Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts.

Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.

Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3.

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.

Compartmentalized and binary behavior of terminal dendrites in hippocampal pyramidal neurons.

The dendritic arbor of pyramidal neurons is not a monolithic structure. We show here that the excitability of terminal apical dendrites differs from that of the apical trunk. In response to fluorescence-guided focal photolysis of caged glutamate, individual terminal apical dendrites generated cadmium-sensitive all-or-none responses that were subthreshold for somatic action potentials. Calcium transients produced by all-or-none responses were not restricted to the sites of photolysis, but occurred throughout individual distal dendritic compartments, indicating that electrogenesis is mediated primarily by voltage-gated calcium channels. Compartmentalized and binary behavior of parallel-connected terminal dendrites can greatly expand the computational power of a single neuron.

Long-term depression in cerebellar Purkinje neurons results from coincidence of nitric oxide and depolarization-induced Ca2+ transients.

The role of nitric oxide (NO) in the induction of long-term depression (LTD) in the cerebellum was explored using a new, organic, membrane-impermeant form of caged NO. NO photolytically released inside Purkinje neurons mimicked parallel fiber (PF) activity in synergizing with brief postsynaptic depolarization to induce LTD. Such LTD required a delay of < 50 ms between the end of photolysis and the onset of depolarization, was prevented by intracellular Ca2+ chelation, and was mutually occlusive with LTD conventionally produced by PF activation plus depolarization. Bath application of NO synthase inhibitor or of myoglobin, a NO trap, prevent LTD induction via PF stimulation, but not that from intracellular uncaged NO, whereas intracellular myoglobin blocked both protocols. NO is therefore an anterograde transmitter in LTD induction. A biochemical requirement for simultaneous NO and elevation of intracellular free Ca2+ would explain why PF activity must coincide with postsynaptic action potentials.

Development of amoxicillin enzyme-linked immunosorbent assay and measurements of tissue amoxicillin concentrations in a pigeon microdialysis model.

A sensitive ELISA was developed for the detection of amoxicillin (AMX) in serum, urine, and milk. The ELISA used an indirect competitive method produced by coating the plate with ovalbumin conjugated with AMX hapten. Antibodies against AMX-BSA were detected by a goat-antirabbit antibody conjugated with peroxidase. Calibration standard curves ranged from 1.28 ng/mL to 20 microg/mL [IC(50) (inhibition concentration 50%) = 100 ng/mL], and the limits of detection were 1.3, 2.7, and 4.8 ng/mL for urine, milk, and serum, respectively. The intra- and interassay variations were less than 4 and 9.6%. The antibody produced against AMX cross-reacted highly with penicillin G (77%); cross-reacted moderately with ampicillin, oxacillin, and cloxacillin (56.9, 51.4, and 48.8%, respectively); but was considered non-cross-reactive with dicloxacillin (7.4%), cefadroxil (<1%), and cefazolin (<1%). Concentrations of AMX were measured simultaneously in venous blood and muscles by using the developed AMX ELISA in an in vivo microdialysis model designed for pigeons. Following i.m. injection (25 mg/kg), AMX attained a peak blood level of 4.74 +/-0.30 mu g/mL and decreased with a half-life of 2.38 +/-0.16 h. In contrast, measurements in pectoral and femoral muscles exhibited delayed appearances, reduced peak concentrations, and prolonged half-lives of 4.07 +/-0.48 (pectoral) and 3.01 +/-0.26 (femoral) that were significantly different from each other and those in the blood (P < 0.05). Blood protein binding was calculated to be 27.9 +/-5.7%. This study demonstrated the semiquantitative application of a selective AMX ELISA in the first microdialysis procedure for continuous monitoring of drug levels in specific tissues of pigeons and maybe useful for related studies in other poultry species.

Retrospective Pathological Studies of Splenic Lesions in Domestic Hamsters (Phodopus spp.).

