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Ca(2+) release from internal stores (sarcoplasmic reticulum or SR) in smooth muscles is initiated either via pharmaco-mechanical coupling due to the action of an agonist and involving IP3 receptors, or via excitation-contraction coupling, mostly involving L-type calcium channels in the plasmalemma (DHPRs), and ryanodine receptors (RyRs), or Ca(2+) release channels of the SR. This work focuses attention on the structural basis for the coupling between DHPRs and RyRs in phasic smooth muscle cells of the guinea-pig urinary bladder. Immunolabeling shows that two proteins of the SR: calsequestrin and the RyR, and one protein the plasmalemma, the L-type channel or DHPR, are colocalized with each other within numerous, peripherally located sites located within the caveolar domains. Electron microscopy images from thin sections and freeze-fracture replicas identify feet in small peripherally located SR vesicles containing calsequestrin and distinctive large particles clustered within small membrane areas. Both feet and particle clusters are located within caveolar domains. Correspondence between the location of feet and particle clusters and of RyR- and DHPR-positive foci allows the conclusion that calsequestrin, RyRs, and L-type Ca(2+) channels are associated with peripheral couplings, or Ca(2+) release units, constituting the key machinery involved in excitation-contraction coupling. Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action.

We recorded Ca2+ sparks and spontaneous transient outward currents (STOCs) simultaneously in smooth muscle cells using whole-cell patch recording and a unique, high-speed widefield digital imaging system to monitor fluo-3 fluorescence in both two and three dimensions (2D and 3D). In 2D imaging, the correlation between the amplitude of a spark and its corresponding STOC was a weak one, and 27 % of the sparks failed to cause STOCs. The STOCless sparks were not significantly different in amplitude from those that caused STOCs. Three-dimensional imaging disclosed that STOCless sparks were located close to the cell surface, and on average their apparent distance from the cell surface was not significantly different from the sparks that cause STOCs. Statistical evaluation of spark clusters disclosed that there were regions of the cell where the probability of spark occurrence was high and others where it was quite low.

This study investigated the relationship between cytoplasmic, mitochondrial, and sarcoplasmic reticulum (SR) [Ca(2+)] in rabbit aorta smooth muscle cells, following cell activation. Smooth muscle cells were loaded with the Ca(2+)-sensitive fluorescent indicator Mag-Fura-2-AM, and then either permeabilized by exposure to saponin, or dialyzed with a patch pipette in the whole-cell configuration to remove cytoplasmic indicator. When the intracellular solution contained millimolar EGTA or BAPTA, activation of SR Ca(2+)release through IP(3)or ryanodine receptors induced a decrease in the [Ca(2+)] reported by Mag-Fura-2. However, when EGTA was present at < or =100 microM, the same stimuli caused an increase in the [Ca(2+)] reported by Mag-Fura-2. The increase in [Ca(2+)] caused by phenylephrine or caffeine was delayed, and prolonged, with respect to the cytoplasmic Ca(2+)transient. Evidence is presented that this Mag-Fura-2 signal reflected a rise in mitochondrial [Ca(2+)]. Agents that inhibit mitochondrial function, such as FCCP or cyanide in combination with oligomycin B, converted the increase in organelle Mag-Fura-2 fluorescence to a decrease, while also prolonging the cytoplasmic Ca(2+)transient. There was considerable similarity between the localization of Mag-Fura-2 fluorescence and the mitochondria-selective indicator tetramethylrhodamine ethyl ester. Thus, we propose that there is close functional integration between the SR and mitochondria in aorta smooth muscle cells, with mitochondria taking up Ca(2+)from the cytoplasm following cell activation., (Copyright 2000 Harcourt Publishers Ltd.)

1. A digital imaging microscope with fura-2 as the Ca2+ indicator was used to determine the sources for the rise in intracellular calcium concentration ([Ca2+]i) that occurs when the membrane in a cell-attached patch is stretched. Unitary ionic currents from stretch-activated channels and [Ca2+]i images were recorded simultaneously. 2. When suction was applied to the patch pipette to stretch a patch of membrane, Ca2+-permeable cation channels (stretch-activated channels) opened and a global increase in [Ca2+]i occurred, as well as a greater focal increase in the vicinity of the patch pipette. The global changes in [Ca2+]i occurred only when stretch-activated currents were sufficient to cause membrane depolarization, as indicated by the reduction in amplitude of the unitary currents. 3. When Ca2+ was present only in the pipette solution, just the focal change in [Ca2+]i was obtained. This focal change was not seen when the contribution from Ca2+ stores was eliminated using caffeine and ryanodine. 4. These results suggest that the opening of stretch-activated channels allows ions, including Ca2+, to enter the cell. The entry of positive charge triggers the influx of Ca2+ into the cell by causing membrane depolarization, which presumably activates voltage-gated Ca2+ channels. The entry of Ca2+ through stretch-activated channels is also amplified by Ca2+ release from internal stores. This amplification appears to be greater than that obtained by activation of whole-cell Ca2+ currents. These multiple pathways whereby membrane stretch causes a rise in [Ca2+]i may play a role in stretch-induced contraction, which is a characteristic of many smooth muscle tissues.

