Rapid Activation Kinetics Research Articles

Ionic currents underlying fast action potentials in the obliquely striated muscle cells of the octopus arm.

The octopus arm provides a unique model for neuromuscular systems of flexible appendages. We previously reported the electrical compactness of the arm muscle cells and their rich excitable properties ranging from fast oscillations to overshooting action potentials. Here we characterize the voltage-activated ionic currents in the muscle cell membrane. We found three depolarization-activated ionic currents: 1) a high-voltage-activated L-type Ca(2+) current, which began activating at approximately -35 mV, was eliminated when Ca(2+) was substituted by Mg(2+), was blocked by nifedipine, and showed Ca(2+)-dependent inactivation. This current had very rapid activation kinetics (peaked within milliseconds) and slow inactivation kinetics (tau in the order of 50 ms). 2) A delayed rectifier K(+) current that was totally blocked by 10 mM TEA and partially blocked by 10 mM 4-aminopyridine (4AP). This current exhibited relatively slow activation kinetics (tau in the order of 15 ms) and inactivated only partially with a time constant of ~150 ms. And 3) a transient A-type K(+) current that was totally blocked by 10 mM 4AP and was partially blocked by 10 mM TEA. This current exhibited very fast activation kinetics (peaked within milliseconds) and inactivated with a time constant in the order of 60 ms. Inactivation of the A-type current was almost complete at -40 mV. No voltage-dependent Na(+) current was found in these cells. The octopus arm muscle cells generate fast (~3 ms) overshooting spikes in physiological conditions that are carried by a slowly inactivating L-type Ca(2+) current.

Requirement of Mitogen-activated Protein Kinase Kinase 3 (MKK3) for Activation of p38α and p38δ MAPK Isoforms by TGF-β1 in Murine Mesangial Cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.

Na + dependence of extracellular Ca 2+-sensing mechanisms leading to activation of an outwardly rectifying Cl − channel in murine osteoclasts

An elevation in the extracellular Ca 2+ concentration ([Ca 2+ o) is a key signal for bone remodeling by inhibiting the resorbing activity of osteoclasts. The [Ca 2+] o-sensing responses include a variety of morphological and functional changes, but the underlying mechanisms are yet to be defined. This study was aimed at investigating the [Ca 2+] o-sensing mechanisms leading to the activation of the Cl − channel in murine osteoclasts. A rise in either Ca 2+ or Gd 3+ activated an outwardly rectifying Cl − (OR cl) channel reversibly and dose-dependently, which was characterized by rapid activation kinetics, little inactivation, and blockage by DIDS. The concentration required for a half-maximal response was estimated to be >20–30 mmol/L for Ca 2+. Intracellular dialysis with an ATP-free pipette solution or application of an actin destabilizer, cytochalasin D, decreased the [Ca 2+] o-activated OR cl current. Substitution of extracellular Na + by an impermeable cation, N-methyl- D-glucamine +, inhibited the [Ca 2+] o-activated OR cl channel, suggesting that the activation depended on extracellular Na +. A blocker for the Na +-Ca 2+ exchanger, 2′4′-dichlorobenzamil hydrochloride (DCB), inhibited the [Ca 2+] o-activated OR cl channel as well. Although 10 mmol/L Ca 2+ activated the OR cl current only slightly at a standard intracellular pH (7.3), decreasing pH by dialyzing cells with an acidic pipette solution (pH 6.6) enhanced the [Ca 2+] o-activated OR cl current. This potentiation by cell acidosis was eliminated by amiloride, a blocker for the Na +-H + exchanger. Zinc ion (0.1 mmol/L) and a polycation, neomycin (0.2 mmol/L), activated the OR cl current at intracellular pH 6.6, whereas the effects of those cations were negligible at intracellular pH 7.3. These results suggest that [Ca 2+] o-sensing mechanisms, leading to activation of the OR cl channel in murine osteoclasts, are regulated by ATP and actin cytoskeletal organization, and are sensitized greatly by cell acidosis. Contributions of Na +-dependent transporters in this activating process are examined in the context of a possible intermediate signal of cell swelling caused by Na + influx.

