Triplex forming oligonucleotide targeted to 3'UTR downregulates the expression of the bcl-2 proto-oncogene in HeLa cells.

The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3'-untranslated region (3'UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3'UTR of the bcl-2 proto-oncogene in the regulation of bcl-2 gene expression. To do this, we have developed a novel cellular system that involves transfection of a Doxycyclin inducible expression plasmid containing the bcl-2 ORF and the 3'UTR together with a TFO targeted to the 3'UTR of the bcl-2 proto-oncogene. Phosphorothioate-modified TFO targeted to the 3'UTR of the bcl-2 gene significantly downregulated the expression of the bcl-2 gene in HeLa cells as demonstrated by western blotting. Our results indicate that blocking the functions of the 3'UTR using the TFO can downregulate the expression of the targeted gene, and suggest that triplex strategy is a promising approach for oligonucleotide-based gene therapy. In addition, triplex-based sequence targeting may provide a useful tool for studying the regulation of gene expression.

Genomic structure and functional expression of a human alpha(2)/delta calcium channel subunit gene (CACNA2).

CACNA2 encodes the alpha(2)/delta subunit of the human voltage-gated calcium channels and is located in the candidate region of malignant hyperthermia susceptibility type 3 (MHS3). We determined the structural organization of CACNA2 by isolation of overlapping genomic DNA clones from a human phage library. The gene consists of at least 40 exons, 2 of which are alternatively spliced, spanning more than 150 kb of genomic DNA. Exons range from 21 to 159 bp, and introns range from 98 bp to at least more than 20 kb. We constructed a full-length cDNA and cloned it into a mammalian expression vector. Cotransfection of the CACNA2 cDNA with alpha(1A) and beta(4) cDNA into HEK293 cells led to the expression of Q-type calcium currents. The alpha(2)/delta subunit enhanced the current density 18-fold compared to cells transfected with only alpha(1A) and beta(4) cDNA. The sequence analysis provides the basis for comprehensive mutation screening of CACNA2 for putative MHS3 individuals and patients with other channelopathies.

Receptor-mediated modulation of recombinant neuronal class E calcium channels.

The modulation of a cloned neuronal calcium channel was studied in a human embryonic kidney cell line (HEK293). The HEK293 cells were stably transfected with the alpha1Ed cDNA, containing the pore forming subunit of a neuronal class E calcium channel. Inward currents of 25 +/- 1.9 pA/pF (n = 79) were measured with the cloned alpha1Ed-subunit. The application of the peptide hormone somatostatin, carbachol, ATP or adenosine reduced the amplitude of Ca2+ and Ba2+ inward currents and exhibited a slowing of inactivation. This inhibitory effect by somatostatin was significantly impaired after pre-incubating the transfected cell line with pertussis toxin (PTX). Internal perfusion of the cells with the G-protein-inactivating agent GDP-beta-S or with the permanently activating agent GTP-gamma-S also attenuated the somatostatin effect. The inhibition indicates that modulation of the alpha1Ed-mediated Ca2+ current involves pertussis toxin-sensitive G-proteins. The block of Ca2+ and Ba2+ inward currents by somatostatin is also found in cells expressing a truncated alpha1Ed-subunit which lacks a 129-bp fragment in the C-terminus. This fragment corresponds to the major structural difference between two native human alpha1E splice variants. As somatostatin inhibits inward currents through both, the cloned alpha1Ed- and the truncated alpha1Ed-DEL-subunit, the hormone-mediated modulation is independent from the presence of the 129-bp insertion in the C-terminus.

Influence of L-type Ca channel alpha 2/delta-subunit on ionic and gating current in transiently transfected HEK 293 cells.

We have measured ionic and gating currents in human embryonic kidney (HEK 293) cells transiently transfected with cDNAs encoding subunits of the cardiac voltage-gated L-type Ca2+ channel. Robust recombinant ionic current and associated nonlinear charge movement could be measured over a broad voltage range without contamination by endogenous channel activity. Coexpression of the alpha 2/delta-subunit along with alpha 1- and beta 2-subunits speeded activation and deactivation kinetics and significantly increased the maximal conductance of ionic current. Charge movement was measured at voltages negative to the threshold for activation of ionic current, and gating charge could be immobilized at positive holding potentials that did not inactivate ionic current. The ratio of maximal ionic conductance to maximal charge moved remained the same in the absence or presence of the alpha 2/delta-subunit. However, the maximal amount of charge moved was increased about twofold in the presence of the alpha 2/delta-subunit. These results suggest that coexpression of the alpha 2/delta-subunit enhances the expression of functional L-type channels and, in addition, provide evidence that most of the L-type channel-associated nonlinear charge movement is caused by transitions between nonconducting states of the channel protein that precede the open and inactivated states.

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation.