Fifteen splenic biopsy specimens from a total of 212 biopsy specimens and necropsy cases of domestic hamsters (Phodopus spp.) from the Division of Wild (Exotic) Animal Medicine, Veterinary Medical Teaching Hospital, National Chung Hsing University, Taiwan, collected between 2010 and 2017, were studied retrospectively. The incidence of lesions in the spleen was 7.1% (15/212). The mean age of affected hamsters was 16.6 months and females were affected more than males. The lesions consisted of 10 neoplasms and five non-neoplastic lesions. The most common tumours were histiocytic sarcoma (HS), lymphoma, malignant fibrous histiocytoma (MFH) and hemangiosarcoma. Immunohistochemistry revealed the HSs and MFHs to express lysozyme. The lymphomas were negative for CD20; however, one case was positive for CD3 and another was positive for CD79a. The hemangiosarcoma expressed von Willebrand factor. The non-neoplastic lesions were all fibrotic nodules and these were all identified in ageing female hamsters. The nodules consisted of collagen fibres identified with Masson's trichrome stain, and they were related to repair of trauma in the spleen.

Organization of intracortical circuits in relation to direction preference maps in ferret visual cortex.

Neurons in the primary visual cortex are selective for the direction of movement of a visual stimulus. Like other stimulus features, direction preference is mapped on the cortical surface in a systematic manner. Intracortical synaptic circuits, in particular inhibitory connections, have been implicated in the emergence of direction selectivity. Whether intracortical inhibition specifically suppresses responses to the nonpreferred direction or has a nonspecific "thresholding" effect is still controversial. To address these questions we investigated the relationship between patterns of intracortical synaptic connections and direction domains in ferret primary visual cortex (area 17) using a combined in vivo-in vitro approach. Excitatory synaptic inputs were iso-direction-tuned. The majority of local inhibitory inputs were also iso-direction-tuned. However, approximately 40% of inhibitory connections originated in regions preferring the opposite direction. These findings indicate that specific inhibitory interactions between cortical regions of opposite direction preference may contribute to the emergence and sharpening of direction selectivity.

Cytosolic Ca2+ oscillations in REF52 fibroblasts: Ca(2+)-stimulated IP3 production or voltage-dependent Ca2+ channels as key positive feedback elements.

Oscillations in cytosolic free calcium concentrations ([Ca2+]i) can be elicited in REF52 fibroblasts by three different modes of stimulation. We have previously demonstrated that [Ca2+]i oscillations result when these cells are simultaneously depolarized and stimulated with a hormone linked to phosphoinositide breakdown. Further evidence is now presented that such oscillations are linked to fluctuations in the concentration of IP3 and the Ca2+ content of an IP3-sensitive Ca2+ store. [Ca2+]i oscillations can also be generated in REF52 cells either by direct stimulation of G-proteins with GTP gamma S or AlF4- or by destabilizing the membrane potential and opening voltage-dependent calcium channels. This report compares the different types of oscillations and their mechanisms.

Dopamine withdrawal elicits prolonged calcium rise to support prolactin rebound release.

Dopamine (DA) acts directly on pituitary lactotropes to inhibit the release of PRL. Removal of DA elicits a pronounced transient rise in PRL release to values exceeding pre-DA rates (PRL rebound). Electrophysiological studies have shown that lactotropes exhibit a period of increased Ca2+ action potential activity after DA withdrawal, leading to the proposal that enhanced Ca2+ influx during this period may support the rebound secretion of PRL. In the present studies, we investigated the effect of DA application and removal on the cytosolic free calcium concentration ([Ca2+]i) monitored by fura-2 in single rat lactotropes. Unchallenged lactotropes fell into two functionally distinct groups: those with stable [Ca2+]i that was not acutely sensitive to extracellular Ca2+, and those with spontaneous fluctuations in [Ca2+]i that were dependent upon influx of external Ca2+. There was striking variability in the [Ca2+]i patterns of the latter group, ranging from irregular, low amplitude fluctuations to rhythmic, repetitive oscillations with definable rise and decay kinetics. Application of DA resulted in a rapid decrease in [Ca2+]i concomitant with the cessation of these spontaneous [Ca2+]i fluctuations. After DA removal, these cells resumed oscillatory [Ca2+]i activities similar to those observed before DA application. In quiescent lactotropes, acute application of DA exerted no effect on resting [Ca2+]i, but quiescent cells could be activated to produce [Ca2+]i fluctuations by the application and withdrawal of DA. Again, the character of the induced [Ca2+] activity showed significant cell to cell variation. In contrast, the pattern of [Ca2+]i fluctuations was remarkably characteristic in a given cell in response to repeated challenges. A composite [Ca2+]i profile of 13 cells paralleled the PRL secretory rebound after application and removal of DA. The oscillatory rise in [Ca2+]i is functionally linked to the rebound release of PRL after DA removal, as both were immediately abolished by blockade of Ca2+ influx. These data demonstrate that the rebound secretion of PRL is dependent upon enhanced influx of extracellular Ca2+ after cells recover from DA-induced hyperpolarization and support the hypothesis that a population of inactivated Ca2+ channels has been recruited in response to application and withdrawal of DA.

