles (freeze-fracture electron microscopy). Such interaction between hormonal and junctional intercellular communication may provide a mechanism for physiological regulation of junctional communication and (perhaps as part of that) for physiological coordination of responses of cells in organs and tissues to hormones.     

Dominant regulation of interendothelial cell gap formation by calcium-inhibited type 6 adenylyl cyclase

Acute transitions in cytosolic calcium ([Ca2+]i) through store-operated calcium entry channels catalyze interendothelial cell gap formation that increases permeability. However, the rise in [Ca2+]i only disrupts barrier function in the absence of a rise in cAMP. Discovery that type 6 adenylyl cyclase (AC6; EC 4.6.6.1) is inhibited by calcium entry through store-operated calcium entry pathways provided a plausible explanation for how inflammatory [Ca2+]i mediators may decrease cAMP necessary for endothelial cell gap formation. [Ca2+]i mediators only modestly decrease global cAMP concentrations and thus, to date, the physiological role of AC6 is unresolved. Present studies used an adenoviral construct that expresses the calcium-stimulated AC8 to convert normal calcium inhibition into stimulation of cAMP, within physiologically relevant concentration ranges. Thrombin stimulated a dose-dependent [Ca2+]i rise in both pulmonary artery (PAECs) and microvascular (PMVEC) endothelial cells, and promoted intercellular gap formation in both cell types. In PAECs, gap formation was progressive over 2 h, whereas in PMVECs, gap formation was rapid (within 10 min) and gaps resealed within 2 h. Expression of AC8 resulted in a modest calcium stimulation of cAMP, which virtually abolished thrombin-induced gap formation in PMVECs. Findings provide the first direct evidence that calcium inhibition of AC6 is essential for endothelial gap formation.