1. The Ca(2+)-sensitive fluorescent indicator rhod-2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura-2 was used in combination with rhod-2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. 2. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half-time (t1/2) to peak for the increase in [Ca2+]m was considerably longer than the t1/2 to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. 3. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t1/2 to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. 4. Using a wide-field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. 5. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. 6. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR.

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.

Localized, transient elevations in cytosolic Ca2+, known as Ca2+ sparks, caused by Ca2+ release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca2+-activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca2+ concentration within the sarcoplasmic reticulum and a Ca2+ spark and that between a Ca2+ spark and a STOC are not well defined or fully understood. To address these problems, we have employed two approaches using single patch-clamped smooth muscle cells freshly dissociated from toad stomach: a high speed, wide-field imaging system to simultaneously record Ca2+ sparks and STOCs, and a method to simultaneously measure free global Ca2+ concentration in the sarcoplasmic reticulum ([Ca2+]SR) and in the cytosol ([Ca2+]CYTO) along with STOCs. At a holding potential of 0 mV, cells displayed Ca2+ sparks and STOCs. Ca2+ sparks were associated with STOCs; the onset of the sparks coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in [Ca2+]CYTO at the time and location of the spark peak was approximately 100 nM above a resting concentration of approximately 100 nM. The frequency and amplitude of spontaneous Ca2+ sparks recorded at -80 mV were unchanged for a period of 10 min after removal of extracellular Ca2+ (nominally Ca2+-free solution with 50 microM EGTA), indicating that Ca2+ influx is not necessary for Ca2+sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in [Ca2+]SR in association with a surge in [Ca2+]CYTO and a fusion of STOCs, followed by a fast restoration of [Ca2+]CYTO and a gradual recovery of [Ca2+]SR and STOCs. The return of global [Ca2+]CYTO to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca2+. After the global [Ca2+]CYTO was fully restored, recovery of STOC frequency and amplitude were correlated with the level of [Ca2+]SR, even though the time for refilling varied greatly. STOC frequency did not recover substantially until the [Ca2+]SR was restored to 60% or more of resting levels. At [Ca2+]SR levels above 80% of rest, there was a steep relationship between [Ca2+]SR and STOC frequency. In contrast, the relationship between [Ca2+]SR and STOC amplitude was linear. The relationship between [Ca2+]SR and the frequency and amplitude was the same for Ca2+ sparks as it was for STOCs. The results of this study suggest that the regulation of [Ca2+]SR might provide one mechanism whereby agents could govern Ca2+ sparks and STOCs. The relationship between Ca2+ sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca2+ release site and the Ca2+-activated potassium channels responsible for a STOC.

In response to a chemotactic gradient, leukocytes extravasate and chemotax toward the site of pathogen invasion. Although fundamental in the control of many leukocyte functions, the role of cytosolic free Ca2+ in chemotaxis is unclear and has been the subject of debate. Before becoming motile, the cell assumes a polarized morphology, as a result of modulation of the cytoskeleton by G protein and kinase activation. This morphology may be reinforced during chemotaxis by the intracellular redistribution of Ca2+ stores, cytoskeletal constituents, and chemoattractant receptors. Restricted subcellular distributions of signaling molecules, such as Ca2+, Ca2+/calmodulin, diacylglycerol, and protein kinase C, may also play a role in some types of leukocyte. Chemotaxis is an essential function of most cells at some stage during their development, and a deeper understanding of the molecular signaling and structural components involved will enable rational design of therapeutic strategies in a wide variety of diseases.

The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP3)-gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca2+ signaling.

Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule, and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single messenger RNA molecules. Analysis of beta-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and messenger RNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.

Pericentrin and gamma-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin-gamma-tubulin complex was distinct from the previously described gamma-tubulin ring complex (gamma-TuRC) as purified gamma-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.

Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose influence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.

We have demonstrated the feasibility of preparing caged peptides by derivatizing a single amino acid side chain in peptides up to 20 amino acids long. Two peptides are illustrated whose activities are reduced by nearly 2 orders of magnitude using this caging approach. The specific strategy described here of derivatizing tyrosine side chains with a charged caging moiety should be generally applicable in the preparation of caged peptides that have a critical tyrosine residue (e.g., LSM1) or that have critical hydrophobic patches (e.g., RS-20). Other amino acid side chains are also accessible via this caging strategy. Derivatives of threonine, serine, lysine, cysteine, glutamate, aspartate, glutamine, and asparagine can be prepared and site specifically inserted into peptides in an analogous manner. The caged peptides synthesized and purified by the methods described here are compatible with biological samples, including living cells, and have been used to demonstrate the central importance of calmodulin, MLCK, and, by inference, myosin II in ameboid locomotion in polarized eosinophil cells. Photoactivation of peptides within cells should provide a wealth of new information in future investigations by allowing specific protein activities to be knocked out in an acute and spatially defined way.

