Search PubMed⌕ Search

Biomedical subjects

K Kuba

Publications and source records attributed to K Kuba.

At least 37 records · Page 2Linked to original sources
PubMed1999

Long-term use-dependent enhancement of impulse-induced exocytosis by adrenaline at frog motor nerve terminals.

Adrenaline (5-20 microM) use-dependently increased end-plate potentials (EPPs) in normal Ringer solution (containing d-tubocurarine to partially block acetylcholine receptors) and a low Ca2+, high Mg2+ solution for more than several hours and decreased the coefficient of variation of EPP amplitude in the latter solution in frog neuromuscular junctions. The amplitude and frequency of miniature EPPs and impulse-induced increases in intraterminal Ca2+ concentration were unaffected. Adrenaline thus causes sustained enhancement of impulse-induced exocytosis by acting at a mechanism of exocytosis downstream to Ca2+ entry.

Animals↗
PubMed1998

Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor.

Invasion of various carcinoma cells follows their interaction with stromal cells. Hepatocyte growth factor (HGF), four-kringle-containing growth factor, is a mesenchymal or stromal-derived mediator which affects the growth and the invasiveness of carcinoma cells. We now have evidence that a four-kringle-containing antagonist for HGF, HGF/NK4 inhibits invasion of tumors in vivo, as well as in vitro. HGF/NK4 competitively inhibited the binding of HGF to Met/ HGF receptors on GB-d1 human gallbladder carcinoma cells. HGF induced invasion of the cells through Matrigel basement membrane components and into collagen gels, but HGF-induced invasion was inhibited by HGF/NK4. Invasion of GB-d1 cells was induced by co-cultivation with stromal fibroblasts, which mimics tumor-stromal interaction, but it was almost completely suppressed by HGF/NK4. Likewise, invasive growth induced by HGF in collagen gels in GB-dl cells, HuCC-T1 human cholangiocarcinoma cells, and ME-180 human uterus cervical carcinoma cells was also strongly inhibited by HGF/NK4. When GB-d1 cells were implanted subcutaneously into nude mouse, tumor cells invaded muscular tissue, but the infusion of HGF/NK4 inhibited this invasion. Furthermore, HGF/NK4 increased apoptotic cell death of GB-d1 cells and inhibited tumor growth in vivo. These results indicate that HGF/ NK4 may inhibit growth and invasion of carcinoma cells, as mediated by HGF during tumor-stromal interactions. We propose that there is a unique therapeutic potential for HGF/NK4 to prevent tumor invasion and perhaps even metastasis.

Aged↗
PubMed1998

Two-photon laser-scanning microscopy: tests of objective lenses and Ca2+ probes.

The characteristics of objective lenses and Ca2+-sensitive probes were examined for imaging with a two-photon laser-scanning microscope (TP-LSM). The brightness of the images of beads taken by different objectives greatly varied and depended predominantly on their numerical aperture (NA) and less on transmittance and chirping effects. Lateral and axial resolutions, dx and dz, defined as the half decay length of fluorescence intensity of the image of a spherical bead (0.3 m) were 0.12 and 0.42 microm (objective; 40x/0.75). They are far better than those of confocal microscopes (0.3 and 1.5 microm, respectively) measured similarly (Kuba et al., 1994). dx linearly increased with an increase in 1/NA, while dz linearly increased with an increase in n/(NA)2 (n, refractive index) except for an objective of large NA (1.3). The coverslip compensation of objective lenses greatly affected the shape of the X-Z scanned images of 5.0 microm beads as well as resolutions, indicating a large effect of spherical aberration. Two-photon excitation spectra of Ca2+-sensitive fluorescent probes, indo-1, fura-2 and Oregon Green BAPTA-1, lied in a wavelength range shorter than twice that activated by one-photon absorption, while emission spectra were unchanged. Three-dimensional images of a cultured hippocampal neurone loaded with Oregon Green BAPTA-1 showed fine structures of spines, dendrites and axons, while imaging with FM1-43 localized presynaptic boutons and demonstrated synaptic vesicle turnover. Dyes bleached little during the recording of 100 sectioned images. These characteristics of TP-LSM as well as its ability to image deeper tissues provide excellent means to study dynamic, spatial changes in intracellular substances and structures. To achieve the good performance of a TP-LSM, however, the relevant usage of appropriate objectives and fluorescent probes are required.

