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Mark T Nelson

Publications and source records attributed to Mark T Nelson.

46 records · Page 3Linked to original sources
PubMed2002

Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes.

We examined the role of the Ca(2+)-regulated protein phosphatase calcineurin in controlling Ca(2+) signalling in mouse ventricular myocytes. Membrane currents and voltage were measured in single myocytes using the patch-clamp technique. Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was measured in cells loaded with the fluorescent Ca(2+) indicators fluo-4 or fura-2 using a confocal or epifluorescence microscope. Inhibition of calcineurin with cyclosporin A (CsA, 100 nM) or the calcineurin auto-inhibitory peptide (CiP, 100 microM), increased the amplitude and rate of decay of the evoked [Ca(2+)](i) transient and also prolonged the action potential (AP) of ventricular myocytes to a similar extent. The effects of CsA (100 nM) and 100 microM CiP on the [Ca(2+)](i) transient and AP were not additive. Calcineurin inhibition did not modify the K(+) currents responsible for repolarisation of the mouse ventricle. Instead, inhibition of calcineurin increased the amplitude of the Ca(2+) current (I(Ca)) and the evoked calcium transient normalized to the I(Ca). Calcium sparks, which underlie the [Ca(2+)](i) transient, had a higher frequency and amplitude, suggesting an elevation of SR calcium load. Inhibition of protein kinase A (PKA) prevented the effects of calcineurin inhibition, indicating that calcineurin opposes the actions of PKA. Finally, immunofluorescence images suggest that calcineurin and PKA co-localize near the T-tubules of ventricular myocytes. We propose that calcineurin and PKA are co-localized to control Ca(2+) influx through calcium channels and calcium release through ryanodine receptors.

Action Potentials↗
PubMed2002

Opposing actions of inositol 1,4,5-trisphosphate and ryanodine receptors on nuclear factor of activated T-cells regulation in smooth muscle.

The nuclear factor of activated T-cells (NFAT), originally identified in T-cells, has since been shown to play a role in mediating Ca(2+)-dependent gene transcription in diverse cell types outside of the immune system. We have previously shown that nuclear accumulation of NFATc3 is induced in ileal smooth muscle by platelet-derived growth factor in a manner that depends on Ca(2+) influx through L-type, voltage-dependent Ca(2+) channels. Here we show that NFATc3 is also the predominant NFAT isoform expressed in cerebral artery smooth muscle and is induced to accumulate in the nucleus by UTP and other G(q/11)-coupled receptor agonists. This induction is mediated by calcineurin and is dependent on sarcoplasmic reticulum Ca(2+) release through inositol 1,4,5-trisphosphate receptors and extracellular Ca(2+) influx through L-type, voltage-dependent Ca(2+) channels. Consistent with results obtained in ileal smooth muscle, depolarization-induced Ca(2+) influx fails to induce NFAT nuclear accumulation in cerebral arteries. We also provide evidence that Ca(2+) release by ryanodine receptors in the form of Ca(2+) sparks may exert an inhibitory influence on UTP-induced NFATc3 nuclear accumulation and further suggest that UTP may act, in part, by inhibiting Ca(2+) sparks. These results are consistent with a multifactorial regulation of NFAT nuclear accumulation in smooth muscle that is likely to involve several intracellular signaling pathways, including local effects of sarcoplasmic reticulum Ca(2+) release and effects attributable to global elevations in intracellular Ca(2+).

Active Transport, Cell Nucleus↗
PubMed2002

Differential regulation of SK and BK channels by Ca(2+) signals from Ca(2+) channels and ryanodine receptors in guinea-pig urinary bladder myocytes.