1. [Ca2+] was recorded in voltage-clamped gastric myocytes from Bufo marinus. Repolarization to -110 mV following a 300 ms depolarization to +10 mV led to triphasic [Ca2+]i decay, with a fast-slow-fast pattern. After a conditioning train of repetitive depolarizations the duration of the second, slow phase of decay was shortened, while the rate of decay during the third, faster phase was increased by 34 +/- 6% (mean +/- S.E.M., n = 21) when compared with unconditioned transients. 2. [Ca2+]i decay was biphasic in cells injected with the calmodulin-binding peptide RS20, with a prolonged period of fast decay followed by a slow phase. There was no subsequent increase in decay rate during individual transients and no acceleration of decay following the conditioning train (n = 8). Decline of [Ca2+]i in cells injected with the control peptide NRS20 was triphasic and the decay rate during the third phase was increased by 50 +/- 19% in conditioned transients (n = 6). 3. Cell injection with CK3AA, a pseudo-substrate inhibitor of calmodulin-dependent protein kinase II, prevented the increase in the final rate of decay following the conditioning train (n = 6). In cells injected with an inactive peptide similar to CK3AA, however, there was a 45 +/- 17% increase after the train (n = 5). 4. Inhibition of Ca2+ uptake by the sarcoplasmic reticulum with cyclopiazonic acid or thapsigargin did not prevent acceleration of decay. 5. These results demonstrate that [Ca2+]i decay is accelerated by Ca(2+)-calmodulin and calmodulin-dependent protein kinase II. This does not depend on Ca2+ uptake by the sarcoplasmic reticulum but may reflect upregulation of mitochondrial Ca2+ removal.

Ca2+ currents (ICa) and cytoplasmic Ca2+ concentration ([Ca2+]c) were measured in isolated gastric myocytes from Bufo marinus using whole cell voltage clamp and fura 2, respectively. After a conditioning train of depolarizing pulses, high-voltage-activated ICa (test potential of +10 mV) was increased, returning to control values after approximately 85 s. This enhancement was [Ca2+]c dependent, with a maximal increase at approximately 600 nM [Ca2+]c. During the conditioning train, ICa measured at 70 ms, which provides a measure of high-voltage-activated current, initially decreased with each successive pulse to a minimum of 56 +/- 5% of the first pulse in the train. Thereafter, the 70-ms current showed considerable recovery. Blockade of calmodulin activity with a peptide (RS20) or calmidazolium did not affect the early inhibition but did abolish current recovery. A peptide inhibitor of calmodulin-dependent protein kinase II (CK3AA) had similar effects. Substraction of currents measured in the presence and absence of RS20 revealed a 2-s delay between the start of the train and the onset of current enhancement. It was also observed that low-voltage-activated current (test potential of -17 mV) was reduced to 76 +/- 7% of control 5 s after the conditioning train; this inhibition recovered to 92 +/- 4% after 35 s and was not dependent on [Ca2+]c elevation.

A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle.

Digital imaging microscopy was used to analyze the spatial distribution and levels of newly synthesized RNA in relation to steady-state poly(A) RNA and to the splicing factor SC35. Transcription was monitored over time after microinjection of BrUTP and was detected using antibodies. Poly(A) RNA was detected with probes directly conjugated to fluorochromes, allowing direct detection of the hybrids. Objective methods were used to determine genuine signal. A defined threshold level to separate signal from noise was established for each nucleus. The nucleolus was used to determine poly(A) and SC35 background and the juxtanuclear cytoplasm was used for the BrUTP background. The remaining signal was segmented into high (concentrated) and low (diffuse) levels. Surprisingly, for all probes examined, most of the signal was not in concentrated areas, but rather was diffusely spread throughout the nucleoplasm. A minority (20-30%) of the SC35 signal was in concentrated areas ("speckles") and the rest was dispersed throughout the nucleoplasm. In addition, the concentrated areas had a mean intensity only twice the average. The amount and significance of the colocalization of the diffuse, or concentrated, areas of SC35 [or poly(A)] with BrUTP incorporation were analyzed. The image from one probe was translated with respect to the other in three dimensions to compare colocalization with random alignments. Both poly(A) and SC35 were found to have low colocalization with the total BrU signal. Sites of transcription were determined using an algorithm to find maxima of BrUTP signal within clusters. From 849 to as many as 3888 sites per nucleus were detected. A rim of hybridization to poly(A) coinciding with the nuclear envelope was eliminated by actinomycin treatment, suggesting that these transcripts were exiting from the nucleus. These results emphasize the importance of utilizing the full dynamic range of the image before drawing conclusions as to the distribution of nuclear components.

