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H C Hartzell

Publications and source records attributed to H C Hartzell.

At least 37 records · Page 2Linked to original sources
PubMed1994

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii.

The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751-1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation.

Animals↗
PubMed1994

Developmental changes in modulation of calcium currents of rabbit ventricular cells by phosphodiesterase inhibitors.

BACKGROUND: We have previously shown major differences in beta-adrenergic and muscarinic modulation of L-type calcium currents (ICa) in newborn and adult rabbit heart. However, little is known about developmental changes in modulation of ICa by phosphodiesterases (PDEs), which also regulate intracellular cAMP concentration by its hydrolysis. METHODS AND RESULTS: Enzymatically isolated adult and newborn (1- to 3-day-old) rabbit ventricular myocytes were used to study the effects of PDE inhibitors on ICa measured by the whole-cell patch-clamp method. 3-Isobutyl-1-methyl-xanthine (IBMX), a nonselective PDE inhibitor, increased ICa in a dose-dependent manner for both groups. The maximal effect of IBMX, expressed as percentage increase in ICa over control levels, was greater for newborn myocytes than for adult myocytes, but the effects of IBMX applied alone were observed only at concentrations > 10 mumol/L. The concomitant use of 0.1 mumol/L isoproterenol produced a significant potentiation of the IBMX effect on ICa, with a significant additive effect of IBMX in newborn myocytes even at 0.05 mumol/L IBMX. The concomitant use of a subthreshold concentration of IBMX (0.1 mumol/L) did not potentiate the dose dependence of adult ICa on isoproterenol but did markedly potentiate the dose dependence of newborn ICa on isoproterenol. The Emax and EC50 of isoproterenol in the presence of 0.1 mumol/L IBMX on newborn ICa were 235% and 8 nmol/L, respectively, whereas the Emax and EC50 of isoproterenol in the absence of IBMX on newborn ICa were 111% and 81 nmol/L, respectively. The addition of 50 mumol/L IBMX to 10 mumol/L isoproterenol markedly increased the newborn ICa density up to a level equivalent to that reached with 200 mumol/L cAMP in the pipette (14.9 +/- 1.2 versus 13.4 +/- 0.7 pA/pF). Our data suggest that the inhibition constant (Ki) of IBMX for inhibiting PDEs that participate in the regulation of ICa is much lower in newborn than in adult myocytes. Milrinone 1 mumol/L, a selective PDE III inhibitor, increased the 0.1 mumol/L isoproterenol-stimulated ICa of adult myocytes but had no significant additive effect for the 0.1 mumol/L isoproterenol-stimulated ICa of newborn myocytes. Rolipram 1 mumol/L, a selective PDE IV inhibitor, increased the 0.1 mumol/L isoproterenol-stimulated ICa for newborn myocytes but had no significant additive effect for the 0.1 mumol/L isoproterenol-stimulated ICa for adult myocytes. CONCLUSIONS: These results suggest that the most important PDE isozyme for regulation of ICa of rabbit myocytes changes from PDE IV to PDE III during the postnatal period.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Molecular physiology of norepinephrine and serotonin transporters.

Cocaine- and antidepressant-sensitive norepinephrine and serotonin transporters (NETs and SERTs) are closely related members of the Na+/Cl- transporter gene family, whose other members include transporters for inhibitory amino acid transmitters, neuromodulators, osmolytes and nutrients. Availability of cloned NET and SERT cDNAs has permitted rapid progress in the definition of cellular sites of gene expression, the generation of transporter-specific antibodies suitable for biosynthetic and localization studies, the examination of structure-function relationships in heterologous expression systems and a biophysical analysis of transporter function. In situ hybridization and immunocytochemical studies indicate a primary expression of NET and SERT genes in brain by noradrenergic and serotonergic neurons, respectively. Both NET and SERT are synthesized as glycoproteins, with multiple glycosylation states apparent for SERT proteins in the brain and periphery. N-glycosylation of NET and SERT appears to be essential for transporter assembly and surface expression, but not for antagonist binding affinity. Homology cloning efforts have revealed novel NET and SERT homologs in nonmammalian species that are of potential value in the delineation of the precise sites for substrate and antagonist recognition, including a Drosophila melanogaster SERT with NET-like pharmacology. Electrophysiological recording of human NETs and SERTs stably expressed in HEK-293 cells reveals that both transporters move charge across the plasma membrane following the addition of substrates; these currents can be blocked by NET-and SERT-selective antagonists as well as by cocaine.