Acute actions of tumor necrosis factor-alpha on intracellular Ca(2+) and K(+) currents in human microglia.

The effects of acute application of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) on levels of intracellular Ca(2+) ([Ca(2+)]i) and on whole-cell outward and inward K(+) currents were studied in cultured human microglia. TNFalpha elicited a linear increase in [Ca(2+)]i to a plateau level in microglia bathed in either standard physiological saline solution or Ca(2+)-free physiological saline solution. The rate of increase of [Ca(2+)]i or the level of [Ca(2+)]i attained was not significantly altered in the absence of external Ca(2+) indicating that Ca(2+) influx did not contribute appreciably to the cytokine-induced rise in [Ca(2+)]i. This point was directly confirmed using Mn(2+) quenching where no change in signal fluorescence was observed with TNFalpha treatment of microglia in Ca(2+)-free physiological saline solution. The rate of increase of [Ca(2+)]i induced by TNFalpha in Ca(2+)-free physiological saline solution was not altered by prior application of ATP to deplete inositol triphosphate stores indicating that these stores did not contribute to the cytokine response. In whole-cell patch clamp recordings, the acute treatment of human microglia with TNFalpha led to the expression of an outward K(+) current in one-third (14 of 41) of cells. This current was activated at potentials positive to -30 mV, showed rapid kinetics of activation with no evident inactivation and had an I-V relation exhibiting outward rectification. Analysis of tail currents showed reversal of the outward K(+) current near -70 mV and tetraethylammonium (10 mM) inhibited the outward K(+) current to 24% of control level. Acute application of TNFalpha had no effect to alter inward rectifier currents generated from voltage ramps. The signaling pathways involving TNFalpha modulation of [Ca(2+)]i and K(+) channels in human microglia may contribute to functional and pathological actions of the cytokine in the brain.

Developmental changes in hyperpolarization-activated currents I(h) and I(K(IR)) in isolated rat intracardiac neurons.

The hyperpolarization-activated nonselective cation current, I(h), was investigated in neonatal and adult rat intracardiac neurons. I(h) was observed in all neurons studied and displayed slow time-dependent rectification. I(h) was isolated by blockade with external Cs(+) (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I(h) was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I(h) was sensitive to changes in external Na(+) and K(+) concentrations. An inwardly rectifying K(+) current, I(K(IR)), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba(2+). I(K(IR)) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I(K(IR)) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K(+) rectifier described in other mammalian autonomic neurons. I(K(IR)) was sensitive to changes in external K(+), whereby raising the external K(+) concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na(+) had no effect on the reversal potential or amplitude of I(K(IR)). I(K(IR)) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I(h) may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.

Cystic Fibrosis Pathogens Activate Ca2+-dependent Mitogen-activated Protein Kinase Signaling Pathways in Airway Epithelial Cells

Much of the pulmonary disease in cystic fibrosis is associated with polymorphonuclear leukocyte-dominated airway inflammation caused by bacterial infection. Respiratory epithelial cells express the polymorphonuclear chemokine interleukin-8 (IL-8) in response to ligation of asialylated glycolipid receptors, which are increased on damaged or regenerating cells and those with cystic fibrosis transmembrane conductance regulator mutations. Because both Pseudomonas aeruginosa and Staphylococcus aureus, the most common pathogens in cystic fibrosis, bind asialylated glycolipid receptors such as asialoGM1, we postulated that diverse bacteria can activate a common epithelial signaling pathway to elicit IL-8 expression. P. aeruginosa PAO1 but not pil mutants and S. aureus RN6390 but not the agr mutant RN6911 stimulated increases in [Ca(2+)](i) in 1HAEo- airway epithelial cells. This response stimulated p38 and ERK1/2 mitogen-activated protein kinase (MAPK) signaling cascades resulting in NF-kappaB activation and IL-8 expression. Ligation of the asialoGM1 receptor or thapsigargin-elicited Ca(2+) release activated this pathway, whereas P. aeruginosa lipopolysaccharide did not. The rapid kinetics of epithelial activation precluded bacterial invasion of the epithelium. Recognition of asialylated glycolipid receptors on airway epithelial cells provides a common pathway for Gram-positive and Gram-negative organisms to initiate an epithelial inflammatory response.