1. Decay in intracellular calcium concentration ([Ca2+]i) was recorded following step depolarizations in voltage clamped gastric myocytes from Bufo marinus. 2. Depolarizations (300 ms) to +10 mV were followed by three phases of [Ca2+]i decay with repolarization to both -110 and -50 mV. The decline was initially rapid (mean fractional decay rate = 81 +/- 11%s-1 at -110 mV), then slowed (decay rate = 14 +/- 2%s-1) and finally accelerated again (decay rate = 24 +/- 3%s-1; n = 19). 3. The initial phase of rapid decay became shorter as the length of the depolarizing pulse increased but was unaffected by changes in pulse voltage. 4. The delayed acceleration in [Ca2+]i decay was no longer seen when the duration of the depolarizing pulses was reduced to 100 ms, but was clearly evident following 500 ms pulses. This phase was abolished when the depolarizing voltage was altered to minimize the rise in [Ca2+]i. 5. Ryanodine and caffeine had no effect on the temporal profile of [Ca2+]i decay. 6. Removal of extracellular Na+ decreased the decay rate during all three phases at -110 mV, but this effect was particularly marked for the initial rapid phase of decay, the rate of which was reduced by 75%. A delayed increase in decay rate was still seen. 7. Inhibition of mitochondrial Ca2+ uptake with cyanide, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or Ruthenium Red had no effect on the initial rate of [Ca2+]i decay but blocked the delayed acceleration. 8. These results are discussed in terms of a model in which rapid influx of Ca2+ produces a high subsarcolemmal [Ca2+], favouring rapid Ca2+ removal by near-membrane mechanisms, particularly Na(+)-Ca2+ exchange. Mitochondrial Ca2+ removal produces a delayed increase in [Ca2+]i decay if the global [Ca2+]i is raised high enough for long enough.

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.

Recent evidence, from a variety of cell types, suggests that mitochondria play an important role in shaping the change in intracellular calcium concentration ([Ca2+]i) that occurs during physiological stimulation. In the present study, using a range of inhibitors of mitochondrial Ca2+ uptake, we have examined the contribution of mitochondria to Ca2+ removal from the cytosol of smooth muscle cells following stimulation. In voltage-clamped single smooth muscle cells, we found that following a 8-s train depolarizing pulses, the rate of Ca2+ extrusion from the cytosol was reduced by more than 50% by inhibitors of cytochrome oxidase or exposure of cells to the protonophore carbonyl cyanide P-trifluoromethoxy-phenylhydrazone. Using the potential-sensitive indicator-tetramethyl rhodamine ethyl ester, we confirmed that the effect of these agents was associated with depolarization of the mitochondrial membrane. Since, the primary function of the mitochondria is to provide the cell's ATP, it could be argued that it is the ATP supply to the ion pumps which is limiting the rate of Ca2+ removal. However, experiments carried out with the mitochondrial Ca2+ uniporter inhibitor ruthenium red produced similar results, while the ATP synthetase inhibitor oligomycin had no effect, suggesting that the effect was not due to ATP insufficiency. These results establish that mitochondria in smooth muscle cells play a significant role in removing Ca2+ from the cytosol following stimulation. The uptake of Ca2+ into mitochondria is proposed to stimulate mitochondrial ATP production, thereby providing a means for matching increased energy demand, following the cell's rise in [Ca2+]i, with increased cellular ATP production.

Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distribution of hexokinase in living cells, fluorophore-labeled functional hexokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitochondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an increase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and diminished hexokinase binding to mitochondria in single cells. 3-O-methylglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexokinase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energy state as monitored by ATP. On the other hand, only in the presence of 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distribution observed with the fluorescent-injected hexokinase, cyanide caused only a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentrations of specific metabolites can alter the binding of hexokinase I to specific sites on mitochondria. Moreover, the apparent difference in sensitivity of injected and endogenous hexokinase to changes in metabolites may reflect the presence of at least two classes of binding mechanisms for hexokinase, with differential sensitivity to metabolites.

Calcium release from intracellular stores occurs in a graded manner in response to increasing concentrations of either inositol 1,4,5-trisphosphate or caffeine. To investigate the mechanism responsible for this quantal release phenomenon, [Ca2+] changes inside intracellular stores in isolated single smooth muscle cells were monitored with mag-fura 2. Following permeabilization with saponin or alpha-toxin the dye, loaded via its acetoxymethyl ester, was predominantly trapped in the sarcoplasmic reticulum (SR). Low caffeine concentrations in the absence of ATP induced only partial Ca2+ release; however, after inhibiting the calcium pump with thapsigargin the same stimulus released twice as much Ca2+. When the SR Ca(2+)-ATPase was rendered non-functional by depleting its "ATP pool," submaximal caffeine doses almost fully emptied the stores of Ca2+. We conclude that quantal release of Ca2+ in response to caffeine in these smooth muscle cells is largely due to the activity of the SR Ca(2+)-ATPase, which appears to return a portion of the released Ca2+ back to the SR, even in the absence of ATP. Apparently the SR Ca(2+)-ATPase is fueled by ATP, which is either compartmentalized or bound to the SR.