Small-conductance (SK) and large-conductance (BK) Ca(2+)-activated K(+) channels are key regulators of excitability in urinary bladder smooth muscle (UBSM) of guinea-pig. The overall goal of this study was to define how SK and BK channels respond to Ca(2+) signals from voltage-dependent Ca(2+) channels (VDCCs) in the surface membrane and from ryanodine-sensitive Ca(2+) release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) membrane. To characterize the role of SK channels in UBSM, the effects of the SK channel blocker apamin on phasic contractions were examined. Apamin caused a dose-dependent increase in the amplitude of phasic contractions over a broad concentration range (10(-10) to 10(-6) M). To determine the effects of Ca(2+) signals from VDCCs and RyRs to SK and BK channels, whole cell membrane current was measured in isolated myocytes bathed in physiological solutions. Depolarization (-70 to +10 mV for 100 ms) of isolated myocytes caused an inward Ca(2+) current (I(Ca)), followed by an outward current. The outward current was reduced in a dose-dependent manner by apamin (10(-10) to 10(-6) M), and designated I(SK). I(SK) had a mean amplitude of 53.8 +/- 6.1 pA or approximately 1.4 pA pF(-1) at +10 mV. The amplitude of I(SK) correlated with the peak I(Ca). Blocking I(Ca) abolished I(SK). In contrast, I(SK) was insensitive to the RyR blocker ryanodine (10 microM). These data indicate that Ca(2+) signals from VDCCs, but not from RyRs, activate SK channels. BK channel currents (I(BK)) were isolated from other currents by using the BK channel blockers tetraethylammonium ions (TEA(+); 1 mM) or iberiotoxin (200 nM). Voltage steps evoked transient and steady-state I(BK) components. Transient BK currents have previously been shown to result from BK channel activation by local Ca(2+) release through RyRs ('Ca(2+) sparks'). Transient BK currents were inhibited by ryanodine (10 microM), as expected, and had a mean amplitude of 152.6 pA at +10 mV. The mean number of transient BK currents during a voltage step (range 0 to 3) correlated with I(Ca). There was a long delay (52.4 +/- 2.7 ms) between activation of I(Ca) and the first transient BK current. In contrast, ryanodine did not affect the steady-state BK current (mean amplitude 135.4 pA) during the voltage step. The steady-state BK current was reduced 95 % by inhibition of VDCCs, suggesting that this process depends largely on Ca(2+) entry through VDCCs and not Ca(2+) release through RyRs. These results indicate that Ca(2+) entry through VDCCs activates both BK and SK channels, but Ca(2+) release (Ca(2+) sparks) through RyRs activates only BK channels.

Algorithms↗
PubMed2002

Transient receptor potential channels regulate myogenic tone of resistance arteries.

Elevation of intravascular pressure causes depolarization and constriction (myogenic tone) of small arteries and arterioles, and this response is a key element in blood flow regulation. However, the nature of pressure-induced depolarization has remained elusive. In the present study, we provide evidence that a transient receptor potential channel (TRPC6) homologue has a major role in this depolarizing response to pressure. Antisense oligodeoxynucleotides to TRPC6 decreased TRPC6 protein expression and greatly attenuated arterial smooth muscle depolarization and constriction caused by elevated pressure in intact cerebral arteries. Suppressing the expression of this channel protein also reduced the current density of a major cation current in resistance artery smooth muscle cells. We propose that TRPC6 channels play an essential role in regulation of myogenic tone.

Animals↗
PubMed2002

Ca(2+) sparks and BK currents in gallbladder myocytes: role in CCK-induced response.

We sought to elucidate the regulation of gallbladder smooth muscle (GBSM) excitability by localized Ca(2+) release events (sparks) and large-conductance Ca(2+)-dependent (BK) channels by determining whether sparks exist in GBSM and, if so, whether they activate BK channels. Sparks were identified in isolated GBSM loaded with fluo 4. Each spark was associated with a transient outward current, suggesting communication of ryanodine receptor (RyR) channels with BK channels. This was confirmed by the inhibition of outward currents with iberiotoxin (100 nM), thapsigargin (200 nM), and ryanodine (10 microM). In current clamp mode, the transient BK currents were associated with brief membrane hyperpolarizations (10.9 +/- 1.3 mV). Because transient BK currents could dampen GBSM excitability, we tested whether CCK attenuates these events. CCK (10 nM) reduced the amplitude and frequency of transient BK currents, and subsequent caffeine application restored transient BK current activity. These results support the concept that RyRs and BK channels contribute to the regulation of GBSM excitability and that CCK can act in part by inhibiting this pathway.

Aniline Compounds↗
PubMed2002

Alkaline pH shifts Ca2+ sparks to Ca2+ waves in smooth muscle cells of pressurized cerebral arteries.