Estradiol enhances excitatory gamma-aminobutyric [corrected] acid-mediated calcium signaling in neonatal hypothalamic neurons.

Contrary to the situation in adulthood, gamma-aminobutyric [corrected] acid (GABA)(A) receptor activation during early brain development depolarizes neurons sufficiently to open L-type voltage-gated Ca(2+) channels. Because GABA is excitatory during the sensitive period of steroid-mediated brain sexual differentiation, we investigated whether estradiol modulates excitatory GABA during this period, by examining two parameters: 1) magnitude of GABA-induced calcium transients; and 2) developmental duration of excitatory GABA. Dissociated hypothalamic neurons from embryonic-day-15 rat embryos were loaded with the Ca(2+) indicator, fura-2, and transient rises in [Ca(2+)](i) (Ca(2+) transient) were measured after application of 10 microM muscimol, a GABA(A) receptor agonist. Cells were treated with 10(-10) M estradiol or vehicle from 0-3 days in vitro (DIV) and imaged on 4 DIV, whereas others were treated from 3-6 DIV and imaged on 7 DIV. The mean amplitude of Ca(2+) transients after muscimol administration were 68% and 61% higher in estradiol-treated neurons on 4 DIV and 7 DIV, respectively, relative to controls. Consistent with GABA becoming inhibitory in mature neurons, 50% fewer control neurons responded on DIV 7, relative to DIV 4. However, estradiol treatment maintained excitatory GABA on DIV 7 (72% in estradiol-treated vs. 35% in control). This is the first report of hormonal modulation of excitatory GABA, and it suggests that estradiol may mediate sexual differentiation by enhancing GABA-induced increases in intracellular Ca(2+).

Nitric oxide regulates spike frequency accommodation in nodose neurons of the rabbit.

A Ca(2+)-dependent slow spike after hyperpolarization (AHPslow) is present in about 35% of the neurons in the nodose ganglion. Although the AHPslow profoundly affects spike frequency accommodation of these neurons, the mechanisms that control the generation and the duration of the AHPslow are unclarified. N omega-Nitro-L-arginine methyl ester (L-NAME; 10 microM), a specific inhibitor of nitric oxide synthase (NOS), reduced the AHPslow by more than 92%. The L-NAME block of the AHPslow was antagonized by application of 50 microM S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor. The fast, Ca(2+)-dependent, spike after hyperpolarization preceding the AHPslow and the elevation of intracellular Ca2+ accompanying the AHPslow were unaffected by L-NAME treatment. These findings indicate that products of NOS activity might directly or indirectly activate the AHPslow K+ channels at a step beyond Ca2+ influx or intracellular Ca2+ mobilization.

The molecular and stereochemical structures of photoproducts generated by UV-irradiation of 4'-hydroxymethyl-4,5',8'-trimethylpsoralen in aqueous solution.

UV irradiation of 4'-hydroxymethyl-4,5',8-trimethyl psoralen (HMT) in aqueous solution yields three major photoproducts. We have isolated and characterized (1) a cyclobutane-bridged dimer in which a cis-syn linkage occurs between the furan end of one HMT moiety and the pyrone end of the other; (2) a cyclobutane-bridged dimer wherein both HMT moieties are linked at their pyrone ends with probable cis-syn configuration; and (3) an isomer of HMT for which we have proposed a structure in which the furan and pyrone ring oxygens assume a para orientation via photoisomerization.

Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements.

The Ca2+-binding kinetics of fura-2 and azo-1 were studied using temperature-jump relaxation methods. In 140 mM KCl at 20 degrees C, the association and dissociation rate constants for fura-2 were 6.02 x 10(8) M-1s-1 and 96.7 s-1, respectively. The fura-2 kinetics were insensitive to pH over the range 7.4 to 8.4. Azo-1 was studied in 140 mM KCl, at pH 7.4, at 10 degrees and 20 degrees C. At 10 degrees C, azo-1 exhibited association and dissociation rate constants of 1.43 x 10(8) M-1s-1 and 777.9 s-1, respectively; while at 20 degrees C, the corresponding values were 3.99 x 10(8) M-1s-1 and 1,177 s-1. The kinetic results demonstrate that fura-2 and azo-1 are well suited to monitoring rapid changes in intracellular [Ca2+].

Nmoc-DBHQ, a new caged molecule for modulating sarcoplasmic/endoplasmic reticulum Ca2+ ATPase activity with light flashes.

We report the synthesis and characterization of O[o-nitromandelyloxycarbonyl]-2,5-di(tert-butyl)hydroquinone (Nmoc-DBHQ), a new "caged" reagent for photoreleasing DBHQ, a membrane-permeant, reversible inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). The Nmoc group is a new caging group developed for the current application. Photolysis of Nmoc-DBHQ proceeds with t1/2 = 126 +/- 2 micros, and t1/2 for subsequent release of DBHQ is estimated to be approximately 5 ms. Nmoc-DBHQ thus allows rapid and reversible modulation of SERCA activity in living cells. Through its acetoxymethyl ester, Nmoc-DBHQ can be loaded into cells easily by incubation. We demonstrate the use of Nmoc-DBHQ for photomodulating SERCA activity in fibroblasts and vagal sensory neurons. We further demonstrate the utility of pulsed DBHQ photorelease for probing and manipulating dynamic phenomena such as [Ca2+] oscillations in fibroblasts.

Practical method for the multigram separation of the 5- and 6-isomers of carboxyfluorescein.

An efficient preparative method for separating 5- and 6-carboxyfluorescein is presented. 6-Carboxyfluorescein dipivalate is isolated as its diisopropylamine salt, which can be converted to the free acid or used directly in coupling reactions. The 5-isomer is isolated from the acidified mother liquor. Isomerically pure carboxyfluoresceins are prepared by hydrolysis of the corresponding dipivalates.

Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.

A new group of fluorescent indicators with visible excitation and emission wavelengths has been synthesized for measurements of cytosolic free Ca2+. The five compounds, "rhod-1," "rhod-2," "fluo-1," "fluo-2," and "fluo-3" (Figs. 2 and 3), combine the 8-coordinate tetracarboxylate chelating site of 1,2-bis(2-amino-phenoxyethane-N,N,N',N'-tetraacetic acid with a xanthene chromophore to give a rhodamine-like or fluorescein-like fluorophore. Binding of Ca2+ increases the fluorescence by up to 40-fold. The Ca2+ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators should give better resolution of high [Ca2+] levels than previously obtainable with quin-2 or fura-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescence microscopy and flow cytometry than the UV required by previous indicators. However, the new dyes' increase in fluorescence upon binding calcium is not accompanied by a wavelength shift, so they are unsuitable for measurements using ratios at two wavelengths. The most promising dye of this series is fluo-3, whose initial biological testing in fibroblasts is described in the following paper (Kao, J. P. Y., Harootunian, A. T., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178).

Photochemically generated cytosolic calcium pulses and their detection by fluo-3.