Fluorescent probes offer insight into the highly localized and rapid molecular events that underlie cell function. However, methods are required that can efficiently transform the limited signals from such probes into high-resolution images. An algorithm has now been developed that produces highly accurate images of fluorescent probe distribution inside cells with minimal light exposure and a conventional light microscope. This method provides resolution nearly four times greater than that currently available from any fluorescence microscope and was used to study several biological problems.

The role of Ca2+ in chemotaxis of eosinophils from the newt Taricha granulosa was investigated using fluorescent indicators and digital imaging microscopy. In response to serum chemoattractant, cytoplasmic Ca2+ concentration ([Ca2+]i) rises prior to polarization. In polarized locomoting cells [Ca2+]i gradients (tail-high-front-low) are always seen, and when cells turn [Ca2+]i rises transiently and falls fastest and furthest in the new direction of cell motion. These Ca2+ signals, which are required for polarization and locomotion, arise from Ca2+ derived from internal stores released in response to inositol 1,4,5-trisphosphate (InsP3) (because microinjected heparin fully blocks them). 1,2-Diacyl-sn-glycerol (DAG), which is co-produced with InsP3, has an inhibitory effect on Ca2+ signals, an effect apparently mediated by protein kinase C. Studies with caged InsP3 reveal that InsP3-responsive stores appear to be concentrated in the nuclear and microtubule-organizing centre regions and that InsP3 moves so rapidly within the cell that it is effectively a global secondary messenger. Thus, stable [Ca2+] gradients observed during unidirectional migration appear to result from the concentration of InsP3-responsive Ca2+ stores in the rear of the cell. By contrast, we propose that reorientation of the [Ca2+] gradient prior to a change in direction of motion results from the joint actions of InsP3 and DAG, with InsP3 acting as a global secondary messenger stimulating Ca2+ release and DAG, through protein kinase C, acting as a spatially restricted secondary messenger inhibiting [Ca2+] increases locally near the site of chemotactic stimulation.

Calcium, a ubiquitous second messenger, stimulates the activity of several mitochondrial dehydrogenases. This has led to the suggestion that the same messenger that signals cell activation could also activate mitochondrial electron/proton transport, thereby meeting demands for increased cellular energy. To test this in live cells, quantitative three-dimensional microscopy and ratio imaging were used to measure membrane potential of individual mitochondria and cytosolic calcium distribution. Mitochondria reversibly depolarized as cytosolic calcium rose and then fell following physiological stimulation. Thus, the dominant response of the mitochondrion to a rise in cytosolic [Ca2+] is to draw on the electrochemical potential, possibly to accelerate processes directly involved in ATP synthesis and calcium homeostasis.

During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]i increased rapidly from < or = 100 nM to > or = 400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of approximately 200 nM. The initial Ca2+ spike was insensitive acutely to removal of extracellular Ca2+ but was abolished by treatments expected to deplete internal Ca2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lamellipod activity and suppressed the Ca2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca2+ spike on stimulation. Although cell flattening and lamellipod activity were induced by chemoattractant when the normal Ca2+ response was blocked, cells failed to polarize and crawl, indicating that Ca2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.

Regulatory light chain phosphorylation is required for assembly of smooth and non-muscle myosins in vitro, but its effect on polymerization within the cell is not understood. Relaxed smooth muscle cells contain dephosphorylated thick filaments, but this does not exclude the presence of a pool of folded myosin monomers which could be recruited to assemble when phosphorylated, thus forming part of smooth muscle's activation pathway. To test this hypothesis, relaxed and contracted avian gizzard cryosections were labeled with a fluorescently conjugated monoclonal antibody specific for the folded monomeric conformation, or with an antibody against the tip of the tail whose epitope is accessible in the monomeric but not the filamentous state. Fluorescence intensity observed in the two physiological states was quantitated by digital imaging microscopy. Only trace amounts of folded monomeric myosin were detected in both the relaxed and contracted states. The amount of monomer also did not increase when alpha-toxin permeabilized gizzard was equilibrated in a solvent that disassembles filaments in vitro. Assembly/disassembly is therefore unlikely to play a major role in regulating the contraction/relaxation cycle in smooth muscle cells.