Animals↗
PubMed1998

A Ca2+-induced Ca2+ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals.

The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20-50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05-0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N, N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN-. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.

Action Potentials↗
PubMed1998

A synergistic interaction between histamine and vasoactive intestinal peptide (VIP) on cyclic AMP production in the chick pineal gland.

The effects of vasoactive intestinal peptide (VIP) and histamine, alone and in combination, on cyclic AMP formation have been studied in selected tissues of the central nervous system (CNS) of chick. VIP strongly stimulated cyclic AMP production in the pineal gland, moderately in the retina (maintained in "eye-cup" preparations), and had no effect in cerebral cortical slices. Combination of VIP with forskolin produced a synergistic response in the pineal gland; in the cerebral cortex VIP did not influence the elevation of cyclic AMP production evoked by forskolin, histamine and isoprenaline. Histamine stimulated cyclic AMP synthesis in the all tested CNS tissues, showing the following order of sensitivity to the amine: pineal gland > cerebral cortex > retina. The effects of histamine were stronger in the presence of forskolin. A combination of VIP and histamine produced the cyclic AMP response in the pineal gland clearly more than additive; such a synergistic interaction was antagonized by aminopotentidine, an accepted in mammals H2-histamine receptor blocker. It is concluded that both VIP and histamine can be considered as functionally important neuromodulators in avians (similar to mammalian species). These two substances may play in concert to regulate the pineal physiology in avian species.

Amino Acid Sequence↗
PubMed1997

Evidence for the calcium-dependent potentiation of M-current obtained by the ratiometric measurement of the fura-2 fluorescence in bullfrog sympathetic neurons.

Intracellular Ca2+ concentration ([Ca]i) was measured following the activation of an inward Ca2+ current and subsequent potentiation of an M-type K+ current (IM) in bullfrog sympathetic neurons. Fura-2 was used as an indicator for [Ca]i. The fluorescence ratio at 340 and 380 nm (F340/F380) was elevated from 0.36 to 1.22 when IM was potentiated by 68% following the Ca2+ current. Based on the in vivo calibration curve obtained from cells permeabilized with digitonin (20 microM), the F340/F380 value of 1.22 was equivalent to a [Ca]i of 0.97 microM. We therefore propose that a rise in [Ca]i into the micromolar range can lead to the potentiation of IM in amphibian autonomic neurons.

Action Potentials↗
PubMed1995

Ca(2+)-induced Ca2+ release and its activation in response to a single action potential in rabbit otic ganglion cells.

1. Ryanodine-sensitive intracellular Ca2+ release activated by Ca2+ entry was studied with fura-2 fluorescence and intracellular voltage recording techniques in rabbit otic ganglion cells. 2. The removal of extracellular Ca2+ reduced sustained, transient or oscillatory rises in intracellular Ca2+ ([Ca2+]i) induced at high extracellular K+ and abolished the [Ca2+]i oscillation in cultured neurones. 3. Ryanodine (10 microM) transiently increased [Ca2+]i and reduced the amplitude and rate of rise of the high-K(+)-induced rise in [Ca2+]i, while caffeine (5 mM) produced a few transient rises in [Ca2+]i in most cultured cells and [Ca2+]i oscillation only in one cell. 4. The two components of the slow after-hyperpolarization (AHP) of an action potential in neurones of freshly isolated ganglia were dependent on extracellular Ca2+ and abolished by Ca2+ channel blockers, Cd2+ or Co2+. 5. The late component of AHP (LAHP), but not the initial component, in 'fresh' neurones increased in area with an increase in the preceding interval, was abolished by ryanodine (10 microM) and intracellularly injected EGTA, and mimicked by intracellular injection of Ca2+. 6. A ryanodine-sensitive Ca(2+)-induced Ca2+ release thus exists, operates in response to an action potential-induced Ca2+ entry and underlies LAHP in rabbit otic ganglion cells.