Animals↗
PubMed1993

Mechanisms of flagellar excision. I. The role of intracellular acidification.

It has previously been shown that exposure of Chlamydomonas to low pH induces the cells to shed their flagella. In the present paper we report that a 30-s treatment with 20 mM acetic, carbonic, formic, or benzoic acid at pH 4.0 will induce flagellar excision. In contrast, 20 mM concentrations of the stronger aspartic, phosphoric, citric, and tartaric acids (pH 4) do not induce excision. Further, the excision efficacy of acetate is a function of the concentration of protonated acetate. Thus, excision correlates with the presence of a protonated, membrane-permeant species of acid. Relative to acetate, the more permeant benzoate induces excision at a much lower concentration of protonated acid. We conclude that a flux of acid into the cell is the signal for excision. Previous work has shown that detergent-permeabilized cells excise their flagella in response to calcium but not in response to low pH. This suggests that the acidification of intact cells triggers excision by stimulating an increase in intracellular calcium. We have previously reported that the source of this calcium might be IP3-sensitive. In our model for the mechanism of pH-induced flagellar excision, a flux of acid into the cell activates phospholipase C, leading to IP3 production, the activation of an IP3-gated calcium channel (located on either an intracellular or surface membrane), and an increase in cytosolic calcium, which is the trigger of flagellar excision.

Acids↗
PubMed1993

A comparative analysis of the time course of cardiac Ca2+ current response to rapid applications of beta-adrenergic and dihydropyridine agonists.

A fast perfusion system was used to analyze the kinetics of the response of L-type calcium current (ICa) to rapid exposures to beta-adrenergic or dihydropyridine agonists in whole-cell patch-clamped frog ventricular myocytes. The perfusion system was based on the lateral motion of an array of plastic capillary tubes from which solutions flowed at a velocity of approximately 5 cm/s. Movement from one capillary to the adjacent one occurred in < 20 ms and complete exchange of extracellular solution was achieved in < 50 ms as demonstrated by the block of ICa by fastflow application of Cd during a depolarizing pulse. Fastflow applications of increasing concentrations of isoprenaline (Iso) led to a dose-dependent stimulation of ICa at [Iso] > 1 nM. The response of ICa to Iso always started after a delay of several seconds. The delay duration decreased as [Iso] increased, and was typically approximately 3 s at 10 microM Iso. The rising phase of ICa increase was monophasic and independent of [Iso] > 100 nM. For short applications of Iso (8.8 s), half maximal and maximal stimulation of ICa occurred approximately 20 s and approximately 40 s after the beginning of Iso application, respectively. When Iso was applied during a depolarizing pulse (with Ba as the charge carrier), IBa never increased during that pulse. The kinetics of the ICa response to Iso were not affected by varying the voltage clamp protocols or the ionic composition of intracellular and extracellular solutions. In comparison with the effects of Iso, the stimulatory effect of the dihydropyridine agonist (-)Bay K 8644 on ICa was approximately 15 times faster: delay, half-time to maximal and time to maximal responses were 15 times shorter with (-)Bay K 8644 than with Iso. It is concluded that frog ventricular myocytes respond slowly to a quick application of beta-adrenergic agonists.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Regulation of the cardiac delayed rectifier K current by neurotransmitters and magnesium.

