Line-scanning Confocal Microscopy Research Articles

Long-Term Intrauterine Hypoxia Disrupts Calcium Sparks in Middle Cerebral Arterial Myocytes of Fetal Sheep

Local activation of ryanodine receptors (RyRs) on the sarcoplasmic reticulum in vascular smooth muscle cells (VSMCs) generates rapid subcellular changes in cellular Ca2+ that are commonly known as Ca2+ sparks. These Ca2+ sparks are well regarded for their role in the autoregulation of vascular contractility and cerebral blood flow through activation of potassium channels and feedback modulation of the membrane potential. Gestational hypoxia disrupts cerebrovascular development, increasing the risk of intracranial hemorrhage and stroke in the newborn. Due to the role of RyR’s in regulating neonatal cerebrovascular blood flow, understanding intracellular Ca2+ release patterns in VSMCs can offer insight into hypoxia-related perinatal cerebrovascular disease. The central aim of this study is to determine the extent to which gestational hypoxia disrupts RyR activity. This was achieved through examination of RyR activity in VSMCs of fetal ovine middle cerebral arteries (MCAs). MCAs were isolated from term fetal sheep (~140 days of gestation) delivered by cesarean section from ewes held at low- (700 m) and high-altitude conditions (3801 m) for 100+ days of gestation. Arteries were bathed in a physiological buffer with 5 mm K+ (control) or were treated with either 30 mM K+ (30K), to depolarize myocytes and increase Ca2+ spark events, in the presence or absence of 10 μM ryanodine, to block RyR mediated Ca2+ release. Ca2+ spark activity was measured in situ using line scan confocal microscopy fluorescence imaging techniques. Automated analysis of Ca2+ sparks in the records was conducted using SparkLab 5.8. Membrane depolarization with 30K increased Ca2+ spark activity. Long term hypoxia, however, reduced spark activity in the 30K group. Ryanodine attenuated Ca2+ spark activity in both low- and high-altitude groups. Hypoxia’s influence on the spatial and temporal aspects of Ca2+ sparks in fetal myocytes varied. These findings corroborate previous evidence demonstrating that long-term hypoxia attenuates Ca2+ spark activity. The disruption in Ca2+ spark activity could impact brain blood flow in the neonate, potentially impacting brain development and the likelihood of a traumatic event. R01HL155295, R01HL149608, Advanced Imaging and Microscopy Core Loma Linda University School of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low-cost, minimalistic line-scanning confocal microscopy

Confocal microscopy provides optical sectioning that is invaluable for many applications, most notably imaging into thick samples. However, the high cost of commercial confocal microscopes limits uses to specialized research and clinical settings. We present a minimalistic line-scanning confocal microscope costing less than $6,500 with optical performance comparable to a commercial laser scanning system. The optical sectioning and imaging performance are shown through measurement of the axial line-spread function and imaging of biological samples of varying thickness. Comparison is made to commercial widefield and confocal microscopes. The low cost of goods and optical sectioning capability of this microscope will allow the use of confocal microscopy in additional research and educational settings.

Fast volumetric imaging with line-scan confocal microscopy by electrically tunable lens at resonant frequency.

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and usually suffers from a reduced acquisition rate, which is critical to properly document rapid neurophysiological dynamics. In this study, we implemented an electrically tunable lens (ETL) in the line-scan confocal microscopy (LSCM), enabling the volumetric acquisition at the rate of 20 frames per second with a maximum volume of interest of 315 × 315 × 80 µm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 µm and 90.4 ± 2.1 µm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth axial penetration by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of both cleared mouse brain ex vivo samples and in vivo brains. The current study showed a successful application of resonant ETL for constructing a high-performance 3D axially scanning LSCM (asLSCM) system. Such advances in rapid volumetric imaging would significantly enhance our understanding of various dynamic biological processes.

Confocal laser speckle autocorrelation imaging of dynamic flow in microvasculature

Laser speckle imaging has been widely used for in-vivo visualization of blood perfusion in biological tissues. However, existing laser speckle imaging techniques suffer from limited quantification accuracy and spatial resolution. Here we report a novel design and implementation of a powerful laser speckle imaging platform to solve the two critical limitations. The core technique of our platform is a combination of line scan confocal microscopy with laser speckle autocorrelation imaging, which is termed Line Scan Laser Speckle Autocorrelation Imaging (LS-LSAI). The technical advantages of LS-LSAI include high spatial resolution (~4.4 μm) for visualizing and quantifying blood flow in microvessels, as well as video-rate imaging speed for tracing dynamic flow.