The effects of external pH (7.0-8.0) on intracellular Ca(2+) signals (Ca(2+) sparks and Ca(2+) waves) were examined in smooth muscle cells from intact pressurized arteries from rats. Elevating the external pH from 7.4 to 7.5 increased the frequency of local, Ca(2+) transients, or "Ca(2+) sparks," and, at pH 7.6, significantly increased the frequency of Ca(2+) waves. Alkaline pH-induced Ca(2+) waves were inhibited by blocking Ca(2+) release from ryanodine receptors but were not prevented by inhibitors of voltage-dependent Ca(2+) channels, phospholipase C, or inositol 1,4,5-trisphosphate receptors. Activating ryanodine receptors with caffeine (5 mM) at pH 7.4 also induced repetitive Ca(2+) waves. Alkalization from pH 7.4 to pH 7.8-8.0 induced a rapid and large vasoconstriction. Approximately 82% of the alkaline pH-induced vasoconstriction was reversed by inhibitors of voltage-dependent Ca(2+) channels. The remaining constriction was reversed by inhibition of ryanodine receptors. These findings indicate that alkaline pH-induced Ca(2+) waves originate from ryanodine receptors and make a minor, direct contribution to alkaline pH-induced vasoconstriction.

Alkalies↗
PubMed2002

Ca2+ sparks and their function in human cerebral arteries.

BACKGROUND AND PURPOSE: Local Ca2+ release events (Ca2+ sparks) caused by the opening of ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum have been suggested to oppose constriction in cerebral arteries through the activation of large-conductance Ca2+-activated K+ (BK) channels. We report the first identification and characterization of Ca2+ sparks and associated BK channel currents in smooth muscle cells isolated from human cerebral arteries. METHODS: Membrane currents and intracellular Ca2+ were measured with the use of the patch-clamp technique and laser scanning confocal microscopy. RESULTS: Ca2+ sparks with a peak fractional fluorescence change (F/F0) of 2.02 +/- 0.04 and size of 8.2 +/- 0.5 microm2 (n=108) occurred at a frequency of approximately 1 Hz in freshly isolated, cerebral artery myocytes from humans. At a holding potential of -40 mV, the majority of, but not all, Ca2+ sparks (61 of 85 sparks) were associated with transient BK currents. Consistent with a role for Ca2+ sparks in the control of cerebral artery diameter, agents that block Ca2+ sparks (ryanodine) or BK channels (iberiotoxin) were found to contract human cerebral arteries. CONCLUSIONS: This study provides evidence for local Ca2+ signaling in human arterial myocytes and suggests that these events may play an important role in control of cerebral artery diameter in humans.

Adult↗
PubMed2002

Sarcoplasmic reticulum and membrane currents.

Local and global Ca2+ signals from voltage-dependent Ca2+ channels (VDCCs) and ryanodine-sensitive Ca2+ release (RyRs) channels in the sarcoplasmic reticulum (SR) encode information to different Ca2+-sensitive targets including the large- (BK) and small-conductance (SK) Ca2+-activated K+ channels in the surface membrane. In smooth muscle, unlike cardiac muscle, Ca2+ signalling to RyRs is not local, exhibiting a significant lag between VDCC activation and subsequent RyR stimulation, measured as Ca2+ sparks and associated BK currents. However, Ca2+ signalling from RyRs (Ca2+ sparks) to BK channels appears to be local in arterial (ASM) and urinary bladder smooth muscle (UBSM), consistent with a close proximity of SR RyRs to BK channels. The response of BK channels in ASM and UBSM depends on the tuning of the Ca2+/voltage sensitivity of the BK channel by its accessory subunit, the beta1 subunit. UBSM, in contrast to ASM, has both BK and SK channels. SK channels in UBSM are solely activated by Ca2+ signals from VDCCs, whereas BK channels are activated by Ca2+ from both VDCCs and RyRs. The differential regulation of BK and SK channels by Ca2+ signals underlies their roles in regulating action potential duration and membrane potential (BK channels) and after-hyperpolarizations (SK channels) in smooth muscle.

Animals↗