The delayed rectifier K current plays an important role in cardiac electrophysiology: It is involved in the repolarization of the action potential and in frequency-dependent changes in action potential duration and waveform. The delayed rectifier current IK is regulated by the autonomic nervous system: Beta-adrenergic agonists increase IK. This increase is due to an increase in the maximally activatable current as well as a shift of the activation curve to more negative potentials. Thus, in response to sympathetic nerve stimulation, the action potential would be expected to repolarize more rapidly as a result of activation of more IK current and its activation at more negative potentials. Single-channel analysis suggests that the increase in IK is due to an increase in the availability of IK channels to respond to depolarization. IK is also regulated by internal free Mg2+. When the internal solution contains high [Mg2+], IK decreases, whereas low [Mg2+] results in an increase in current. The effect of Mg2+ is not detectably voltage dependent, suggesting that the mechanism of Mg2+ action involves an allosteric or enzymatic effect. Mg2+ also affects the rate of washout of the response to beta-adrenergic agonists, suggesting that Mg2+ may be affecting the activity of a protein phosphatase.

Adrenergic beta-Agonists↗
PubMed1993

Rate-limiting steps in the beta-adrenergic stimulation of cardiac calcium current.

Fast-flow perfusion and flash photolysis of caged compounds were used to study the activation kinetics of L-type calcium current (ICa) in frog cardiac myocytes. Rapid exposure to isoproterenol (Iso) for 1 s or approximately 1 min produced similar kinetics of increase in ICa with an initial lag period of approximately 3 s, followed by a monophasic rise in current with a half-time of approximately 20 s. Epinephrine, as well as caged Iso, produced increases with similar kinetics. The fact that ICa increased significantly even after short Iso applications suggests that agonist binding to the receptor is rapid and that the increase in ICa is independent of free agonist. To dissect the kinetic contributions of various steps in the cAMP-phosphorylation cascade, the kinetics of the responses to caged cAMP and caged GTP gamma S and fast perfusion of forskolin, acetylcholine, and propranolol were compared. The response to caged cAMP exhibited no lag period, but otherwise increased at a rate similar to that produced by Iso and reached a peak at approximately 40 s after flash photolysis. This suggests that the lag period itself is due to a step before cAMP accumulation, but that activation of protein kinase and phosphorylation of the calcium channel are relatively slow. A lag period was also observed when ICa was stimulated by flash photolysis of caged GTP gamma S and when adenylyl cyclase was activated directly by rapid perfusion with forskolin. The lag period observed with forskolin may be due to slow binding of forskolin. The lag period was not due to the time required for cAMP to reach a threshold concentration, because a similar lag was observed in response to Iso in cells having ICa previously stimulated submaximally by internal perfusion with a low concentration of cAMP. These results suggest that the lag period can be attributed to a step associated with activation of adenylyl cyclase and cAMP accumulation.

Acetylcholine↗
PubMed1993

Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

Acetylcholine↗
PubMed1993

Opposite effects of phosphatase inhibitors on L-type calcium and delayed rectifier currents in frog cardiac myocytes.

1. Application of the phosphatase inhibitors okadaic acid (OA) and microcystin (MC) to frog cardiomyocytes caused large increases in L-type calcium current (ICa) in the absence of beta-adrenergic agonists. The increase occurred without effects on the peak current-voltage relation or voltage-dependent inactivation. OA and MC caused a decrease in amplitude of delayed rectifier current (IK), which is opposite to the increase produced by cAMP-dependent phosphorylation. The decrease occurred without effects on voltage-dependent activation or reversal potential. 2. Analysis of the dose-response relations for OA and MC on ventricular cell ICa were best fitted with a single-site relationship with a K1/2 of 1.58 microM and 0.81 microM, respectively. These data suggest the predominant form of phosphatase active on ICa in this cell type is produced by protein phosphatase 1. Inhibition of phosphatase 2B (calcineurin) was without appreciable effect. 3. Reducing intracellular ATP levels was without effect on basal ICa suggesting that calcium channels may not need to be phosphorylated to open. ATP depletion was able to block completely the ICa increase induced by OA or MC. This demonstrates that the effects of OA and MC on ICa are mediated by a phosphorylation reaction. In contrast, ATP depletion totally abolished IK, suggesting either a requirement for ATP or phosphorylation for basal function of the delayed rectifier channel. 4. Internal perfusion of a peptide inhibitor (PKI(5-22)) of protein kinase A (PK-A) was without effect on basal current levels of ICa or IK, suggesting that this kinase is not phosphorylating these channels under basal conditions. Furthermore, although PKI is capable of completely blocking the response of ICa to isoprenaline or forskolin, PKI does not affect the increase in ICa induced by MC or OA. Inhibition of adenylate cyclase with acetylcholine or inhibition of PK-A with adenosine cyclic 3',5'-(Rp)-phosphothioate (Rp-cAMPS) also had no effect on the response to OA or MC. 5. Application of beta-adrenergic agonist, forskolin or cAMP all produced additional increases in the presence of saturating doses of MC or OA. This supports the hypothesis that PK-A is not mediating the OA response and that phosphatase inhibition does not result in complete phosphorylation of PK-A sites. 6. To attempt to identify the protein kinase activity responsible for OA effects on ICa and IK, several types of protein kinase inhibitors were internally perfused.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1992