Fast, multiplane line-scan confocal microscopy using axially distributed slits.

The inherent constraints on resolution, speed and field of view have hindered the development of high-speed, three-dimensional microscopy techniques over large scales. Here, we present a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples.

Large field of view line-scanning confocal holographic microscopy

Large field of view line-scanning confocal holographic microscopy

Optimizing the performance of multiline-scanning confocal microscopy

Line-scanning (LS) confocal microscopy provides high imaging speed and moderate optical sectioning strength, which makes it a useful tool for imaging various biospecimens ranging from living cells to fixed tissues. Conventional LS systems have only used a single excitation line and slit, and thus have not fully exploited the benefits of parallelization. Here we investigate the optical performance of multi-LS confocal microscopy (mLS) by employing a digital micro-mirror device that provides programmable patterns of the illumination beam and the detection slit. Through experimental results and optical simulations, we assess the depth discrimination of mLS under different optical parameters and compare it with multi-point systems such as spinning disk confocal microscopy (SDCM). Under the same illumination duty cycle, we find that mLS has better optical sectioning than SDCM at a high degree of parallelization. The optimized mLS provides a low photobleaching rate and video-rate imaging while its optical sectioning is similar to single LS confocal microscopy.

Low-photobleaching line-scanning confocal microscopy using dual inclined beams.

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line-scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2-fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single-molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three-dimensional single-molecule RNA imaging in mammalian cells.

Measurements of Triadic Calcium in Differentiated Muscle Fibers using a Gcamp Probe Targeted to the Junctional Sr Membrane

With the aim of gaining further insights into sarcoplasmic reticulum (SR) Ca2+ release function in skeletal muscle, we used a domain consisting of the 306 residues (T306) of the N-terminal end of triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane of muscle fibers. Muscles from mouse were electroporated with plasmid constructs encoding GCaMP6f or T306-GCaMP6f. Muscle fibers were isolated 1-2 weeks later and voltage-activated fluorescence changes were studied with line-scan confocal microscopy under voltage-clamp control. GCaMP6f fluorescence was homogenous throughout the transfected fibers and experienced large changes upon membrane depolarization in the absence of exogenous Ca2+ buffer in the intracellular solution (e.g. mean relative change of ∼20 in response to a 0.5 s-long pulse from −80 to −15 mV). When compared with simultaneously measured rhod-2 Ca2+ transients, GCaMP6f transients showed obvious signs of kinetic delays with, for instance, the time constant of decay after the end of short depolarizing pulses being 4-5 times that of the rhod-2 transients. In contrast, T306-GCaMP6f expression was localized to the vicinity of one or of a few nuclei. Within these spots, fluorescence yielded a transverse banded pattern consistent with triadic localization. Upon membrane depolarization, T306-GCaMP6f generated fluorescence transients of faster kinetics and much lower relative amplitude than T306-free GCaMP6f (e.g. mean relative change of 3.2 in response to a 0.5 s-long pulse to −15 mV), suggesting reduced affinity and faster kinetics of the SR membrane-bound probe as compared to the free probe. In several fibers, voltage-induced fluorescence changes at different positions of the scanned line showed time-course discrepancies which may reflect distinct behaviors of ryanodine receptor activation. Supported by CNRS, INSERM, Université Lyon 1 and AFM-Téléthon MyoNeurAlp 2.3.1.3.

Superresolution fluorescence microscopy for 3D reconstruction of thick samples

Three-dimensional (3D) reconstruction of thick samples using superresolution fluorescence microscopy remains challenging due to high level of background noise and fast photobleaching of fluorescence probes. We develop superresolution fluorescence microscopy that can reconstruct 3D structures of thick samples with both high localization accuracy and no photobleaching problem. The background noise is reduced by optically sectioning the sample using line-scan confocal microscopy, and the photobleaching problem is overcome by using the DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography). As demonstrations, we take 3D superresolution images of microtubules of a whole cell, and two-color 3D images of microtubules and mitochondria. We also present superresolution images of chemical synapse of a mouse brain section at different z-positions ranging from 0 μm to 100 μm.