Activation of ryanodine receptors on the sarcoplasmic reticulum of single smooth muscle cells from the stomach muscularis of Bufo marinus by caffeine is accompanied by a rise in cytoplasmic [Ca2+] ([Ca2+]i), and the opening of nonselective cationic plasma membrane channels. To understand how each of these pathways contributes to the rise in [Ca2+]i, one needs to separately monitor Ca2+ entry through them. Such information was obtained from simultaneous measurements of ionic currents and [Ca2+]i by the development of a novel and general method to assess the fraction of current induced by an agonist that is carried by Ca2+. Application of this method to the currents induced in these smooth muscle cells by caffeine revealed that approximately 20% of the current passing through the membrane channels activated following caffeine application is carried by Ca2+. Based on this information we found that while Ca2+ entry through these channels rises slowly, release of Ca2+ from stores, while starting at the same time, is much faster and briefer. Detailed quantitative analysis of the Ca2+ release from stores suggests that it most likely decays due to depletion of Ca2+ in those stores. When caffeine was applied twice to a cell with only a brief (30 s) interval in between, the amount of Ca2+ released from stores was markedly diminished following the second caffeine application whereas the current carried in part by Ca2+ entry across the plasma membrane was not significantly affected. These and other studies described in the preceding paper indicate that activation of the nonselective cation plasma membrane channels in response to caffeine was not caused as a consequence of emptying of internal Ca2+ stores. Rather, it is proposed that caffeine activates these membrane channels either by direct interaction or alternatively by a linkage between ryanodine receptors on the sarcoplasmic reticulum and the nonselective cation channels on the surface membrane.

The effects of caffeine on cytoplasmic [Ca2+] ([Ca2+]i) and plasma membrane currents were studied in single gastric smooth muscle cells dissociated from the toad, Bufo marinus. Experiments were carried out using Fura-2 for measuring [Ca2+]i and tight-seal voltage-clamp techniques for recording membrane currents. When the membrane potential was held at -80 mV, in 15% of the cells studied caffeine increased [Ca2+]i without having any effect on membrane currents. In these cells ryanodine completely abolished any caffeine induced increase in [Ca2+]i. In the other cells caffeine caused both an increase in [Ca2+]i and activation of an 80-pS nonselective cation channel. In this group of cells ryanodine only partially blocked the increase in [Ca2+]i induced by caffeine; moreover, the change in [Ca2+]i that did occur was tightly coupled to the time course and magnitude of the cation current through these channels. In the presence of ryanodine, blockade of the 80-pS channel by GdCl3 or decreasing the driving force for Ca2+ influx through the plasma membrane by holding the membrane potential at +60 mV almost completely blocked the increase in [Ca2+]i induced by caffeine. Thus, the channel activated by caffeine appears to be permeable to Ca2+. Caffeine activated the cation channel even when [Ca2+]i was clamped to below 10 nM when the patch pipette contained 10 mM BAPTA suggesting that caffeine directly activates the channel and that it is not being activated by the increase in Ca2+ that occurs when caffeine is applied to the cell. Corroborating this suggestion were additional results showing that when the membrane was depolarized to activate voltage-gated Ca2+ channels or when Ca2+ was released from carbachol-sensitive internal Ca2+ stores, the 80-pS channel was not activated. Moreover, caffeine was able to activate the channel in the presence of ryanodine at both positive and negative potentials, both conditions preventing release of Ca2+ from stores and the former preventing its influx. In summary, in gastric smooth muscle cells caffeine transiently releases Ca2+ from a ryanodine-sensitive internal store and also increases Ca2+ influx through the plasma membrane by activating an 80-pS cation channel by a mechanism which does not seem to involve an elevation of [Ca2+]i.

Although a role for the ryanodine receptor (RyR) in Ca2+ signaling in smooth muscle has been inferred, direct information on the biochemical and functional properties of the receptor has been largely lacking. Studies were thus carried out to purify and characterize the RyR in stomach smooth muscle cells from the toad Bufo marinus. Intracellular Ca2+ measurements with the Ca(2+)-sensitive fluorescent indicator fura-2 under voltage clamp indicated the presence of a caffeine- and ryanodine-sensitive internal store for Ca2+ in these cells. The (CHAPS)-solubilized, [3H]ryanodine-labeled RyR of toad smooth muscle was partially purified from microsomal membranes by rate density centrifugation as a 30-S protein complex. SDS/PAGE indicated the comigration of a high molecular weight polypeptide with the peak attributed to 30-S RyR, which had a mobility similar to the cardiac RyR and on immunoblots cross-reacted with a monoclonal antibody to the canine cardiac RyR. Following planar lipid bilayer reconstitution of 30-S stomach muscle RyR fractions, single-channel currents (830 pS with 250 mM K+ as the permeant ion) were observed that were activated by Ca2+ and modified by ryanodine. In vesicle-45Ca2+ efflux measurements, the toad channel was activated to a greater extent at 100-1000 microM than 1-10 microM Ca2+. These results suggest that toad stomach muscle contains a ryanodine-sensitive Ca2+ release channel with properties similar but not identical to those of the mammalian skeletal and cardiac Ca(2+)-release channels.