Direct regulation of cardiac Ca2+ channels by G proteins: neither proven nor necessary?

The cardiac L-type Ca2+ channel has served as a model for ion channel regulation for over a decade. The Ca2+ current is increased by beta-adrenoceptor stimulation and this effect is inhibited by muscarinic acetylcholine receptor stimulation. It is well established that beta-adrenoceptor stimulation increases this current largely by cAMP-dependent phosphorylation but recently data have been presented that suggest that this channel may also be regulated directly by G proteins. This review by Criss Hartzell and Rodolphe Fischmeister evaluates evidence for this second regulatory pathway and concludes that, although G proteins affect cardiac Ca2+ channels in bilayers and excised patches, there is little evidence that this pathway is physiologically significant.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Differences in effects of forskolin and an analog on calcium currents in cardiac myocytes suggest intra- and extracellular sites of action.

The effects of forskolin (FO) and a water-soluble derivative of FO, L858051 (7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino)-butyryl] forskolin), were compared on calcium currents (ICa) studied by the whole-cell patch-clamp technique in frog ventricular cardiac myocytes. Both FO and L858051 increased ICa, with half-times of 160 +/- 20 sec and 343 +/- 22 sec, respectively. The stimulation was blocked by internal perfusion with inhibitors of protein kinase A. The EC50 for stimulation of ICa was 0.3 microM for FO and 1.0 microM for L858051. The maximal stimulated current was the same for both drugs, 20.3 microA/cm2 and 23.1 microA/cm2, respectively. Internal perfusion with 30-500 microM guanylyl 5'-imidodiphosphate [Gpp(NH)p] suppressed ICa stimulation by low concentrations of FO or L858051. This suppression was due to a rightward shift in the concentration-response curve, with increases in the EC50 values to 11.4 microM for FO and 28.4 microM for L858051. Isoproterenol (ISO) was ineffective in increasing ICa after the FO-stimulated ICa had been reduced by Gpp(NH)p and FO had been washed out. In contrast, after the L858051-stimulated current had been reduced by Gpp(NH)p, ISO stimulated ICa significantly. This stimulation was blocked by inhibitors of protein kinase A and was due to a positive effect of L858051 not shared by FO. A brief application of L858051 after Gpp(NH)p had blocked the ISO response restored the ISO response for at least 30 min. This effect was mimicked by internal perfusion with low concentrations of L858051. We conclude that the ability of brief exposure of L858051, but not FO, to restore the response to ISO after Gpp(NH)p is due to the accumulation of L858051 intracellularly, due to its hydrophilicity. Because internal L858051 and FO are very ineffective in stimulating adenylyl cyclase, whereas internal L858051 can restore the ISO response blocked by Gpp(NH)p, we propose that FO compounds can affect adenylyl cyclase at two sites, one site that is accessible only from the extracellular side that stimulates catalytic activity and another that is accessible from the intracellular side that increases beta-agonist efficacy in the presence of Gpp(NH)p.