Enhanced nucleoplasmic Ca2 + signaling in ventricular myocytes from young hypertensive rats

Arterial hypertension causes left ventricular (LV) myocyte hypertrophy. Alterations in nuclear Ca2+ may be involved in regulation of histone acetylation, transcription and hypertrophy. Regulation of nuclear Ca2+ in hypertension, however, is unknown. Therefore, we elucidated cellular mechanisms underlying nuclear Ca2+ regulation in LV myocytes from hypertensive versus normotensive rats and evaluated possible consequences for Ca2+-dependent regulation of histone acetylation. LV myocytes and myocyte nuclei were isolated from young spontaneously hypertensive rats (SHR) shortly after development of hypertension. Normotensive Wistar-Kyoto rats (WKY) served as controls. Cytoplasmic and nucleoplasmic Ca2+ transients (CaTs) were imaged simultaneously using linescan confocal microscopy and Fluo-4. LV myocytes and nuclei from SHR exhibited hypertrophy. Cytoplasmic and nucleoplasmic CaTs were increased in SHR. The increase in nucleoplasmic Ca2+, however, exceeded the increase in cytoplasmic Ca2+, indicating enhanced nuclear Ca2+ signaling in SHR. Ca2+ load of sarcoplasmic reticulum and perinuclear Ca2+ stores was also increased in SHR, while fractional release from both stores remained unchanged. Intranuclear Ca2+ propagation was accelerated in SHR, associated with preserved density of nuclear envelope invaginations and elevated nuclear expression of nucleoporins and SR-Ca2+-ATPase, SERCA2a. Nuclear Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) expression was elevated and histone deacetylases exhibited redistribution from nucleus to cytosol associated with increased histone acetylation in SHR. Thus, in early hypertension, there is remodeling of nuclear Ca2+ handling resulting in enhanced nuclear Ca2+ signaling. Enhanced nuclear Ca2+ signaling, in turn, increases nuclear localization and activity of CaMKIIδ driving nuclear export of histone deacetylases and increased histone acetylation.

Ischemia–reperfusion destabilizes rhythmicity in immature atrioventricular pacemakers: A predisposing factor for postoperative arrhythmias in neonate rabbits

Postoperative arrhythmias such as junctional ectopic tachycardia and atrioventricular block are serious postoperative complications for children with congenital heart disease. We hypothesize that ischemia-reperfusion (I/R) related changes exacerbate these postoperative arrhythmias in the neonate heart and administration of postoperative inotropes is contributory. The purpose of this study was to study the effects of I/R and postischemic dopamine application on automaticity and rhythmicity in immature and mature pacemaker cells and whole heart preparations. Single pacemaker cells and whole heart models of postoperative arrhythmias were generated in a rabbit model encompassing 3 primary risk factors: age, I/R exposure, and dopamine application. Single cells were studied using current clamp and line scan confocal microscopy, whereas whole hearts were studied using optical mapping. Four responses were observed in neonatal atrioventricular nodal cells (AVNCs): slowing of AVNC automaticity (from 62±10 to 36 ± 12 action potentials per minute, P<.05); induction of arrhythmicity or increased beat-to-beat variability (0.08 ± 0.04 to 3.83 ± 1.79, P<.05); altered automaticity (subthreshold electrical fluctuations); and disruption of calcium transients. In contrast, these responses were not observed in mature AVNCs or neonatal sinoatrial cells. In whole heart experiments, neonatal hearts experienced persistent postischemia arrhythmias of varying severity, whereas mature hearts exhibited no arrhythmias or relatively transient ones. Neonatal pacemaker cells and whole hearts demonstrate a susceptibility to I/R insults resulting in alterations in automaticity, which may predispose neonates to postoperative arrhythmias such as junctional ectopic tachycardia and atrioventricular block.

Line-scanning fiber bundle endomicroscopy with a virtual detector slit.

Coherent fiber bundles can be used to relay the image plane from the distal tip of an endomicroscope to an external confocal microscopy system. The frame rate is therefore determined by the speed of the microscope's laser scanning system which, at 10-20 Hz, may be undesirably low for in vivo clinical applications. Line-scanning allows an increase in the frame rate by an order of magnitude in exchange for some loss of optical sectioning, but the width of the detector slit cannot easily be adapted to suit different imaging conditions. The rolling shutter of a CMOS camera can be used as a virtual detector slit for a bench-top line-scanning confocal microscope, and here we extend this idea to endomicroscopy. By synchronizing the camera rolling shutter with a scanning laser line we achieve confocal imaging with an electronically variable detector slit. This architecture allows us to acquire every other frame with the detector slit offset by a known distance, and we show that subtracting this second image leads to improved optical sectioning.

High-speed line-field confocal holographic microscope for quantitative phase imaging.

