cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. C436-C450 ◽  
Author(s):  
Alan S. Lader ◽  
Yihan Wang ◽  
G. Robert Jackson ◽  
Steven C. Borkan ◽  
Horacio F. Cantiello
Keyword(s):  
Monoclonal Antibody ◽  
Cardiac Myocytes ◽  
Single Channel ◽  
Cell Model ◽  
Neonatal Mouse ◽  
Intracellular Camp ◽  
Mouse Heart ◽  
Whole Cell ◽  
Anion Conductance ◽  
Camp Stimulation

In this study, patch-clamp techniques were applied to cultured neonatal mouse cardiac myocytes (NMCM) to assess the contribution of cAMP stimulation to the anion permeability in this cell model. Addition of either isoproterenol or a cocktail to raise intracellular cAMP increased the whole cell currents of NMCM. The cAMP-dependent conductance was largely anionic, as determined under asymmetrical (low intracellular) Cl− conditions and symmetrical Cl−in the presence of various counterions, including Na+, Mg2+, Cs+, and N-methyl-d-glucamine. Furthermore, the cAMP-stimulated conductance was also permeable to ATP. The cAMP-activated currents were inhibited by diphenylamine-2-carboxylate, glibenclamide, and an anti-cystic fibrosis transmembrane conductance regulator (CFTR) monoclonal antibody. The anti-CFTR monoclonal antibody failed, however, to inhibit an osmotically activated anion conductance, indicating that CFTR is not linked to osmotically stimulated currents in this cell model. Immunodetection studies of both neonatal mouse heart tissue and cultured NMCM revealed that CFTR is expressed in these preparations. The implication of CFTR in the cAMP-stimulated Cl−- and ATP-permeable conductance was further verified with NMCM of CFTR knockout mice [ cftr(−/−)] in which cAMP stimulation was without effect on the whole cell currents. In addition, stimulation with protein kinase A and ATP induced Cl−-permeable single-channel activity in excised, inside-out patches from control, but not cftr(−/−) NMCM. The data in this report indicate that cAMP stimulation of NMCM activates an anion-permeable conductance with functional properties similar to those expected for CFTR, thus suggesting that CFTR may be responsible for the cAMP-activated conductance. CFTR may thus contribute to the permeation and/or regulation of Cl−- and ATP-permeable pathways in the developing heart.

scholarly journals cAMP activates an ATP-permeable pathway in neonatal rat cardiac myocytes

2000 ◽  
Vol 279 (1) ◽  
pp. C173-C187 ◽  
Author(s):  
Alan S. Lader ◽  
Yong-Fu Xiao ◽  
Catherine R. O'Riordan ◽  
Adriana G. Prat ◽  
G. Robert Jackson ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Single Channel ◽  
Atp Release ◽  
Neonatal Rat ◽  
Luciferase Assay ◽  
Neonatal Rat Cardiac Myocytes ◽  
Single Channel Currents ◽  
Rat Cardiac Myocytes ◽  
Camp Stimulation ◽  
R Domain

The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl− currents in either symmetrical MgCl2 or NaCl. ATP, adenosine 5′- O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl− and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMP-activated ATP transport in NRCM.

cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells

1997 ◽  
Vol 272 (2) ◽  
pp. C466-C475 ◽  
Author(s):  
H. F. Cantiello ◽  
G. R. Jackson ◽  
A. G. Prat ◽  
J. L. Gazley ◽  
J. N. Forrest ◽  
...  
Keyword(s):  
Single Channel ◽  
Primary Cultures ◽  
Disulfonic Acid ◽  
Rectal Gland ◽  
Bathing Solution ◽  
Whole Cell ◽  
Extracellular Milieu ◽  
Conductive Pathway ◽  
Shark Rectal Gland ◽  
Camp Stimulation