The Ca2+ channel subunits alpha 1C-a and alpha 1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba2+ current (IBa) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 microM cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 microM PKA and 1 microM okadaic acid. The activity of the alpha 1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 microM H 89 and was not increased by superfusion with 5 microM forskolin plus 20 microM isobutyl-methylxanthine (IBMX). The alpha 1C-a.beta 2.alpha 2/delta complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 microM H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the alpha 1C-a.beta 2.alpha 2/delta channel with 10 microM PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca2+ current (ICa) of cardiac myocytes increased threefold during internal dialysis with 5 microM PKA or 25 microM microcystin and during external superfusion with 0.1 microM isoproterenol or 5 microM forskolin plus 50 microM IBMX. These results indicate that the L-type Ca2+ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.

The Ca(++)-channel blocker Ro 40-5967 blocks differently T-type and L-type Ca++ channels.

The effects of Ro 40-5967, a nondihydropyridine Ca++ channel blocker, on low-voltage activated (T-type) and high-voltage activated (L-type) Ca++ channels were compared. L-type barium currents were measured in Chinese hamster ovary cells stably transfected with the alpha 1 subunit of the class Cb Ca++ channel. T-type barium currents were investigated in human medullary thyroid carcinoma cells. The Ba++ currents of human medullary thyroid carcinoma cells were transient, activated at a threshold potential of -50 mV with the maximum at -14 +/- 3.2 mV and blocked by micromolar Ni++. The T- and L-type current inactivated with time constants of 33.4 +/- 4.1 and 416 +/- 26 msec at maximum barium currents, respectively. Ro 40-5967 inhibited reversibly the T- and L-type currents with IC50 values of 2.7 and 18.6 microM, respectively. The inhibition of the L-type current was voltage-dependent, whereas that of the T-type current was not. Ro 40-5967 blocked T-type current already at a holding potential of -100 mV. The different types of block, i.e., voltage-dependent vs. tonic block, may contribute to the pharmacological profile of Ro 40-5967 in intact animals.

Sustained hyperpolarization of cultured guinea pig coronary endothelial cells induced by adenosine.

Electrophysiological experiments on cultured monolayers of guinea pig coronary endothelial cells were performed to substantiate the hypothesis that variation of the endothelial membrane potential may be a functionally important mechanism that contributes to the regulation of coronary blood flow. The endothelial cells were loaded with sodium by superfusion with K(+)-free solution at 37 degrees C for 5-30 min. Readmission of external K+ produced a transient hyperpolarization of up to 85 mV, which was due to stimulation of the electrogenic pump current. In most but not all of the monolayers, superfusion with 2 microM adenosine elicited a transient or a sustained hyperpolarization. The transient hyperpolarization had an amplitude of 15 +/- 6 mV. The sustained hyperpolarization had an amplitude of 11 +/- 3 mV. Our results are in line with the hypothesis that the hyperpolarization of the endothelium induced by release of adenosine into the perivascular space of the capillaries may be conducted electronically to the terminal arterioles and may cause vasodilation via current flow through myoendothelial gap junctions.

Effects of vasoactive agonists on the membrane potential of cultured bovine aortic and guinea-pig coronary endothelium.

1. The effects of bradykinin, ATP, adenosine, histamine and thrombin on the membrane potential of confluent monolayers of cultured bovine aortic endothelial cells (BAECs) and guinea-pig coronary endothelial cells (GCECs) were studied at 37 degrees C using the whole-cell mode of the patch-clamp technique. 2. The amplitude histogram of the resting potentials of BAEC monolayers showed a bimodal distribution with one peak around -25 mV and another peak around -85 mV. Transitions from one potential level to the other were observed. The bistable membrane potential can be explained by an N-shaped current-voltage relation of the endothelial cell membrane. 3. When BAECs with a low resting potential (-10 to -30 mV) were superfused with maximally effective concentrations of ATP (2-10 microM) an initial hyperpolarization of -80 to -90 mV was observed which decayed to a plateau of about -60 mV within 1 min. When ATP was removed after 2-3 min the membrane potential returned to control level within 1 min. This was followed by a second hyperpolarization of 10-20 mV, which decayed within 15 min. 4. In the absence of extracellular calcium, ATP produced only a brief transient hyperpolarization in aortic endothelium. The plateau and the secondary hyperpolarization were abolished. These findings are consistent with the idea that the changes in membrane potential reflect changes in intracellular free Ca2+ and that the initial peak is due to release of Ca2+ from intracellular stores, whereas the plateau and the secondary hyperpolarization depend on transmembrane Ca2+ influx. 5. Bradykinin evoked potential changes similar to ATP in BAECs, except that the secondary hyperpolarization during wash-out was absent. When the membrane potential was more negative than -80 mV, ATP and bradykinin induced only a small initial hyperpolarization followed by a depolarization of up to 20 mV. 6. In aortic endothelium, ADP (10 microM) evoked a much smaller response than ATP. Adenosine (10 microM), thrombin (2 units/ml), acetylcholine (10 microM) and histamine (10 microM) had only a very small effect on the membrane potential, if any. 7. The amplitude histogram of the membrane potential of GCECs showed only one peak around -35 mV. In coronary endothelium, application of bradykinin, ATP, histamine, thrombin, acetylcholine and adenosine all evoked a transient hyperpolarization of 10-40 mV lasting 1 min or less, which then turned into a depolarization. 8. The K+ channel openers cromakalim (BRL 34915) and lemakalim (BRL 38227) did not affect the membrane potential of GCECs or BAECs.(ABSTRACT TRUNCATED AT 400 WORDS)

