Changes of beta2-adrenergic stimulation induced by hyperosmosis in human atrium.

PURPOSE: The purpose of the present study was to determine whether extracellular osmotic pressure modulates beta2-adrenergic stimulation of the contraction force and L-type Ca2+ current in human atrial myocytes. MATERIAL AND METHODS: Experiments were performed on human atrial trabeculae and myocytes isolated from the right atrium. The concentration dependent effect of salbutamol (SAL), a beta2-adrenoreceptor agonist, on peak tension (P) and L-type calcium current (ICaL) under isoosmolar (345 mOsm) and hyperosmolar (405 or 525 mOsm was achieved by adding of mannitol) conditions was studied. RESULTS: Salbutamol (10 nmol/L-10 micromol/L) added to the control solution increased P by 180.6 +/- 45.8% over control with a half-stimulation constant EC50 = 27 +/- 6 nmol/L. Under isoosmolar conditions SAL (0.1/10(3)nmol/L) increased ICaL by 182.3 +/- 19.8% over control with an EC50 2.9 +/- 0.9 nmol/L. In hyperosmolar solutions the same concentrations of SAL increased P and ICaL by 57.2 +/- 12.6% and 217.2 +/- 70.5% over control with EC50 = 640 +/- 260 nmol/L and 12 +/- 5 nmol/L respectively. CONCLUSIONS: These results indicated that hyperosmolarity reduced the effect of beta2-adrenergic stimulation, i.e. the dose-response curve of salbutamol on L-type calcium current was shifted to the higher concentration range and maximal increase in contraction force was diminished in human atrial cells.

Activation of metabotropic glutamate receptor 1 accelerates NMDA receptor trafficking.

Regulation of neuronal NMDA receptors (NMDARs) by group I metabotropic glutamate receptors (mGluRs) is known to play a critical role in synaptic transmission. The molecular mechanisms underlying mGluR1-mediated potentiation of NMDARs are as yet unclear. The present study shows that in Xenopus oocytes expressing recombinant receptors, activation of mGluR1 potentiates NMDA channel activity by recruitment of new channels to the plasma membrane via regulated exocytosis. Activation of mGluR1alpha induced (1) an increase in channel number times channel open probability, with no change in mean open time, unitary conductance, or reversal potential; (2) an increase in charge transfer in the presence of NMDA and the open channel blocker MK-801, indicating an increased number of functional NMDARs in the cell membrane; and (3) increased NR1 surface expression, as indicated by cell surface Western blots and immunofluorescence. Botulinum neurotoxin A or expression of a dominant negative mutant of synaptosomal associated protein of 25 kDa molelcular mass (SNAP-25) greatly reduced mGluR1alpha-mediated potentiation, indicating that receptor trafficking occurs via a SNAP-25-mediated form of soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor-dependent exocytosis. Because group I mGluRs are localized to the perisynaptic region in juxtaposition to synaptic NMDARs at glutamatergic synapses in the hippocampus, mGluR-mediated insertion of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C.

Potentiation of ionotropic glutamate receptor activity by metabotropic glutamate receptors (mGluRs) is thought to modulate activity at glutamatergic synapses in the hippocampus. However, the precise pathway by which this modulation occurs is not well understood. The present study tests the hypothesis that mGluR1-mediated potentiation of N-methyl-D-aspartate receptors (NMDARs) occurs via a phospholipase C (PLC)-initiated cascade. NMDAR functional activity was examined by whole-cell recording from Xenopus oocytes expressing recombinant NMDARs and mGluR1alpha. The mGluR1 agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) significantly potentiated NMDA-elicited currents. mGluR1alpha-mediated potentiation of NMDA responses was eliminated by the PLC inhibitor U-73122. Buffering of intracellular Ca2+ by BAPTA-AM or depletion of intracellular Ca2+ by the Ca2+/ATPase inhibitor thapsigargin greatly reduced ACPD potentiation. ACPD potentiation was reduced by the specific protein kinase C (PKC) inhibitor Ro-32-0432 and eliminated by the broad spectrum kinase inhibitor staurosporine. ACPD produced no further potentiation after potentiation of NMDARs by the PKC-activating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Thus, Group I mGluRs potentiate NMDA responses via activation of PLC; at least part of the potentiation is due to rise in intracellular Ca2+ and stimulation of PKC. Cytochalasin D, which disrupts the actin cytoskeleton, blocked ACPD-elicited chloride currents and ACPD-induced potentiation of NMDAR currents, consistent with a role for cytoskeletal protein(s) in the signaling pathway. As Group I mGluRs are localized to the perisynaptic region in juxtaposition to NMDARs at glutamatergic synapses, mGluR-mediated potentiation of NMDAR activity may play a role in synaptic transmission and plasticity including LTP.

Protein kinase C modulates NMDA receptor trafficking and gating.

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.

Insulin promotes rapid delivery of N-methyl-D- aspartate receptors to the cell surface by exocytosis.

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nP(o)) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or P(o); (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in P(o); and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes.

The whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. The beta-2 adrenergic agonist zinterol increased ICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 microM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 microM), a nonselective phosphodiesterase inhibitor, or forskolin (10 microM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 microM zinterol. Zinterol had a long lasting action on frog ICa because after washout of the drug, ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 microM) during this recovery phase promptly reduced ICa back to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 microM, respectively. In all three preparations, intracellular perfusion with 20 microM PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol on ICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase in ICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation.

Pharmacological characterization of the receptors involved in the beta-adrenoceptor-mediated stimulation of the L-type Ca2+ current in frog ventricular myocytes.

1. The whole-cell patch-clamp was used for studying the effects of various beta1- and beta2-adrenoceptor agonists and antagonists on the L-type Ca current (Ica) in frog ventricular myocytes. 2. Dose-response curves for the effects of isoprenaline (non selective beta-agonist), salbutamol (beta2-agonist), dobutamine (beta1-agonist) on ICa were obtained in the absence and presence of various concentrations of ICI 118551 (beta2-antagonist), metoprolol (beta1-antagonist) and xamoterol (partial beta1-agonist) to derive EC50 (i.e. the concentration of beta-agonist at which the response was 50% of the maximum) and Emax (the maximal response) values by use of a Michaelis equation. Schild regression analysis was performed to examine whether the antagonists were competitive and to determine the equilibrium dissociation constant (K(B)) for the antagonist-receptor complex. 3. Isoprenaline increased ICa with an EC50 of 20.0 nM and an Emax of 597%. ICI 118551 and metoprolol competitively antagonized the effect of isoprenaline with a K(B) of 3.80 nM and 207 nM, respectively. 4. Salbutamol increased ICa with an EC50 of 290 nM and an Emax of 512%. ICI 118551 and metoprolol competitively antagonized the effect of salbutamol with a K(B) of 1.77 nM and 456 nM, respectively. 5. Dobutamine increased ICa with an EC50 of 2.40 microM and an Emax of 265%. ICI 118551 and metoprolol competitively antagonized the effect of dobutamine with a K(B) of 2.84 nM and 609 nM, respectively. 6. Xamoterol had no stimulating effect on ICa. However, xamoterol competitively antagonized the stimulating effects of isoprenaline, salbutamol and dobutamine on ICa with a K(B) of 58-64 nM. 7. We conclude that a single population of receptors is involved in the beta-adrenoceptor-mediated regulation of ICa in frog ventricular myocytes. The pharmacological pattern of the response of ICa to the different beta-adrenoceptor agonists and antagonists tested suggests that these receptors are of the beta2-subtype.