Heterogeneity of the Memory CD4 T Cell Response: Persisting Effectors and Resting Memory T Cells

Defining the cellular composition of the memory T cell pool has been complicated by an inability to distinguish effector and memory T cells. We present here an activation profile assay, using anti-CD3 and antigenic stimuli, that clearly distinguishes effector and memory CD4 T cells and defines subsets of long-lived memory CD4 T cells based on CD62 ligand (CD62L) expression. The CD62L(low) memory subset functionally resembles effector cells, exhibiting hyper-responsiveness to antigenic and anti-CD3 mediated stimuli, high proliferative capacity, and rapid activation kinetics. The CD62L(high) memory subset functionally resembles resting memory cells, exhibiting hyporesponsiveness to anti-CD3 stimuli, lower proliferative capacity, and slower activation kinetics. Our results indicate that the memory CD4 T cell pool is heterogeneous, consisting of persisting effectors and resting memory T cells.

Morphology of local axon collaterals of electrophysiologically characterised neurons in the rat medial septal/ diagonal band complex.

Neurons in the medial septal/diagonal band complex (MS/DB) in vivo exhibit rhythmic burst-firing activity that is phase-locked with the hippocampal theta rhythm. The aim was to assess the morphology of local axon collaterals of electrophysiologically identified MS/DB neurons using intracellular recording and biocytin injection in vitro. Cells were classified according to previous criteria into slow-firing, fast-spiking, regular-spiking, and burst-firing neurons; previous work has suggested that the slow-firing neurons are cholinergic and that the other types are GABAergic. A novel finding was the existence of two types of burst-firing neuron. Type I burst-firing neurons had significantly longer duration after hyperpolarisation potentials when held at -60 mV, and at -75 mV, type I neurons exhibited a low-threshold spike with more rapid activation and inactivation kinetics than those of type II neurons. We have, also for the first time, described the main features of the local axon collaterals of the five neuron types. All filled neurons possessed a main axon that gave forth 1-12 local primary axon collaterals. All electrophysiological types, except for the type I burst-firing neuron, had a main axon that coursed toward the fornix. Myelination of the main axon was a prominent feature of all but the slow-firing neurons. Branching of the primary axon collaterals of the fast-spiking and type I burst-firing neurons was more extensive than that of the other cell types, with those of the slow-firing neurons exhibiting the least branching. All cell types possessed axon collaterals of the en passant type, and some in addition had twiglike or basketlike axon terminals. All cell types made synapses on distal dendrites; a proportion of the fast-spiking and burst-firing cells in addition had basketlike terminals that made synaptic contacts on proximal dendrites and on somata. Two morphological types of somata were postsynaptic to the basket cells: large (20-30-microm) oval cells with dark cytoplasm, and large oval cells with paler cytoplasm, often with an apical dendrite. The presence of lamellar bodies in the large dark neurons suggests that they may be cholinergic neurons, because previous work has localised these structures in some neurons that stain for choline acetyltransferase. Our work suggests therefore that there may be GABAergic neurons in the MS/DB that form basket synaptic contacts on at least two types of target cell, possibly cholinergic and GABAergic neurons, which means that the basket cells could play a key role in the generation of rhythmic activity in the MS/DB.