Fluo-3, one member of a family of new fluorescent Ca2+ indicators excitable at wavelengths in the visible (Minta, A., Kao, J. P. Y., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8171-8178), has been tested in living cells. We demonstrate that fluo-3 can be loaded into fibroblasts and lymphocytes by incubation with the pentaacetoxymethyl ester of the dye and that the ester is hydrolyzed intracellularly to yield genuine fluo-3 capable of indicating changes in [Ca2+]i induced by agonist stimulation. Fluo-3 can also be microinjected into fibroblasts along with photolabile compounds such as nitr-5 and caged inositol trisphosphate for photorelease experiments. Fluo-3 permits continuous monitoring of [Ca2+]i without interference with use of UV-sensitive caged compounds. A procedure for combined use of ionophore and heavy metal ions in end-of-experiment calibration of fluo-3 intensities to give [Ca2+]i is also described.

A novel Ca(2+) influx pathway in mammalian primary sensory neurons is activated by caffeine.

Single-cell microfluorimetry and electrophysiology techniques were used to identify and characterize a novel Ca(2+) influx pathway in adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion neurons (NGNs) exhibit robust Ca(2+)-induced Ca(2+) release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) is considered diagnostic evidence of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca(2+) stores or pharmacological blockade of intracellular Ca(2+) release channels (ryanodine receptors), caffeine still elicited a significant rise in [Ca(2+)](i) in approximately 50% of NGNs. The same response was not elicited by pharmacological agents that elevate cyclic nucleotide concentrations. Moreover, extracellular Ca(2+) was obligatory for such caffeine-induced [Ca(2+)](i) rises in this population of NGNs, suggesting that Ca(2+) influx is responsible for this rise. Simultaneous microfluorimetry with whole cell patch-clamp studies showed that caffeine activates an inward current that temporally parallels the rise in [Ca(2+)](i). The inward current had a reversal potential of +8.1 +/- 6.1 (SE) mV (n = 4), a mean peak amplitude of -126 +/- 24 pA (n = 4) at E(m) = -50 mV, and a slope conductance of 1.43 +/- 0.79 nS (n = 4). Estimated EC(50) values for caffeine-induced CICR and for caffeine-activated current were 1.5 and approximately 0.6 mM, respectively. These results indicate that caffeine-induced rises in [Ca(2+)](i), in the presence of extracellular Ca(2+), can no longer be interpreted as unequivocal diagnostic evidence for CICR in neurons. These results also indicate that sensory neurons possess a novel Ca(2+) influx pathway.

Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes.

New fluorescent Na+ indicators, SBFI (short for sodium-binding benzofuran isophthalate) and SBFP (short for sodium-binding benzofuran phthalate) (Minta, A., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 19449-19457), were tested in Jurkat tumor lymphocytes and in REF52 rat embryo fibroblasts. Both dyes could be introduced by direct microinjection. However, when cells were incubated with the tetra(acetoxymethyl) esters of the dyes, only SBFI gave intracellular loading that was reasonably responsive to [Na+]i. Because some compartmentation of the SBFI was visible and because the indicator properties are somewhat affected by cytoplasm, the relationship between intracellular free Na+ [( Na+]i and the 340/385 nm excitation ratio of the indicator was calibrated in situ using poreforming antibiotics to equilibrate cytosolic [Na+] [( Na+]i) with extracellular [Na+]. The excitation ratio was sufficiently sensitive to resolve small changes, less than or equal to 1 mM, in [Na+]i in single cells. Basal [Na+]i values in lymphocytes and serum-starved fibroblasts were 9.4 and 4.2 mM, respectively. As expected, large increases in [Na+]i were elicited by blocking the Na+ pump with ouabain or withdrawal of extracellular K+. Mitogens such as phytohemagglutinin acting on the lymphocytes, or serum or vasopressin in fibroblasts, caused [Na+]i to increase up to 2-fold. In fibroblasts, the rise in [Na+]i was due at least partly to a stimulation of Na+ influx, which was not wholly through the Na+/H+ exchanger. The mitogen-induced increases in [Na+]i and the rate of Na+ influx are consistent with earlier estimates based on measurements of total [Na+] or tracer fluxes. However, the absolute values for free [Na+]i are much lower than previous values for total intracellular Na+, suggesting that much of the latter is bound or sequestered.