The electrical response of cultured guinea-pig coronary endothelial cells to endothelium-dependent vasodilators.

1. Primary cultures of coronary endothelial cells were obtained by enzymatic dispersion of isolated guinea-pig hearts and separation of different cardiac cell types by density gradient centrifugation. The cells were grown to confluency and the membrane potential of the monolayer was recorded using the whole-cell-clamp mode of the patch-clamp technique. 2. When superfused with physiological salt solution at 37 degrees C the average resting potential of the monolayers was -35 +/- 9 mV. A 2 min application of bradykinin (0.1-20 nM) induced a transient hyperpolarization of up to 40 mV (median 33 mV), which was followed by a sustained depolarization of up to 28 mV (median 10 mV). The average duration of the hyperpolarization, measured midway between the resting potential and peak negativity, was 48 s with 20 nM-bradykinin. 3. The concentration of bradykinin producing a half-maximal hyperpolarization was 2.5 nM. When high concentrations of bradykinin (greater than 10 nM) were applied for several minutes voltage oscillations of low amplitude with periodicity of 2-3 min were observed. 4. The peak of the hyperpolarization depended on the extracellular potassium concentration ([K+]o). The limiting slope of the relation between membrane potential and log [K+]o was 52 mV per 10-fold change in [K+]o. With 50 mM [K+]o the hyperpolarization was abolished and with 100 mM [K+]o the hyperpolarization turned into a depolarization. 5. After removal of external Ca2+ the first transient hyperpolarization elicited by bradykinin had the same amplitude as under control conditions, but its duration was reduced to about 72%. The second application of bradykinin in Ca2(+)-free solution produced only a depolarization. The hyperpolarizing response to bradykinin could be re-primed by exposing the preparation to Ca2(+)-containing solution for 2 min. 6. The transient hyperpolarization elicited by 4 nM-bradykinin could be inhibited by d-tubocurarine, a blocker of Ca2(+)-activated potassium channels. On average, 1 mM-tubocurarine reduced the hyperpolarization by 49 +/- 18%. Apamin (10 microM) reduced the hyperpolarization by 15 +/- 11%. 7. ATP (1 microM) produced a hyperpolarization of similar amplitude to that produced by bradykinin, but of shorter duration (average 29 s), and a very small (less than 5 mV) or no sustained depolarization. Histamine (10 microM) produced an even shorter transient hyperpolarization, followed by a depolarization of up to 15 mV. 8. Most of the monolayers of coronary endothelial cells responded to adenosine in a similar way as to bradykinin.(ABSTRACT TRUNCATED AT 400 WORDS)

Hypoxic dilation of coronary arteries is mediated by ATP-sensitive potassium channels.

The function of the heart depends critically on an adequate oxygen supply through the coronary arteries. Coronary arteries dilate when the intravascular oxygen tension decreases. Hypoxic vasodilation in isolated, perfused guinea pig hearts can be prevented by glibenclamide, a blocker of adenosine triphosphate (ATP)-sensitive potassium channels, and can be mimicked by cromakalim, which opens ATP-sensitive potassium channels. Opening of potassium channels in coronary smooth muscle cells and the subsequent drop in intracellular calcium is probably the major cause of hypoxic and ischemic vasodilation in the mammalian heart.

Passive electrical properties and electrogenic sodium transport of cultured guinea-pig coronary endothelial cells.