Sodium channels in central neurons of the tobacco budworm, Heliothis virescens: basic properties and modification by scorpion toxins

Voltage-activated sodium channels in central neurons of larval and adult Heliothis virescens were characterized using whole-cell patch clamp techniques. Macroscopic currents showing rapid activation and inactivation kinetics were uniformly sensitive to tetrodotoxin ( IC 50=1.9 nM). Currents began to activate at voltage steps to −45 mV and reached half maximal at −30 mV. Fast inactivation was evident at voltages as negative as −75 mV and reached half maximal at −50 mV. Full recovery from inactivation occurred within 1 to 2 ms. Currents in larval neurons exhibited similar properties to those of adult neurons, except for the rate of fast inactivation ( t 1), which was significantly slower in larval neurons. The biophysical properties of sodium channels remained unchanged for up to 3 days in culture. Two insecticidal neurotoxins, LqhαIT and AaIT, produced distinctly different modifications of H. virescens sodium channels. LqhαIT slowed channel inactivation, while AaIT specifically shifted voltage-dependent activation to more negative potentials. Overall, the results indicate that sodium channels in H. virescens neurons exhibit biophysical characteristics similar to those of vertebrates, yet possess pharmacological uniqueness with respect to scorpion toxin modification.

Characterization of outward currents in neurons of the avian nucleus magnocellularis.

Characterization of outward currents in neurons of the avian nucleus magnocellularis. J. Neurophysiol. 80: 2824-2835, 1998. Neurons of the nucleus magnocellularis (NM) preserve the timing of auditory signals through the convergence of a variety of voltage- and ligand-gated ion channels. To understand better how these channels interact, we have characterized the kinetics, voltage sensitivity, and pharmacology of outward currents of NM neurons in brain slices. The reversal potential (Erev) of outward currents varied with potassium concentration as expected for currents carried by potassium. However, Erev was consistently more positive than the Nernst potential for potassium (EK). Deviation of Erev from the calculated EK most likely arose from potassium accumulation in extracellular spaces by potassium conductances active at rest and during depolarizing steps. Three outward potassium currents were studied that varied in voltage and pharmacological sensitivity. A tetraethylammonium (TEA)-sensitive, high-threshold current was activated within 1-5 ms of the onset of depolarization, with a half-maximal activation voltage (V1/2) of -19 mV. It was blocked partially by 4-aminopyridine (4-AP) and was the dominant ionic conductance of NM neurons. A dendrotoxin-I (DTX) and 4-AP-sensitive, low-threshold current had a V1/2 of -58 mV, rapid activation kinetics, and only partial inactivation, with decay time constants between 20 and 100 ms. A rapidly inactivating current was observed that was resistant to TEA and DTX and was blocked by intracellular Cs+. The transient current was inactivated almost completely at the resting potential. The onset of inactivation was fastest at potentials negative to those that caused activation. When intracellular K+ was replaced by Cs+, large inward and outward currents were obtained that corresponded respectively to the above-mentioned DTX- and TEA-sensitive currents. Outward, TEA-sensitive current was carried by Cs+, with a PCs/PK of approximately 0.1. In current-clamped neurons, DTX induced repetitive firing and increased membrane time constant near rest but had little effect on action potential duration. These studies indicate that a low-threshold, DTX-sensitive current plays a key role in making NM neurons highly responsive to the onset and offset of synaptic stimuli.

Heterologous Expression of the Kv3.1 Potassium Channel Eliminates Spike Broadening and the Induction of a Depolarizing Afterpotential in the Peptidergic Bag Cell Neurons

The bag cell neurons of Aplysia are a cluster of cells that control egg laying behavior. After brief synaptic stimulation, they depolarize and fire spontaneously for up to 30 min. During the first few seconds of this afterdischarge, the action potentials of the bag cell neurons undergo pronounced broadening. Single bag cell neurons in culture also show spike broadening in response to repeated depolarizations. This broadening is frequency-dependent and associated with the induction of a depolarizing afterpotential lasting minutes. In some neurons the depolarizing afterpotential is sufficient to trigger spontaneous firing. To test the possibility that spike broadening during stimulation is required to trigger the depolarizing afterpotential, we eliminated frequency-dependent broadening by heterologous expression of the Kv3.1 potassium channel. This channel has rapid activation and deactivation kinetics and no use-dependent inactivation. Expression of Kv3.1 prevented spike broadening and also eliminated the depolarizing afterpotential. Measurements of the integral of calcium current during voltage commands, which simulated the action potentials of the control neurons and those expressing Kv3.1, indicate that spike broadening produces up to a fivefold increase in calcium entry. Manipulations that limit calcium entry during action potentials or chelation of intracellular calcium using BAPTA AM prevented the induction of the depolarizing afterpotential. We conclude that spike broadening is essential for the induction of the depolarizing afterpotential probably by regulating calcium influx and suggest that one of the physiological roles of spike broadening may be to regulate long-term changes in neuronal excitability.