The molecular mechanisms associated with ATP transport and release into the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) cells, patch-clamp techniques were applied to primary cultures of SRG cells. Whole cell currents were obtained with either intracellular tris(hydroxymethyl)aminomethane (Tris) or Mg2+ salts of ATP (200 mM nominal ATP) and 280 mM NaCl bathing solution. Basal currents showed a sizable ATP permeability for outward movement of MgATP. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation significantly increased the whole cell conductance (with either intracellular Tris-ATP or MgATP). Symmetrical whole cell ATP currents were also observed after cAMP activation, thus consistent with ATP as the main charge carrier. The cAMP-inducible ATP currents were insensitive to the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylate, and anthracene-9-carboxylic acid but were readily blocked by nifedipine (400 microM) and glibenclamide (400 microM). The nature of the electrodiffusional ATP movement was further assessed by single-channel analysis of either MgATP or Tris-ATP currents in excised inside-out patches, both spontaneous and after activation with protein kinase A. Single-channel ATP currents were inhibited by either nifedipine or glibenclamide. Thus SRG cells express endogenous ATP-permeable pathways both before and after cAMP stimulation. Electrodiffusional ATP movement by SRG cells may play a significant role in the transport and delivery of cellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.

A1 Adenosine Receptors Modulate Respiratory Activity of the Neonatal Mouse Via the cAMP-Mediated Signaling Pathway

1999 ◽  
Vol 81 (1) ◽  
pp. 247-255 ◽  
Author(s):  
S. L. Mironov ◽  
K. Langohr ◽  
D. W. Richter
Keyword(s):  
Signaling Pathway ◽  
Adenosine Receptor ◽  
Adenosine Receptors ◽  
Single Channel ◽  
Respiratory Activity ◽  
Respiratory Rhythm ◽  
Neonatal Mouse ◽  
Intracellular Camp ◽  
Open Probability ◽  
Adenosine Receptor Agonist

Mironov, S. L., K. Langohr, and D. W. Richter. A1 adenosine receptors modulate respiratory activity of the neonatal mouse via the cAMP-mediated signaling pathway. J. Neurophysiol. 81: 247–255, 1999. The effects of adenosine and its analogs on the function of the respiratory center were studied in the spontaneously active rhythmic slice of neonatal and juvenile mice (4–14 days old). Whole cell, spontaneous postsynaptic currents (sPSCs) and single channel KATP currents were recorded in inspiratory neurons of the pre-Bötzinger complex. Adenosine (50–600 μM) inhibited the respiratory rhythm. This was accompanied by increase in the activity of KATP channels in cell-attached patches. The A1 adenosine receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA, 0.3–2 μM), inhibited the respiratory rhythm, sPSCs, and enhanced activity of KATP channels. The A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1–3 μM), showed opposite effects and occluded the CCPA actions. Agents specific for A2 adenosine receptors (CGS 21860 and NECA, both applied at 1–10 μM) were without effect. Elevation of intracellular cAMP concentration ([cAMP]i) by 8-Br-cAMP (200–500 μM), forskolin (0.5–2 μM), or isobutylmethylxantine (IBMX, 30–90 μM) reinforced the rhythm, whereas NaF (100–800 μM) depressed it. The open probability of single KATP channels in cell-attached patches decreased after application of forskolin and increased in the presence of NaF. [cAMP]i elevation reversed the effects of A1 receptors both on the respiratory rhythm and KATP channels. A1 receptors and [cAMP]i modified the hypoxic respiratory response. In the presence of A1 agonists the duration of hypoxic augmentation shortened, and depression of the respiratory rhythm occurred earlier. Elevation of [cAMP]i prolonged augmentation and delayed the development of the depression. We conclude that A1 adenosine receptors modulate the respiratory rhythm via inhibition of intracellular cAMP production and concomitant activation of KATP channels.