A Ca2+ indicator has been synthesized and characterized which can be used to monitor rapid changes in the free Ca2+ concentration ([Ca2+]) immediately adjacent to cell membranes. This indicator, referred to as C18-Fura-2, consists of a Fura-2 molecule conjugated to a lipophilic alkyl chain which will insert into cell membranes. When associated with cell membranes in low concentrations, C18-Fura-2 exhibits an excitation spectrum with a large Stokes shift and a single isobestic point, thus [Ca2+] can be calculated ratiometrically. The apparent Ca2+ dissociation constant of cell-associated C18-Fura-2 is around 150 nM. C18-Fura-2 orients in the cell membrane so that the fluorophore is facing the side to which it was applied. C18-Fura-2 was used to record rapid changes in intracellular [Ca2+] which occurred in response to membrane depolarization in isolated smooth muscle cells. The initial rise of the [Ca2+] transient reported by C18-Fura-2 was four to six times faster than the rise of the [Ca2+] transient reported by cytosolic Fura-2. This result suggests that C18-Fura-2 was located at the plasma membrane near sites of Ca2+ influx and indicates that membrane-associated Ca2+ indicators can be used to detect rapid, localized changes in [Ca2+] which are obscured in signals recorded using water-soluble, bulk cytosolic fluorescent Ca2+ indicators.

Human platelet-derived growth factor receptors (PDGFRs) expressed in human Hep G2 cells internalized and concentrated in a juxtanuclear region near the Golgi network within 10 minutes after the cells were treated with PDGF. A PDGFR mutant (F5) that lacks high-affinity binding sites for the Src homology 2 domain-containing proteins phosphatidylinositol-3 kinase (PI-3 kinase), Ras guanosine triphosphatase activating protein, phospholipase C-gamma, and a phosphotyrosine phosphatase (Syp) remained at the cell periphery. Restoration of the PI-3 kinase binding sites on F5 completely restored the ability of the receptor to concentrate intracellularly. A PDGFR mutant lacking only PI-3 kinase binding sites failed to concentrate intracellularly. Thus, PI-3 kinase binding sites appear both necessary and sufficient for the normal endocytic trafficking of the activated PDGFR.

The sodium/calcium (Na+/Ca2+) exchanger is often considered to be a key regulator of the cytoplasmic calcium concentration ([Ca2+]) in smooth muscle but neither its precise role in Ca2+ homeostasis nor even its existence in smooth muscle are generally agreed upon. Here we directly assessed the role Na+/Ca2+ exchange plays in regulating [Ca2+] in single voltage-clamped smooth muscle cells. Following an elevation of [Ca2+], its decline was found to have both voltage-dependent and voltage-independent components. The voltage-dependent component was abolished when Na+ was removed from the external bathing solution. During the fall of [Ca2+] a small and declining Na(+)-dependent inward current was observed of a magnitude predicted by 3:1 Na+/Ca2+ exchange stoichiometry. At [Ca2+] above 400 nM the principal efflux of Ca2+ above rest was attributed to this Na(+)-dependent removal mechanism. These results establish that a Na+/Ca2+ exchanger exists in smooth muscle and argue that it can regulate [Ca2+] at physiological Ca2+ concentrations.

Local chemical events underlying chemotaxis were characterized in a new model cell, the newt eosinophil. These cells exhibit a chemotactic response to a trypsin-sensitive component of newt serum. Ca2+ plays a role in this process, since treatments expected to diminish Ca2+ availability from the medium [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, Co2+, and verapamil], to break down transmembrane Ca2+ gradients (ionomycin), or to interfere with the function of intracellular Ca2+ stores (caffeine and neomycin) inhibited cell polarization and movement. Using imaging techniques we found that cytosolic Ca2+ concentration ([Ca2+]i) increased in response to newt serum. Migrating newt eosinophils exhibited a dynamic heterogeneous distribution of [Ca2+]i. [Ca2+]i was elevated in cells undergoing a change of direction relative to cells migrating persistently in one direction. Migrating cells contained gradients of [Ca2+]i along their long axis, with the front of the cell having consistently lower [Ca2+]i than the rear. When cells were loaded with the cell-permeant form of fura 2, fura 2 acetoxymethyl ester, a caffeine-sensitive membrane-delimited region of elevated [Ca2+]i was seen associated with the microtubule organizing center. A model is proposed relating the distribution of [Ca2+]i and the location of the external stimulus to the generation and interaction of substances within the cell that both simulate and inhibit increases in [Ca2+]i.

Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have not been possible because of their small size, their intrinsic motility, and the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dimensional imaging microscopy and mathematical modeling to determine accurate distributions of a potentiometric fluorescent probe between the cytosol and individual mitochondria inside a living cell. Analysis of this distribution via the Nernst equation permits assignment of potentials to each of the imaged mitochondrial membranes. The mitochondrial membrane potentials are distributed over a narrow range centered at -150 mV within the neurites of differentiated neuroblastoma cells. We find that the membrane potential of a single mitochondrion is generally remarkably stable over times of 40-80 s, but significant fluctuations can occasionally be seen. The motility of individual mitochondria is not directly correlated to membrane potential, but mitochondria do become immobile after prolonged treatment with respiratory inhibitors or uncouplers. Thus, three spatial dimensions, a key physiological parameter, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be readily extended to permit investigations of how mitochondrial function is integrated with other processes in the intact cell.