TRP trapped in fly signaling web

In the Drosophila eye, photoactivation of rhodopsin leads to the opening of the light-sensitive cation influx channels TRP and TRPL. This response is extremely rapid and results in depolarization of the photoreceptor cells followed by Ca2+-mediated feedback regulation of the visual signaling cascade. The mechanisms that facilitate the rapid kinetics of activation and feedback regulation are poorly understood. However, the recent discovery that most of the proteins that function in fly phototransduction associate into a supramolecular complex permits a re-evaluation of the mechanisms underlying the activation and regulation of the cascade. The central player in the signaling complex is INAD, a protein with five protein-interaction motifs known as PDZ domains. The INAD complex does not appear to be a particle, but a massive signaling web composed of an INAD polymer with which some of the target proteins associate through complex multivalent interactions.

Contribution of the Kv3.1 potassium channel to high-frequency firing in mouse auditory neurones.

1. Using a combination of patch-clamp, in situ hybridization and computer simulation techniques, we have analysed the contribution of potassium channels to the ability of a subset of mouse auditory neurones to fire at high frequencies. 2. Voltage-clamp recordings from the principal neurones of the medial nucleus of the trapezoid body (MNTB) revealed a low-threshold dendrotoxin (DTX)-sensitive current (ILT) and a high-threshold DTX-insensitive current (IHT). 3. IHT displayed rapid activation and deactivation kinetics, and was selectively blocked by a low concentration of tetraethylammonium (TEA; 1 mM). 4. The physiological and pharmacological properties of IHT very closely matched those of the Shaw family potassium channel Kv3.1 stably expressed in a CHO cell line. 5. An mRNA probe corresponding to the C-terminus of the Kv3.1 channel strongly labelled MNTB neurones, suggesting that this channel is expressed in these neurones. 6. TEA did not alter the ability of MNTB neurones to follow stimulation up to 200 Hz, but specifically reduced their ability to follow higher frequency impulses. 7. A computer simulation, using a model cell in which an outward current with the kinetics and voltage dependence of the Kv3.1 channel was incorporated, also confirmed that the Kv3.1- like current is essential for cells to respond to a sustained train of high-frequency stimuli. 8. We conclude that in mouse MNTB neurones the Kv3.1 channel contributes to the ability of these cells to lock their firing to high-frequency inputs.

Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells.

A member of a new subclass of the voltage-activated sodium channel genes has been cloned from the human medullary thyroid carcinoma (hMTC) cell line. The cDNA of hNE-Na (human neuroendocrine sodium channel) encodes a 1977 amino acid protein which phylogenetically represents a link between sodium channels isolated from skeletal muscle and brain. The hNE-Na alpha subunit was transiently expressed in human embryonic kidney cells either alone or in combination with the human sodium channel beta 1 subunit. The channel exhibited rapid activation and inactivation kinetics, and was blocked by tetrodotoxin and cadmium with IC50 values of 24.5 nM and 1.1 mM, respectively. Action potentials were generated in cells expressing high levels of hNE-Na. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated its expression in hMTC cells, in a C-cell carcinoma, and in thyroid and adrenal gland. Transcripts were not identified in pituitary gland, brain, heart, liver or kidney, indicating that the hNE-Na is a sodium channel solely expressed in neuroendocrine cells.