Peptidomics Analysis of Transient Regeneration in the Neonatal Mouse Heart

2017 ◽  
Vol 118 (9) ◽  
pp. 2828-2840 ◽  
Author(s):  
Yi Fan ◽  
Qijun Zhang ◽  
Hua Li ◽  
Zijie Cheng ◽  
Xing Li ◽  
...  
Keyword(s):  
Neonatal Mouse ◽  
Mouse Heart

Abstract 17185: Sympathetic Reinnervation is Required for Mammalian Cardiac Regeneration

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Ian A White ◽  
Julie Gordon ◽  
Wayne Balkan ◽  
Joshua M Hare
Keyword(s):  
Sympathetic Innervation ◽  
Cardiac Regeneration ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Neonatal Mouse ◽  
Left Ventricular Myocardium ◽  
Autonomic Nerves ◽  
Mouse Heart ◽  
Minimal Scarring ◽  
Age Related

Rationale: Established animal models of tissue and limb regeneration demonstrate a critical dependence on concurrent reinnervation by the peripheral nervous system. The abundance of autonomic nerves in the mammalian heart suggests they play a similar role in the response to cardiac injury. Objective: To test the hypothesis that reinnervation is required for innate neonatal cardiac regeneration. Methods and Results: Crossing Wnt1:cre transgenic mice with a double-tandem (td) tomato reporter strain identified all neural crest-derived cell lineages including the peripheral autonomic nerves in the heart. Whole mount epi-fluorescence microscopy facilitated the clear resolution of subepicardial autonomic nerves in the mouse ventricles providing unprecedented detail of the subepicardial neuroanatomy of the mouse heart. We confirmed that sympathetic nerve structures envelop the entire heart, and importantly, exhibit robust re-growth into the regenerating myocardium following resection of the left ventricular apex in neonatal mice. While innervated hearts regenerate with minimal scarring to the left ventricular myocardium, we report that innate cardiac regeneration was inhibited following sympathectomy, as determined by cross-sectional percentage of viable LV myocardium (n=9, 0.87±1.4% vs. n=6, 14.05±4.4% ; p<0.01). Conclusions: Ablation of post-ganglionic sympathetic nerves blocks the innate regenerative capacity of neonatal mouse hearts. Therefore, the innate ability of the neonatal mouse heart to undergo regeneration in response to injury is dependent on sympathetic innervation of the ventricular myocardium. This finding has significant implications for adult regeneration following myocardial infarction where nerve growth is hindered by age related influences and scar tissue.

Desensitization and resensitization rates of glutamate-activated channels may regulate motoneuron excitability

1991 ◽  
Vol 66 (4) ◽  
pp. 1166-1175 ◽  
Author(s):  
D. O. Smith ◽  
C. Franke ◽  
J. L. Rosenheimer ◽  
F. Zufall ◽  
H. Hatt
Keyword(s):  
Ampa Receptors ◽  
Single Channel ◽  
Kainate Receptors ◽  
Whole Cell ◽  
Solution Exchange ◽  
Channel Opening ◽  
Fast Solution ◽  
Agonist Concentration ◽  
Kinetics Of ◽  
Channel Properties

1. Single-channel properties of desensitizing glutamate-activated channels were analyzed in outside-out patch-clamp recordings from a motoneuron-enriched cell fraction from embryonic chick. A piezo-driven device was used to achieve fast solution exchange at the electrode tip, resulting in maximum activation within 2 ms. 2. Quisqualate/AMPA receptors, with a 13-pS conductance, desensitized rapidly; the desensitization rate depended on agonist concentration but not on membrane potential. When quisqualate was applied slowly, the quisqualate-activated channels desensitized without prior channel opening, indicating desensitization from the closed state. After a 10-ms refractory period, resensitization of all channels required up to 300 ms; resensitization rate did not depend on the duration of the preceding quisqualate application. 3. At agonist concentrations less than or equal to 1 mM, kainate receptors, with a 20-pS conductance, did not desensitize. At kainate concentrations greater than or equal to 1 mM, though, kainate receptors desensitized to a low steady-state conductance within approximately 200 ms. Resensitization of all channels required as long as 3 s, which could render kainate receptors inexcitable during high-frequency activation. 4. Desensitization rates of whole-cell currents were similar to those observed in outside-out mode. Glutamate- and quisqualate-activated responses were similar, suggesting that the rapidly desensitizing quisqualate-sensitive receptor type may dominate the kinetics of whole-cell excitatory postsynaptic currents (EPSCs) in this preparation. 5. It may be concluded that the efficacy of glutamate-mediated synaptic transmission is modulated by differences in the rates of desensitization and resensitization.

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