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T C Hwang

Publications and source records attributed to T C Hwang.

27 records · Page 2Linked to original sources
PubMed1992

The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator.

Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined.

Adenosine Triphosphate↗
PubMed1992

Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance.

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.

Acetylcholine↗
PubMed1992

Pipette GTP is essential for receptor-mediated regulation of Cl- current in dialysed myocytes from guinea-pig ventricle.

1. Wide-tipped, low-resistance (approximately 1 M omega) pipettes were used to record the whole-cell Cl- current activated by cAMP-dependent protein kinase (PKA) in guinea-pig ventricular myocytes internally dialysed with or without GTP. Without GTP in the pipette, the response to 1 microM-isoprenaline declined with time and eventually disappeared, usually within approximately 20 min of rupturing the membrane and beginning cell dialysis. 2. This rundown of the isoprenaline response occurred more quickly with wider, lower-resistance pipette tips. 3. After complete rundown of the isoprenaline response, histamine (10 microM), another agonist known to elicit the Cl- current, also had no effect, but extracellular forskolin (1 microM) or intrapipette cAMP (1 mM) could still readily elicit the Cl- current. 4. In contrast, with 100 microM-GTP in the pipette, the response to 1 microM-isoprenaline was well maintained for periods greater than 20 min. But, if GTP was then withdrawn from the pipette, a rundown of the isoprenaline response was seen comparable to that in the experiments begun with GTP-free pipette solution. Moreover, in experiments begun without pipette GTP, the addition of 100 microM-GTP to the pipette solution, after the response to isoprenaline had disappeared, was able to restore that Cl- current response. 5. With GTP in the pipette, the forskolin-induced Cl- current could be suppressed by concurrent exposure to carbachol (10 microM). That inhibition was not seen in myocytes pretreated with pertussis toxin. In untreated myocytes dialysed with GTP-free pipette solution, after disappearance of the isoprenaline response, the muscarinic receptor-mediated inhibition was itself abolished. 6. We confirm that both beta-adrenoceptor-mediated activation of the Cl- current by isoprenaline, and muscarinic receptor-mediated inhibition of the forskolin-induced Cl- current, are mediated by G proteins, and conclude that the disappearance of both receptor-mediated responses during whole-cell recording with GTP-free pipette solution reflects the fall of cellular [GTP] below the level required to maintain G protein-dependent signal transduction.

Animals↗
PubMed1990

A delayed rectifier potassium current in Xenopus oocytes.

A delayed voltage-dependent K+ current endogenous to Xenopus oocytes has been investigated by the voltage-clamp technique. Both activation and inactivation of the K+ current are voltage-dependent processes. The K+ currents were activated when membrane potential was depolarized from a holding potential of -90 to -50 mV. The peak current was reached within 150 ms at membrane potential of +30 mV. Voltage-dependent inactivation of the current was observed by depolarizing the membrane potential from -50 to 0 mV at 10-mV increments. Voltage-dependent inactivation was a slow process with a time constant of 16.5 s at -10 mV. Removal of Ca2+ from the bath has no effect on current amplitudes, which indicates that the current is Ca2+)-insensitive. Tail current analysis showed that reversal potentials were shifted by changing external K+ concentration, as would be expected for a K(+)-selective channel. The current was sensitive to quinine, a K+ channel blocker, with a Ki of 35 microM. The blockade of quinine is voltage-independent in the range of -20 to +60 mV. Whereas oocytes from the same animal have a relatively homogeneous current distribution, average amplitude of the K+ current varied among oocytes from different animals from 30 to 400 nA at membrane potential of +30 mV. Our results indicate the presence of the endogenous K+ current in Xenopus oocytes with characteristics of the delayed rectifier found in some nerve and muscle cells.

Animals↗
PubMed1990

Direct modulation of secretory chloride channels by arachidonic and other cis unsaturated fatty acids.

The effect of fatty acids on Cl- channels and transepithelial Cl- secretion is investigated. Patch-clamp experiments show that arachidonic acid blocks Cl- channels in a dose-dependent manner. Kinetic analysis shows that the mean open time is decreased 10-fold with 25 microM arachidonic acid. There is a linear relationship between the reciprocal of mean open time and blocker concentration within the range of 1 to 25 microM. The reciprocal of mean blocked time does not change with arachidonic acid concentration. Other cis unsaturated fatty acids, including oleic, linoleic, and ricinoleic acids, demonstrate similar blocks. Trans unsaturated acids such as elaidic acid and saturated fatty acids, including stearic, palmitic, and myristic acids, do not inhibit the channel at 20 microM. Ricinoleic acid decreases short circuit current in T84 cells, a colonic carcinoma cell line that secretes Cl-. Our results suggest that the direct effect of arachidonic and other fatty acids on Cl- secretion is to block Cl- channel current.

Arachidonic Acid↗
PubMed1989

Cl- channels in CF: lack of activation by protein kinase C and cAMP-dependent protein kinase.

Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.

Adult↗
PubMed1989

Deciduomal estrogen and progesterone receptors in unilateral pregnant hamsters.

Deciduomal reactions in response to artificial stimuli were induced on the tube-ligated uterine horns of unilateral pregnant hamsters. Serum progesterone levels were determined, and the changes of estrogen and progesterone receptors (ER and PR, respectively) were simultaneously studied in the deciduoma. After stimulation on day 4 of pregnancy (D4), the serum progesterone level rose steadily to about 16 ng/ml on D12 and remained at plateau until D14. A sharp increase followed by a rapid fall in progesterone was observed on D15 and D16. The weight of the deciduoma-bearing horn increased more than 5 times to a maximal level on D10. Both deciduomal and myometrial weights were maintained and remained elevated on D16. Histological observation revealed that the maintenance of decidual cells was heterogenous. Cells at antimesometrial site (AMS) started to regress on D10, whereas cells at mesometrial site (MS) remained recognizable on D16. Cytosol ER was detected during the early stage (D6) of decidualization. The cytosol ER in the deciduoma continuously decreased and became undetectable on D10. Nuclear ER was not detected during the time of study. The nuclear and cytosol PR increased and reached a peak level on D8 and D10, respectively. The concentration of PR declined sharply afterward. The PR levels in deciduomal tissues at MS and AMS were separately studied. PR in AMS decreased gradually after D8 to basal level on D14. PR at MS still remained relatively constant on D12 followed by a precipitous decrease. These data show a prolongation of the deciduomal life span and the heterogeneity of deciduomal maintenance and regression in unilateral pregnant hamsters. This strongly suggests a close relationship between the loss of PR and the regression of the deciduoma.

Animals↗