We present a high-speed and phase-sensitive reflectance line-scanning confocal holographic microscope (LCHM). We achieved rapid confocal imaging using a fast line-scan CCD camera and quantitative phase imaging using off-axis digital holography (DH) on a 1D, line-by-line basis in our prototype experiment. Using a 20 kHz line scan rate, we achieved a frame rate of 20 Hz for 512x512 pixels en-face confocal images. We realized coherent holographic detection two different ways. We first present a LCHM using off-axis configuration. By using a microscope objective of a NA 0.65, we achieved axial and lateral resolution of ~3.5 micrometers and ~0.8 micrometers, respectively. We demonstrated surface profile measurement of a phase target at nanometer precision and the digital refocusing of a defocused confocal en-face image. Ultrahigh temporal resolution M mode is demonstrated by measuring the vibration of a PZT-actuated mirror driven by a sine wave at 1 kHz. We then report our experimental work on a LCHM using an in-line configuration. In this in-line LCHM, the coherent detection is enabled by moving the reference arm at a constant speed, thereby introducing a Doppler frequency shift that leads to spatial interference fringes along the scanning direction. Lastly, we present a unified formulation that treats off-axis and in-line LCHM in a unified joint spatiotemporal modulation framework and provide a connection between LCHM and the traditional off-axis DH. The presented high-speed LCHM may find applications in optical metrology and biomedical imaging.

Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents.

Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4 : Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic.

Spatial heterodyne scanning laser confocal holographic microscopy

Scanning laser confocal holographic microscopy using a spatial heterodyne detection method is presented. Spatial heterodyne detection technique employs a Mach-Zehnder interferometer with the reference beam frequency shifted by two acousto-optic modulators (AOM) relative to the object beam frequency. Different from the traditional temporal heterodyne detection technique in which hundreds temporal samples are taken at each scanning point to achieve the complex signal, the spatial heterodyne detection technique generates spatial interference fringes by use of a linear tempo-spatial relation provided by galvanometer scanning in a typical line-scanning confocal microscope or for the slow-scanning on one dimension in a point-scanning confocal microscope, thereby significantly reducing sampling rate and increasing the signal to noise ratio under the same illumination compared to the traditional temporal heterodyne counterpart. The proposed spatial heterodyne detection scheme applies to both line-scanning and point-scanning confocal microscopes. In this paper, we present the mathematical principles of the spatial heterodyne detection method and experimental schemes for both line-scanning and point-scanning confocal microscopes. Computer experiments are provided to demonstrate the validity of this idea. The presented spatial heterodyne scanning laser confocal holographic microscope (SH-SLCHM) can obtain both amplitude and quantitative phase information of the object field without a significant increase in complexity of optical and electronic design on the basis of a traditional scanning laser confocal microscope (SLCM). SH-SLCHM may find applications in optical metrology, in-vivo tissue imaging, and ophthalmic imaging.

Spatially Localized Disruptions of Voltage Activated Calcium Release in Mtm1-Deficient Muscle Fibers

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin (Mtm1) are responsible for myotubular myopathy. We previously showed that muscle fibers from Mtm1-deficient mouse suffer from defective excitation-contraction coupling. Here we measured voltage-clamp activated Ca2+ signals in fibers from the flexor digitorum brevis muscles of 4 week-old wild type (WT) and Mtm1-ko mice under line-scan confocal microscopy using the dye rhod-2. Ca2+ release in the diseased fibers was deficient over the full range of activation: fitting the voltage dependence of the peak rate of rise of the line-averaged rhod-2 F/F0 signals with a Boltzmann function gave mean values for maximum rate, midpoint voltage and slope of 0.29 ± 0.02 and 0.14 ± 0.02 F/F0.ms−1, −8.8 ± 1.5 and 0.95 ± 2.3 mV, 7.0 ± 0.4 and 9.2 ± 0.7 mV in WT (n=22) and Mtm1-ko fibers (n=15), respectively. Furthermore, the mean time to peak rate was significantly delayed by 5-10 ms in Mtm1-ko as compared to WT fibers. These global alterations were associated with severe spatial inhomogeneity of Ca2+ release in the diseased fibers with rhod-2 transients yielding localized disruptions along the scanned line including reduced peak amplitude but also delayed or slower rate of onset, suggestive of specific alteration of the early peak component of the rate of Ca2+ release. In fibers treated with wortmannin and LY294002, the properties of the line-averaged rhod-2 F/F0 signals were unchanged in WT fibers but the mean maximum rate of rise of the rhod-2 signal was enhanced by 60% in Mtm1-deficient fibers. Results show that Mtm1-deficiency provokes EC coupling failure through accumulation of spatially localized disruptions of Ca2+ release. They also suggest that pharmacological inhibition of PtdIns-3-kinase activity has the potency to alleviate defective EC coupling in the diseased fibers.