GABA‐Ergic interneurons of the striatum express the shaw‐like potassium channel KvS3.1

In addition to numerous GABA-ergic efferent neurons, the striatum contains a subpopulation of fast-firing GABA-ergic interneurons characterized by the presence of immunoreactivity for the calcium-binding protein, parvalbumin. Double-label in situ hybridization with digoxigenin- and radiolabelled cRNA probes was performed on striatal sections of adult rats to identify mRNAs expressed by striatal GABA-ergic interneurons. In the dorsolateral striatum, only parvalbumin mRNA-positive neurons expressed the mRNA encoding the potassium channel Kv3.1, a member of the Shaw family of potassium channels with rapid activation and inactivation kinetics, usually found in fast-firing neurons such as the basket cells of the hippocampus. Colocalization of the parvalbumin and Kv3.1 proteins was confirmed by double-label immunohistochemistry. Parvalbumin mRNA-positive neurons expressed very high levels of the mRNA encoding glutamic acid decarboxylase (Mr 67,000: GAD67) in the dorsolateral striatum. A smaller proportion of double-labelled neurons was found in the ventrolateral striatum. A small number of densely labelled neurons for GAD67 mRNA also expressed the mRNA encoding the dopamine D2 receptor, but none expressed detectable levels of the dopamine D1 receptor mRNA. This indicates major differences in the expression of dopamine receptor mRNA in a majority of GABA-ergic interneurons vs. GABA-ergic efferent neurons of the striatum. The results suggest that distinct molecular characteristics are associated with the distinct electrophysiological properties of striatal GABA-ergic neurons.

Signal transduction through the sphingomyelin pathway

The sphingomyelin pathway is a new signal transduction system initiated by hydrolysis of plasma membrane sphingomyelin to ceramide by the actin of a neutral sphingomyelinase. Ceramide serine/threonine protein kinase termed ceramide-activated protein kinase. This kinase belongs to a family of proline-directed protein kinases that recognize substrates containing the minimal motif, X-Thr/Ser-Pro-X, where the phosphoacceptor site is followed on the carboxyl terminus by a proline residue and X may be any amino acid. Three lines of evidence, rapid kinetics of activation of the sphingomyelin pathway by tumor necrosis factor (TNF) alpha, the ability of cell-permeable ceramide analogs to bypass receptor activation and mimic the effect of TNF alpha, and reconstitution of this cascade in a cell-free system, support the concept that the sphingomyelin pathway serves to signal TNF alpha-induced monocytic differentiation. Hence, the sphingomyelin pathway may represent a signaling system analogous to more well-defined systems such as the cyclic adenosine monophosphate and phosphoinositide pathways.

Growth hormone rapidly activates insulin-like growth factor I gene transcription in vivo.

Many of the growth-promoting properties of GH are mediated by insulin-like growth factor I (IGF-I), a highly conserved circulating 70-amino acid peptide. Recent studies have shown that multiple mechanisms influence IGF-I gene expression, including transcription from two promoters, alternative RNA splicing, and variable polyadenylation. In order to determine how GH regulates IGF-I gene expression we have analyzed the response of hypophysectomized rats to a single ip injection of recombinant GH. A rise in hepatic IGF-I mRNA was detected within 2 h of GH treatment, with peak values of more than 15-fold above untreated animals by 4 h, and a decline by 16 h. A coordinate increase was seen in all IGF-I mRNA splicing and polyadenylation variants, indicating that neither alternative RNA processing nor differential poly A addition were altered by GH. Transcription run-on experiments using isolated hepatic nuclei and direct analysis of nuclear RNA demonstrated a rise in nascent IGF-I mRNA within 30 min of GH treatment, with peak levels reaching more than 10-fold above background by 2 h and declining by 6 h. As determined by RNase protection assays, transcripts directed by each promoter were coordinately and equivalently activated after GH. A single GH-responsive DNase I hypersensitive site was mapped in chromatin to the second IGF-I intron. This site exhibited rapid kinetics of induction which mirrored the pattern of transcriptional stimulation after GH treatment. These experiments show that GH enhances IGF-I expression in vivo by predominantly transcriptional mechanisms. The rapid kinetics of IGF-I gene activation and the temporally associated chromatin changes demonstrate a direct link between a GH-dependent signal transduction pathway and nuclear events.