Action Potentials↗
PubMed1994

A UV laser-scanning confocal microscope for the measurement of intracellular Ca2+.

Modifications to the optics of a conventional confocal laser-scanning microscope were made to allow imaging intracellular Ca(2+)-dependent fluorescence with a UV laser (351 or 364 nm). Modifications included: (1) a chromatic compensation lens in the laser path; (2) the design of a practically achromatic relay lens; (3) a longer tube length for the objective; and (4) highly reflective mirrors maximizing fluorescence measurement. This UV laser-scanning confocal microscope (UV-CLSM) yielded a lateral resolution of < 0.3 micron and an axial resolution of < 1.5 microns and a relevant field size of 100 microns in diameter for a 40X objective). The effects of varying the focal length of a compensation lens, the degree of the correction for the coverglass thickness of objective and the detector aperture size on the quality of image formation were examined. Finally, UV-CLSM revealed optical sections of fine and complex structures of bullfrog sympathetic neurones loaded with a Ca(2+)-sensitive fluorescent probe. Changes in intracellular free Ca2+ distribution in response to high [K+] or caffeine were demonstrated. In addition, an increase in the intracellular concentration of caffeine applied externally was clearly imaged in space and time and distinguished from a resultant rise in [Ca2+]i. Thus, the UV-CLSM developed is suitable for ratiometric intracellular Ca2+ measurements and other biological studies.

Animals↗
PubMed1994

Cyclic ADP-ribose modulates Ca2+ release channels for activation by physiological Ca2+ entry in bullfrog sympathetic neurons.

Although Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors has been found to occur in intact neurons, little is known about the physiological processes that regulate it. We studied the effects of cyclic ADP-ribose (cADPR) on CICR in cultured bullfrog sympathetic neurons by fura-2 fluorescence recording and patch-clamp techniques. cADPR applied through a patch pipette augmented action potential- or depolarizing pulse-induced rises in intracellular Ca2+ without a change in Ca2+ entry initiating the responses, but not in the presence of ryanodine. Likewise, cADPR enhanced a single or oscillatory rise(s) in intracellular Ca2+ induced by caffeine. These results strongly suggest that cADPR can be an endogenous modulator of ryanodine receptors in neurons.

Adenosine Diphosphate Ribose↗
PubMed1993

Mechanical modulation of a voltage-dependent non-inactivating K+ current in cultured bullfrog sympathetic neurones.

Cultured bullfrog sympathetic ganglion cells were voltage-clamped with a whole-cell patch-clamp technique. Local flow of a solution (identical to the bathing solution) from a micropipette to a cell, but not other mechanical stimuli, produced a non-inactivating outward (in 34 cells out of 141) or inward (in 70 cells) current [I(f)(out) or I(f)(in), respectively] depending on cells. Both I(f)(out) and I(f)(in) appeared at voltages more positive than -60 mV. The mechanism, however, was activated even at -70 mV, as I(f)(out) or I(f)(in) appeared on shifting membrane potential to -30 mV immediately after the local flow. I(f)(out) and I(f)(in) were accompanied by increases and decreases, respectively, in the membrane conductance and current relaxation to a voltage jump between -30 mV and -55 mV without a change in its time constant (whose value was similar to that of a voltage-dependent non-inactivating K+ current, IM), and reversed at a membrane potential close to the equilibrium potential for K+. Both I(f)(out) and I(f)(in) were blocked by Ba2+ (4-8 mM), a blocker of IM, and by muscarine (10 microM), which produced either an "apparent inward" or outward current. A transient outward current activated by a voltage jump from -85 mV (or -75 mV) to -30 mV was little affected by a local flow of a solution which produced I(f)(out) or I(f)(in). These results suggest that the local solution flow produced I(f)(in) or I(f)(out) by deactivating or activating IM, respectively.

Animals↗
PubMed1993

Bradykinin-induced ion currents in cultured rat trigeminal ganglion cells.