1. Coronary endothelial cells were isolated from adult guinea-pig hearts (Nees, Gerbes & Gerlach, 1981) and the electrical properties of primary cultures were studied using the tight-seal whole-cell recording mode of the patch clamp technique. 2. On the third or fourth day in culture whole-cell clamp records from single coronary endothelial cells were obtained at 37 degrees C. The resting potential was -33 +/- 6 mV (n = 10). The membrane time constant determined with rectangular current pulses was 68 +/- 22 ms (n = 10). 3. In voltage clamp experiments, no time-dependent membrane conductance changes were found in the range -80 to +40 mV. The current-voltage relation was linear in normal physiological salt solution containing 5.4 mM-K+. The input resistance was 1.7 +/- 0.4 G omega. When the external K+ concentration was increased to 116 mM the cells depolarized to about -3 mV and the clamp currents showed marked inward rectification. 4. Between days four and seven in culture the endothelial cells formed confluent monolayers which showed the characteristic 'cobblestone' morphology. The input resistance of cells in a monolayer was 8 +/- 3 M omega, i.e. a factor of 200 lower than that found in single cells. It was concluded that the cells in the confluent monolayer are coupled electrically by gap junctions. 5. Exposure of coronary endothelial cells to K+-free solution for 5 min produced a depolarization of about 8 mV. Upon readmission of normal external K+ the cells transiently hyperpolarized by up to 20 mV. This transient hyperpolarization decayed with a time constant of 1.9 +/- 0.3 min. 6. The transient hyperpolarization could be abolished by application of 2 x 10(-4) M-dihydro-ouabain (DHO). Application of DHO in the steady state produced a depolarization of 8 +/- 1 mV. From these findings it was concluded that coronary endothelial cells possess an electrogenic sodium pump which contributes about -8 mV to the resting potential. 7. From the passive electrical properties of single cells and the morphological data available it was calculated that endothelium in situ may have a large electrical space constant, probably between 250 and 550 microns. 8. The functional implications of the large space constant of the endothelial monolayer are discussed. It is suggested that intra-endothelial conduction of electrical signals from capillaries to the resistance vessels may be involved in the local regulation of blood flow in the intact heart.

The myotonic mouse mutant ADR: electrophysiology of the muscle fiber.

The hereditary neuromuscular syndrome of the mouse, "arrested development of righting response" (ADR), is characterized by after-contractions of skeletal muscle. In order to analyze the etiology of this hereditary defect, mutant and wildtype muscle fibers were studied by intracellular recording. Direct stimulation of ADR muscle fibers elicited runs of action potentials of 1-5 seconds duration, with declining amplitudes and varying frequencies. The electrical after-activity coincided with after-contractions of the fiber, and both phenomena could be suppressed by the membrane-stabilizing drug tocainide. Since similar runs of APs were observed with wildtype muscle in chloride-free medium, the possible involvement of chloride conductance in the ADR syndrome was studied. Although membrane time constants in wildtype muscle were 4 times prolonged in chloride-free medium, those of ADR muscle were only 1.4 times longer. This finding indicated a drastic reduction of chloride conductance of the ADR muscle membrane. We conclude that ADR is a myotonia, related in its etiology to hereditary myotonias of man.

Development of mammalian nerve-muscle synapses in culture: lack of interference by antibodies to the neural cell adhesion molecule N-CAM and its L2/HNK-1 carbohydrate epitope.

Rat myotubes were cultured with embryonic mouse spinal cord explants. After 4 days of co-culture, (miniature) endplate potentials, (M)EPPs, could be detected in 20-40% of the nerve-contacted myotubes. Polyclonal antibodies against the neural cell adhesion molecule N-CAM and monoclonal antibodies to its L2/HNK-1 carbohydrate epitope were tested for possible effects on synapse formation in this system. Co-cultures that were started and maintained in the presence of Fab fragments of these antibodies developed functional synapses to the same extent as did control cultures. Staining with an anti-IgG antibody demonstrated that added Fab fragments had bound to neurites and that an excess was present in the medium. These findings suggest that even if N-CAM and the L2/HNK-1 epitope were involved in early stages of muscle-nerve contact, they are not essential for the formation of electrophysiologically active synapses.

Synapse formation and synaptic activity in mammalian nerve-muscle co-culture are not inhibited by antibodies to neural cell adhesion molecule L1.

Co-cultures of rat myotubes and spinal cord explants from mouse embryos were maintained in the presence of Fab fragments of polyclonal antibodies to neuronal cell surface antigen L1. Microscopic observation showed that neurite outgrowth was not blocked by anti-L1. By intracellular recording, no effect was observed on the number of myotubes that showed endplate potentials, nor on the efficiency of synaptic contacts. As was demonstrated by indirect immunofluorescence, added Fab fragments remained bound to the neurite surface and were present in the medium for at least two days in culture, after which time antibodies were replaced during the medium change. Taken together, these observations show that L1 antigen is not involved in synapse formation between nerve and muscle.

Beating heart muscle in a skeletal muscle bed.

Minced cardiac muscle from newborn rats was transplanted into a skeletal muscle bed of nude mice. The implanted tissue regenerated into autonomously beating heart muscle, the donor origin of which was shown by immunohistochemistry and isozyme analysis. Host skeletal muscle regeneration was stimulated compared with controls without implanted tissue.