The mode of action of tedisamil on voltage-dependent K+ channels

The mechanism of the interaction of tedisamil with voltage-dependent K+ channels was studied using whole-cell and single-channel recordings in a variety of species and cell types. In K+ channels with rapid activation kinetics (Ito of rat ventricular myocytes; IA of mouse astroglial cells), tedisamil enhanced the kinetics of inactivation of the current without significantly suppressing the amplitude of the initial current. In K+ channels with slower activation/inactivation kinetics, tedisamil had a divergent effect. On IK of the glial cells, which have slow activation and inactivation kinetics, the kinetics of inactivation were enhanced and the initial peak current was reduced. On the other hand, in IK of guinea-pig ventricular myocytes, which have even slower activation kinetics with no inactivation, tedisamil slowed or completely suppressed the activation of the current. Finally, in K+ channels with rapid activation but slow inactivation kinetics (pedestal-type current of rat ventricular myocytes), tedisamil accelerated the inactivation without affecting the initial current. Thus, the prime determinant of the blocking mode of tedisamil appeared to be the kinetics of activation of the K+ channel; that is, the slower the kinetics of activation of the channel, the greater the initial block by the drug. Unitary Ito currents recorded in rat ventricular myocytes showed that tedisamil induced a rapid flicker block of the open channel and prolonged the time between the burst of openings without any effect on the unitary conductance. These effects were modeled by assuming that the drug bound to the open channel at a finite rate. Thus, tedisamil appears to decrease K+ currents by interacting uniformly with the open state of the channel.

Separation and modulation of calcium currents in bullfrog sympathetic neurons.

The calcium current of frog sympathetic neurons has relatively rapid activation kinetics (tau < 3 ms) in response to changes in voltage. Pharmacologically, the current is blocked approximately 90% of omega-conotoxin, but < 10% by dihydropyridine antagonists. This suggests that nearly all of the current is N type. However, inactivation is slow and incomplete even for depolarizations lasting > 1 s, consistent with recent evidence that N-type channels do not always inactivate rapidly. The calcium current is partially inhibited via receptors for acetylcholine, luteinizing hormone releasing hormone, substance P, ATP, and norepinephrine. These effects are mimicked by internal dialysis with GTP-gamma-S, suggesting involvement of a G protein. The transmitters affect the activation kinetics of the calcium current in a voltage-dependent manner, which can be modeled as a reversible shift of some channels to "reluctant" states in which strong depolarization is needed to produce channel opening. The effects of transmitters develop and recover with t1/2 approximately 1-2 s, so if a second messenger is involved in receptor-calcium channel coupling, it must act rapidly.

Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel.

The principal voltage-sensitive sodium channel from human heart has been cloned, sequenced, and functionally expressed. The cDNA, designated hH1, encodes a 2016-amino acid protein that is homologous to other members of the sodium channel multigene family and bears greater than 90% identity to the tetrodotoxin-insensitive sodium channel characteristic of rat heart and of immature and denervated rat skeletal muscle. Northern blot analysis demonstrates an approximately 9.0-kilobase transcript expressed in human atrial and ventricular cardiac muscle but not in adult skeletal muscle, brain, myometrium, liver, or spleen. When expressed in Xenopus oocytes, hH1 exhibits rapid activation and inactivation kinetics similar to native cardiac sodium channels. The single channel conductance of hH1 to sodium ions is about twice that of the homologous rat channel and hH1 is more resistant to block by tetrodotoxin (IC50 = 5.7 microM). hH1 is also resistant to mu-conotoxin but sensitive to block by therapeutic concentrations of lidocaine in a use-dependent manner.