Simultaneous Detection and Colocalization of Calcium Sparks and Ryanodine Receptor Clusters in Cardiac Myocytes

Further understanding of calcium handling and excitation-contraction coupling in cardiac myocytes requires quantitative data analysis methods to characterize calcium release events in terms of structural properties of the cell. Such automatic methods provide a robust, consistent and reproducible characterization of physiological systems from observed experimental data. We present a multilevel analysis that simultaneously focuses on both the occurrence of spontaneous calcium sparks and the distribution of RyR clusters across a cardiac myocyte. The approach localizes the release events and determines the distance to the nearest ryanodine receptor cluster, therefore providing quantitative information on the spatio-temporal distribution of activation sites in the cell. The location of clusters takes into account motion artifacts produced by calcium waves or mini-waves. The method has been validated with line-scan confocal microscopy data from mouse ventricular myocytes and provides a full characterization of the spark morphology including its amplitude, baseline, decay time, upstroke time, Full-Width at Half-Maximum, Full-Duration at Half-Maximum and background noise.The processing is applied to linescan images of both RyR clusters and calcium fluorescence. It includes the following steps: i) Adaptive filtering of background fluorescence fluctuations using a robust estimation of the noise by means of the median absolute deviation of the basal fluorescence signal. ii) Individual sparks are detected by using a modified watershed segmentation method that includes stopping rules for both event size and shape. iii) Location of RyR clusters was implemented by thresholding a continuous wavelet transform of the cluster image (robust to motion and contraction). iV) Measurement of spark morphology properties, including the distance from each spark to the closest RyR cluster.

Abstract 17699: Inositol-1,4,5-Trisphosphate-Mediated Pacemaker Activity in NCX KO Mouse Sinoatrial Nodal Cells

Sinoatrial node (SAN) automaticity is due to the interplay of several membrane currents, including the current produced by Na-Ca exchanger (NCX) in response to Ca cycling. Several lines of evidence suggest that inositol-1,4,5-triphosphate receptors (IP 3 Rs) that mobilize [Ca] i are implicated in generation of automaticity in embryonic and adult cardiomyocytes. Increased IP 3 R expression observed in failing heart may predispose to arrhythmias and could contribute to the elevated heart rates associated with systolic heart failure. However, whether IP 3 R signaling influences SAN pacemaking is still controversial, in part due to the confounding influence of periodic Ca flux through the sarcolemma during every beat. To address this concern, we used NCX knockout (KO) SAN cells to study the role of IP 3 signaling on pacemaker activity. In these cells Ca flux across the sarcolemmal membrane is practically eliminated due to NCX ablation and no Ca flux through L-type Ca channels due to lack of action potentials, yet periodic spontaneous waves are still generated by the localized Ca releases (LCRs) of the “Ca clock”. Thus these waves are “uncoupled” from the membrane because there is no NCX to contribute to the diastolic depolarization in response to the release of [Ca] i . We recorded spontaneous Ca oscillations using line scan confocal microscopy in WT and NCX KO SAN cells in the presence of an IP 3 R blocker or during inhibition of phospholipase C (PLC). We found that superfusion with the IP 3 R blocker, 2-APB (2 μm) decreased the frequency of Ca transients in WT SAN cells by 82.6% (n=9, p&lt;0.05) and Ca waves in NCX KO cells by 66% (n=10, p&lt;0.05). Similar results were also obtained on superfusion with the PLC antagonist, U73122 (1 μm). Alternatively, an increase in IP 3 production using the α-1 adrenergic receptor agonist phenylephrine (10 μm) led to an increase in the frequency of Ca transients in WT SAN cells (n=8. P&lt; 0.05) and Ca waves in NCX KO cells (n=9, p &lt;0.05). This effect was blocked on the subsequent additional application of 2-APB. In summary our results indicate that Ca release from IP 3 Rs can modulate Ca oscillation frequency in NCX KO SAN cells and support our hypothesis that IP 3 signaling modulates the Ca cycling processes that regulate pacemaker frequency in the SAN.

Constitutive PKA activity is essential for maintaining the excitability and contractility in guinea pig urinary bladder smooth muscle: role of the BK channel.

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.