Gates to Gregg High Voltage Transmission Line Study

The usefulness of LANDSAT data in the planning of transmission line routes was assessed. LANDSAT digital data and image processing techniques, specifically a multi-date supervised classification aproach, were used to develop a land cover map for an agricultural area near Fresno, California. Twenty-six land cover classes were identified, of which twenty classes were agricultural crops. High classification accuracies (greater than 80%) were attained for several classes, including cotton, grain, and vineyards. The primary products generated were 1:24,000, 1:100,000 and 1:250,000 scale maps of the classification and acreage summaries for all land cover classes within four alternate transmission line routes

T- and L-type Ca 2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca 2+ currents in smooth muscle cells solated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37°C using the amphotericin B perforated-patch onfiguration of the patch-clamp technique. Currents were recorded using Cs + -rich pipette solutions to block K + currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca 2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a VÎ of _45 ± 5 mV. In contrast, the T current inactivated with a VÎ of _80 ± 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 ± 54 nM) and Ni 2+ (ED50 = 65 ± 16 mM) but were enhanced when external Ca 2+ was substituted with Ba 2+ . The T current was little affected by TTX, reduction in external Na + , application of nifedipine at concentrations below 300 nM or substitution of external Ca 2+ with Ba 2+ , but was reduced by Ni 2+ with an ED50 of 6 ± 1 mM. When cells were stepped from _100 to _30 mV in Ca 2+ -free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg 2+ with an ED50 of 32 ± 16 mM. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca 2+ currents

Contribution of K v 2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra

We have characterized the native voltage-dependent K + (K v ) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K v 2.1 and K v 2.2 channels cloned from the rabbit urethra and stably expressed in HEK 293 cells (HEK Kv2.1 and HEK Kv2.2 ). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch clamp technique with K + -rich pipette solutions. Cells were bathed in 100 nM penitrem A (Pen A) to block large conductance Ca 2+ -activated K + (BK) currents and depolarized to +40 mV for 500 ms to evoke K v currents. These were unaffected by margatoxin, κ-dendrotoxin or α-dendrotoxin (100 nM, n=3-5), but were blocked by stromatoxin-1 (ScTx, IC 50 ~130 nM), consistent with the idea that the currents were carried through K v 2 channels. RNA was detected for K v 2.1 K v 2.2 and the silent subunit K v 9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K v 2 subtypes and K v 9.3 in isolated RUSMC. HEK Kv2.1 and HEK Kv2.2 currents were blocked in a concentration dependent manner by ScTx with estimated IC 50 values of ~150 nM (K v 2.1, n=5) and 70 nM (K v 2.2, n=6). The mean V 1/2 of inactivation of the USMC K v current was – 56±3 mV (n=9). This was similar to the HEK Kv2.1 current (–55 ± 3 mV, n=13) but significantly different from the HEK Kv2.2 currents (-30 ± 3 mV, n=11). Action potentials (AP) evoked from RUSMC studied under current clamp mode were unaffected by ScTx. However when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K v 2.1 channels contribute significantly to the K v current in RUSMC

The effect of high [K(+)]o on spontaneous Ca(2+) waves in freshly isolated interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) act as putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous global Ca(2+) waves that can be increased in frequency by raising external [K(+)]. The purpose of this study was to elucidate the mechanism of this response. Intracellular [Ca(2+)] was measured in fluo-4-loaded smooth muscle cells (SMCs) and ICC using a Nipkow spinning disk confocal microscope. Increasing [K(+)]o to 60 mmol/L caused an increase in [Ca(2+)]i accompanied by contraction in SMCs. Raising [K(+)]o did not cause contraction in ICC, but the frequency of firing of spontaneous calcium waves increased. Reducing [Ca(2+)]o to 0 mmol/L abolished the response in both cell types. Nifedipine of 1 μmol/L blocked the response of SMC to high [K(+)]o, but did not affect the increase in firing in ICC. This latter effect was blocked by 30 μmol/L NiCl2 but not by the T-type Ca(2+) channel blocker mibefradil (300 nmol/L). However, inhibition of Ca(2+) influx via reverse-mode sodium/calcium exchange (NCX) using either 1 μmol/L SEA0400 or 5 μmol/L KB-R7943 did block the effect of high [K(+)]o on ICC. These data suggest that high K(+) solution increases the frequency of calcium waves in ICC by increasing Ca(2+) influx through reverse-mode NCX

Back-illuminated electron multiplying technology: The world's most sensitive CCD for ultra low-light microscopy

ABSTRACT The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera stands to be one the most revolutionary contributions ever to the burgeoning fields of low-light dynamic cellular microscopy and single molecule detection, combining extremely high photon conversion efficiency with the ability to eliminate the readout noise detection limit. Here, we present some preliminary measurements recorded by a very rapid frame rate version of this camera technology, incorporated into a spinning disk confocal microscopy set-up that is used for fast intracellular calcium flux measurements. The results presented demonstrate the united effects of: (a) EMCCD technology in amplifying the very weak signal from these fluorescently labelled cells above the readout noise detection limit, that they would otherwise be completely lost in; (b) back-thinned CCD technology in maximizing the signal/shot noise ratio from such weak photon fluxes. It has also been shown how this innovative development can offer significant signal improvements over that afforded by ICCD technology. Practically, this marked advancement in detector sensitivity affords benefits such as shorter exposure times (therefore faster frame rates), lower dye concentrations and reduced excitation powers and will remove some of the barriers that have been restricting the development of new innovative low-light microscopy techniques

Vasodilation of rat skeletal muscle arteries by the novel BK channel opener GoSlo is mediated by the simultaneous activation of BK and Kv7 channels

BACKGROUND AND PURPOSE: BK channels play important roles in various physiological and pathophysiological processes and thus have been the target of several drug development programs focused on creating new efficacious BK channel openers, such as the GoSlo-SR compounds. However, the effect of GoSlo-SR compounds on vascular smooth muscle has not been studied. Therefore, we tested the hypothesis that GoSlo-SR compounds dilate arteries exclusively by activating BK channels. EXPERIMENTAL APPROACH: Experiments were performed on rat Gracilis muscle, saphenous, mesenteric and tail arteries using isobaric and isometric myography, sharp microelectrodes, digital droplet PCR and the patch-clamp technique. KEY RESULTS: GoSlo-SR compounds dilated isobaric and relaxed and hyperpolarized isometric vessel preparations and their effects were abolished after (i) functionally eliminating K channels by pre-constriction with 50 mM KCl or (ii) blocking all K channels known to be expressed in vascular smooth muscle. However, these effects were not blocked when BK channels were inhibited. Surprisingly, the K(V)7 channel inhibitor XE991 reduced their effects considerably, but neither K(V)1 nor K(V)2 channel blockers altered the inhibitory effects of GoSlo-SR. However, the combined blockade of BK and K(V)7 channels abolished the GoSlo-SR-induced relaxation. GoSlo-SR compounds also activated K(V)7.4 and K(V)7.5 channels expressed in HEK 293 cells. CONCLUSIONS AND IMPLICATIONS: This study shows that GoSlo-SR-compounds are effective relaxants in vascular smooth muscle and mediate their effects by a combined activation of BK and K(V)7.4/K(V)7.5 channels. Activation of K(V)1, K(V)2 or K(V)7.1 channels or other vasodilator pathways seem not to be involved