Animals↗
PubMed1991

Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation.

The positive inotropic effect of the sympathetic nervous system on the heart is partly mediated by an increase in the voltage-gated Ca2+ current (ICa). This increase is generally attributed to beta-adrenergic receptor-stimulated cyclic AMP-dependent phosphorylation of the Ca2+ channel. It has been suggested that cAMP-dependent phosphorylation cannot explain all the effects of beta-adrenergic agonists on ICa and that a parallel membrane-delimited pathway involving the 'direct' action of the G protein Gs also stimulates ICa. A precedent exists for such a membrane-delimited pathway in the activation of a K+ channel by acetylcholine in heart. A membrane-delimited pathway for stimulation of ICa might be important in rapid beat-to-beat regulation of contraction by the sympathetic nervous system, because isoproterenol may produce a biphasic increase in ICa with the rapid phase (tau = 150 ms) putatively mediated by the direct pathway and the slow phase (tau = 35 s) by cAMP-dependent phosphorylation. Here we report that in frog, rat, and guinea pig ventricular myocytes ICa increases slowly and monophasically in response to isoproterenol. The increase is completely blocked by inhibitors of cAMP-dependent phosphorylation. Furthermore, the time course of the increase in ICa closely parallels the increase in contractile force produced by sympathetic nerve stimulation. These data refute earlier suggestions that regulation of Ca2+ channels by the sympathetic nervous system involves or requires a direct G-protein pathway.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Cyclic AMP phosphodiesterases and Ca2+ current regulation in cardiac cells.

At least four different isoforms of phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP in cardiac cells. However, their distribution, localization and functional coupling to physiological effectors (such as ion channels, contractile proteins, etc.) vary significantly among various animal species and cardiac tissues. Because the activity of cardiac Ca2+ channels is strongly regulated by cAMP-dependent phosphorylation, Ca(2+)-channel current (ICa) measured in isolated cardiac myocytes may be used as a probe for studying cAMP metabolism. When the activity of adenylyl cyclase is bypassed by intracellular perfusion with submaximal concentrations of cAMP, effects of specific PDE inhibitors on ICa amplitude are mainly determined by their effects on PDE activity. This approach can be used to evaluate in vivo the functional coupling of various PDE isozymes to Ca2+ channels and their differential participation in the hormonal regulation of ICa and cardiac function. Combined with in vitro biochemical studies, such an experimental approach has permitted the discovery of hormonal inhibition of PDE activity in cardiac myocytes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Alterations in Ca2+ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers.

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.

Animals↗
PubMed1991

Regulation of Ca2+ current in frog ventricular cardiomyocytes by 5'-guanylylimidodiphosphate and acetylcholine.

1. Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. The effect of internal perfusion with the hydrolysis-resistant GTP analogue, GppNHp (5'guanylylimidodiphosphate), on basal ICa and ICa stimulated with forskolin or isoprenaline was examined to gain insight into the role of G proteins in ICa regulation. 2. Without added guanine nucleotides, isoprenaline stimulated ICa approximately 14-fold with an EC50 of 0.09 microM. Forskolin stimulated ICa approximately 10-fold with an EC50 of 0.30 microM. 3. Internal 30 microM-GppNHp produced an approximately 80% decrease in ICa elevated by 0.3 microM-isoprenaline or 3 microM-forskolin. The inhibition of isoprenaline stimulation was due to a decrease in the maximal stimulation from approximately 14-fold to approximately 14-fold without a significant change in the EC50. In contrast, the reduction in forskolin stimulation was due to a 22-fold increase in the EC50 to 11.4 microM, with little change in maximal stimulation. 4. The inhibition of stimulated ICa by GppNHp is likely to be mediated by a G protein, because the effects of GppNHp are irreversible, and are blocked by excess GTP. ICa is affected similarly by GppNHp and by ACh. This suggests that GppNHp activates the same G protein that is normally activated by ACh, but activation by GppNHp occurs in the absence of agonist occupation of the muscarinic receptor. 5. The increase in the EC50 for forskolin produced by internal GppNHp was reversed by exposure to isoprenaline, which itself did not affect ICa amplitude. On average, exposure to isoprenaline in the presence of GppNHp caused an irreversible 81-fold decrease in the EC50 for forskolin to 0.14 microM. Stimulation of ICa by forskolin after internal GppNHp and exposure to isoprenaline was completely blocked by the protein kinase A inhibitor PKI(5-22). 6. These effects do not involve the phospholipase C system, because they are not mimicked by phorbol esters or internal inositol 1,4,5-trisphosphate (IP3) and are not blocked by bromophenacyl bromide or neomycin. 7. Direct effects of G proteins on ICa were not evident, because internal perfusion with PKI(5-22) completely inhibited isoprenaline- or forskolin-stimulated increases in ICa, and neither ACh nor internal GppNHp (30-500 microM) affected basal ICa or ICa elevated by internally perfused cyclic AMP. 8. These results suggest that the predominant site of action of the inhibitory G protein activated by either GppNHp or ACh is adenylyl cyclase. Furthermore, the internally perfused frog cardiomyocytes may provide a useful approach for probing the detailed interactions of G proteins, forskolin, and adenylyl cyclase in an intact cell.