The Na+/Ca2+ exchanger, driven by a transmembrane Na+ gradient, plays a key role in regulating Ca2+ concentration in many cells. Although the exchanger influences Ca2+ concentration, its activity in smooth muscle appears to be closely coupled to Ca2+ availability from intracellular stores. This linkage might result if the exchanger were positioned close to Ca2+ storage sites within the sarcoplasmic reticulum. To test this hypothesis we have developed methods to assess the relative three-dimensional distribution of proteins involved in Na+/K+ pumping, Na+/Ca2+ exchange, Ca2+ storage within the sarcoplasmic reticulum, and attachment of contractile filaments to the membrane in smooth muscle. Here we report that the Na+/Ca2+ exchanger is largely co-distributed with the Na+/K+ pump on unique regions of the plasma membrane in register with, and close to, calsequestrin-containing regions of the sarcoplasmic reticulum in sites distinct from the sites where contractile filaments attach to the membrane. This molecular organization suggests that the plasma membrane is divided into at least two functional domains, and appear to provide a mechanism for the strong linkage seen in smooth muscle between Na+/K+ pumping and Na+/Ca2+ exchange, and between Na+/Ca2+ exchange and Ca2+ release from the sarcoplasmic reticulum.

beta-Agonists cause an inhibition of contractility and a transient stimulation of Na+/K+ pumping in smooth muscle cells of the stomach from the toad Bufo marinus. To determine if the stimulation of Na+/K+ pumping causes changes in intracellular [Na+] ([Na+]i) that might link Na+ pump stimulation to decrease Ca2+ availability for contraction, [Na+]i was measured in these cells with SBFI, a Na(+)-sensitive fluorescent indicator. Basal [Na+]i was 12.8 +/- 4.2 mM (n = 32) and was uniform throughout the cell. In response to isoproterenol, [Na+]i decreased an average of 7.1 +/- 1.1 mM in 3 sec. Since this decrease in [Na+]i could be completely blocked by inhibition of the Na+ pump, or by blockade of the beta-receptor, [Na+]i reduction is the result of occupation of the beta-receptor by isoproterenol and subsequent stimulation of the Na+ pump. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin mimicked the effect of isoproterenol, indicating that the sequence of events linking beta-receptor occupation to Na+ pump stimulation most likely includes activation of adenylate cyclase, production of cAMP, and stimulation of cAMP-dependent protein kinase. The decrease in [Na+]i is sufficiently large and fast that it is expected to stimulate turnover of the Na+/Ca2+ exchanger in the Ca2+ extrusion mode, thereby accounting for the observed linkage between stimulation of the Na+/K+ pump and inhibition of contractility in response to beta-adrenergic agonists.

A quantitative three-dimensional analysis of nuclear components involved in precursor messenger RNA metabolism was performed with a combination of fluorescence hybridization, immunofluorescence, and digital imaging microscopy. Polyadenylate [poly(A)] RNA-rich transcript domains were discrete, internal nuclear regions that formed a ventrally positioned horizontal array in monolayer cells. A dimmer, sometimes strand-like, poly(A) RNA signal was dispersed throughout the nucleoplasm. Spliceosome assembly factor SC-35 localized within the center of individual domains. These data support a nuclear model in which there is a specific topological arrangement of noncontiguous centers involved in precursor messenger RNA metabolism, from which RNA transport toward the nuclear envelope radiates.

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).

Myosin light chain kinase (MLCK) contains the autoinhibitor sequence right next to the N-terminus side of the calmodulin binding region. In this paper, the structural requirement of the inhibition of MLCK activity was studied using synthetic peptide analogs. Peptides Ala-783-Lys-799 and Ala-783-Arg-798 inhibited calmodulin independent MLCK at the same potency as the peptide Ala-783-Gly-804. Deletion of Arg-797-Lys-799 or substitution of these residues to Ala markedly increased the Ki while the substitution of Lys-792 and Lys-793 to Ala and the deletion of Lys-784-Lys-785 did not affect the inhibitory activity of the peptides. The results suggest that Arg-797-Arg-798 are especially important for the inhibitory activity among other basic residues in the autoinhibitory region.

Digital imaging microscopy has been used to visualize the splicing protein PRP6p and three other yeast nuclear proteins. The results show that PRP6p is uniquely localized to discrete subnuclear regions. A combination of cytological and biochemical assays suggests that these sites can be saturated when the protein is overexpressed and likely correspond to the location of U4/U6 snRNPs. The observations indicate that some splicing components are located in discrete subregions of the yeast nucleus, similar to the situation described for the mammalian nucleus.

Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.