Effects of bradykinin (BK) on membrane currents of cultured rat trigeminal ganglion cells were studied with a G omega-sealed discontinuous voltage clamp technique. Bradykinin (0.05 nM-1 microM) produced membrane depolarization in most cells and hyperpolarization in some cells via a variety of ionic mechanisms: (1) activation of a cation current, (2) enhancement or (3) inhibition of a hyperpolarization-activated inwardly rectifying cation current known as IH, (4) reduction or (5) enhancement of an outwardly rectifying outward current (presumably a delayed K+ current), (6) inhibition of a slow-gating voltage-dependent steady-state outward current (at > -55 mV) and/or (7) increase in another slow-gating voltage-dependent outward current (at > or = -70 mV). These components of BK-induced currents appeared in different combinations and extents among cells, explaining complex excitatory and modulatory actions of BK in different regions and types of sensory neurons.

Animals↗
PubMed1993

Characteristics of Ca2+ release induced by Ca2+ influx in cultured bullfrog sympathetic neurones.

1. A rise in intracellular Ca2+ ([Ca2+]i) and a Ca2+ current (ICa) induced by a depolarizing pulse were simultaneously recorded by fura-2 or indo-1 fluorescence and whole-cell patch clamp techniques in cultured bullfrog sympathetic ganglion cells. 2. [Ca2+]i (calculated from the ratio of fura-2 fluorescences excited at 380 and 340 nm and recorded with a photomultiplier at > 492 nm) rose regeneratively (in most cells) during a command pulse (from -60 to 0 mV, 100 ms), continued to rise thereafter, peaked at 666 ms (on average) and decayed slowly with a half-decay time of 22.8 s. 3. Scanning a single horizontal line across the cytoplasm with an ultraviolet argon ion laser (351 nm) and recording indo-1 fluorescences at two wavelengths (peaked at 410 and 475 nm) with a confocal microscope demonstrated that [Ca2+]i beneath the cell membrane rose much faster than that in the deeper cytoplasm. The time course of the spatial integral of [Ca2+]i, however, corresponded well with that recorded with fura-2 fluorescence using a photomultiplier. 4. [Ca2+]i measured by fura-2 fluorescence ratio using a photomultiplier did not increase during a strong depolarizing pulse (-60 to +80 mV), but sometimes rose after the pulse. A depolarization-induced rise in [Ca2+]i ([Ca2+]i transient) was blocked in a Ca(2+)-free, EGTA solution, reduced by lowering the extracellular Ca2+ concentration ([Ca2+]o) to 0.45 or 0.9 mM and enhanced by raising [Ca2+]o to 7.2 or 14.4 nM. 5. The extracellular Ca2+ dependence was non-linear when long depolarizing pulses (up to 500 ms) were applied; the amplitude of [Ca2+]i transient/Ca2+ entry (unit [Ca2+]i transient) increased with an increase in Ca2+ entry. 6. Increasing the duration of depolarization (-50 or -60 to 0 mV) from 20 to 500 ms enhanced asymptotically the integral of ICa (due to inactivation), and progressively the magnitude of [Ca2+]i transients, leading to the apparent non-linear dependence of unit [Ca2+]i transient on Ca2+ entry as well as on the duration of membrane depolarization. The peak time of [Ca2+]i transient was unchanged for pulse durations up to 300 ms, but prolonged with an increase in pulse duration to 500 ms. 7. Inhibitors of Ca2+ release from intracellular Ca2+ reservoirs, dantrolene (10 microM) and ryanodine (50 microM), blocked the [Ca2+]i transient to 56 and 30%, respectively, of the control. 8. The higher the basal [Ca2+]i level, the greater was the magnitude of the [Ca2+]i transients.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Excitation-induced Ca2+ dynamics in sympathetic neurons measured with conventional epifluorescence and confocal UV laser-scanning microscopes.

A depolarization-induced rise in intracellular Ca2+ in cultured bullfrog sympathetic ganglion cells depended non-linearly on Ca2+ influx and membrane depolarization, spread inwardly faster than that without Ca(2+)-induced Ca2+ release (CICR), and was blocked by dantrolene and ryanodine. Ca2+ entry and a graded activation of CICR thus induce the "Ca2+ transient."

Animals↗