Acetylcholine↗
PubMed1991

Effects of Mg2+ on basal and beta-adrenergic-stimulated delayed rectifier potassium current in frog atrial myocytes.

1. The effects of internal Mg2+ ions on the delayed rectifier potassium current (IK) of bull-frog atrial myocytes were studied using the whole-cell configuration of the patch-clamp technique with a perfusable patch electrode. 2. Initial variations in IK amplitude were dependent on [Mg2+]i. With [Mg2+] greater than 1 mM, the amplitude of IK usually decreased after initiating the whole-cell recording configuration (run-down); with [Mg2+]i less than 1 mM, IK usually increased (run-up). Mg2+ blocked IK with an apparent half-maximal effect of 0.6 mM [Mg2+]i. 3. The basal free [Mg2+]i, indicated by the amplitude of IK before run-up or run-down, was estimated from the relationship between [Mg2+]i and IK to be 0.8 mM. 4. The amplitude of both the activation curve and the instantaneous voltage-current relationship was decreased by increasing [Mg2+]i. Under these conditions, the voltage dependence of IK was not affected. 5. The rate of activation of the current at +40 mV was slowed by increasing [Mg2+]i with little effect on the rate of deactivation at -50 mV. This is in contrast to the effects of isoprenaline, which speeded activation and slowed deactivation. 6. Isoprenaline increased IK on average by about 2.5 pA/pF, whether IK had previously run down or not, and regardless of [Mg2+]i. The reversibility of isoprenaline was partially inhibited at [Mg2+]i less than 1 mM. 7. It is concluded that Mg2+ affects IK via several mechanisms that might include a Mg(2+)-dependent phosphatase.

Adrenergic beta-Agonists↗
PubMed1990

Single delayed rectifier channels in frog atrial cells. Effects of beta-adrenergic stimulation.

The patch-clamp technique with two pipettes was used to record single delayed K+ channels (cell-attached electrode) and to control the potential and the composition of the intracellular compartment (whole-cell electrode). With 30 microM cAMP in the cell and physiological potassium concentrations inside and outside the patch, a channel carrying an outward current was characterized. Its open probability was very low and the channel was recorded in only 5% of patches under control conditions. Increasing intracellular cAMP increased the probability of finding a channel in a patch 10-fold. The channel had the characteristics expected of a delayed rectifier channel. The time-course of its ensemble average resembled the whole-cell current in the same cell. The current-voltage relationship exhibited inward rectification, with a slope conductance of 20 pS in the linear portion and a reversal potential close to EK. Both the open- and the closed-time distributions were described by the sum of two exponentials, suggesting a complicated gating scheme involving two closed states and two open states. The beta-adrenergic stimulation did not change the conductance of the channel, but increased its probability of opening.

Animals↗