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1.  NOS Inhibition Enhances Myogenic Tone by Increasing Rho-Kinase Mediated Ca2+ Sensitivity in the Male but Not the Female Gerbil Spiral Modiolar Artery 
PLoS ONE  2013;8(1):e53655.
Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-NG-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca2+ concentration and/or the Ca2+ sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca2+, and Ca2+ sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K+ and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca2+ concentration in smooth muscle cells but increased the Ca2+ sensitivity. This increase in the Ca2+ sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca2+ sensitivity and abolished the LNNA-induced increase in Ca2+ sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca2+ sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca2+ sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.
doi:10.1371/journal.pone.0053655
PMCID: PMC3536759  PMID: 23301097
2.  Reduced Ca2+ spark activity after subarachnoid hemorrhage disables BK channel control of cerebral artery tone 
Intracellular Ca2+ release events (‘Ca2+ sparks') and transient activation of large-conductance Ca2+-activated potassium (BK) channels represent an important vasodilator pathway in the cerebral vasculature. Considering the frequent occurrence of cerebral artery constriction after subarachnoid hemorrhage (SAH), our objective was to determine whether Ca2+ spark and BK channel activity were reduced in cerebral artery myocytes from SAH model rabbits. Using laser scanning confocal microscopy, we observed ∼50% reduction in Ca2+ spark activity, reflecting a decrease in the number of functional Ca2+ spark discharge sites. Patch-clamp electrophysiology showed a similar reduction in Ca2+ spark-induced transient BK currents, without change in BK channel density or single-channel properties. Consistent with a reduction in active Ca2+ spark sites, quantitative real-time PCR and western blotting revealed decreased expression of ryanodine receptor type 2 (RyR-2) and increased expression of the RyR-2-stabilizing protein, FKBP12.6, in the cerebral arteries from SAH animals. Furthermore, inhibitors of Ca2+ sparks (ryanodine) or BK channels (paxilline) constricted arteries from control, but not from SAH animals. This study shows that SAH-induced decreased subcellular Ca2+ signaling events disable BK channel activity, leading to cerebral artery constriction. This phenomenon may contribute to decreased cerebral blood flow and poor outcome after aneurysmal SAH.
doi:10.1038/jcbfm.2010.143
PMCID: PMC3049462  PMID: 20736958
cerebral aneurysm; FKBP12.6; potassium channels; ryanodine receptors; vascular smooth muscle; vasospasm
3.  Gender Differences in Myogenic Regulation along the Vascular Tree of the Gerbil Cochlea 
PLoS ONE  2011;6(9):e25659.
Regulation of cochlear blood flow is critical for hearing due to its exquisite sensitivity to ischemia and oxidative stress. Many forms of hearing loss such as sensorineural hearing loss and presbyacusis may involve or be aggravated by blood flow disorders. Animal experiments and clinical outcomes further suggest that there is a gender preference in hearing loss, with males being more susceptible. Autoregulation of cochlear blood flow has been demonstrated in some animal models in vivo, suggesting that similar to the brain, blood vessels supplying the cochlea have the ability to control flow within normal limits, despite variations in systemic blood pressure. Here, we investigated myogenic regulation in the cochlear blood supply of the Mongolian gerbil, a widely used animal model in hearing research. The cochlear blood supply originates at the basilar artery, followed by the anterior inferior cerebellar artery, and inside the inner ear, by the spiral modiolar artery and the radiating arterioles that supply the capillary beds of the spiral ligament and stria vascularis. Arteries from male and female gerbils were isolated and pressurized using a concentric pipette system. Diameter changes in response to increasing luminal pressures were recorded by laser scanning microscopy. Our results show that cochlear vessels from male and female gerbils exhibit myogenic regulation but with important differences. Whereas in male gerbils, both spiral modiolar arteries and radiating arterioles exhibited pressure-dependent tone, in females, only radiating arterioles had this property. Male spiral modiolar arteries responded more to L-NNA than female spiral modiolar arteries, suggesting that NO-dependent mechanisms play a bigger role in the myogenic regulation of male than female gerbil cochlear vessels.
doi:10.1371/journal.pone.0025659
PMCID: PMC3183064  PMID: 21980520
4.  Calcium sparks in the intact gerbil spiral modiolar artery 
BMC Physiology  2011;11:15.
Background
Calcium sparks are ryanodine receptor mediated transient calcium signals that have been shown to hyperpolarize the membrane potential by activating large conductance calcium activated potassium (BK) channels in vascular smooth muscle cells. Along with voltage-dependent calcium channels, they form a signaling unit that has a vasodilatory influence on vascular diameter and regulation of myogenic tone. The existence and role of calcium sparks has hitherto been unexplored in the spiral modiolar artery, the end artery that controls blood flow to the cochlea. The goal of the present study was to determine the presence and properties of calcium sparks in the intact gerbil spiral modiolar artery.
Results
Calcium sparks were recorded from smooth muscle cells of intact arteries loaded with fluo-4 AM. Calcium sparks occurred with a frequency of 2.6 Hz, a rise time of 17 ms and a time to half-decay of 20 ms. Ryanodine reduced spark frequency within 3 min from 2.6 to 0.6 Hz. Caffeine (1 mM) increased spark frequency from 2.3 to 3.3 Hz and prolonged rise and half-decay times from 17 to 19 ms and from 20 to 23 ms, respectively. Elevation of potassium (3.6 to 37.5 mM), presumably via depolarization, increased spark frequency from 2.4 to 3.2 Hz. Neither ryanodine nor depolarization changed rise or decay times.
Conclusions
This is the first characterization of calcium sparks in smooth muscle cells of the spiral modiolar artery. The results suggest that calcium sparks may regulate the diameter of the spiral modiolar artery and cochlear blood flow.
doi:10.1186/1472-6793-11-15
PMCID: PMC3170618  PMID: 21871098
5.  An epilepsy/dyskinesia-associated mutation enhances BK channel activation by potentiating Ca2+ sensing 
Neuron  2010;66(6):871-883.
Summary
Ca2+-activated BK channels modulate neuronal activities including spike frequency adaptation and synaptic transmission. Previous studies found that Ca2+ binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca2+ binding opens the gate over this distance remains unknown. By studying an Asp to Gly mutation (D434G) associated with human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD), we show that a cytosolic motif immediately following the activation gate S6 helix, known as the AC region, mediates the allosteric coupling between Ca2+ binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca2+ sensitivity is enhanced by increases in solution viscosity that reduce protein dynamics. The GEPD mutation alters such a response, suggesting that a less flexible AC region may be more effective in coupling Ca2+ binding to channel opening.
doi:10.1016/j.neuron.2010.05.009
PMCID: PMC2907746  PMID: 20620873
6.  The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating 
The Journal of General Physiology  2005;126(3):227-241.
Large conductance, voltage- and Ca2+-activated K+ (BKCa) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four α subunits of BKCa may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BKCa gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BKCa activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BKCa channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.
doi:10.1085/jgp.200509321
PMCID: PMC2266574  PMID: 16103277

Results 1-6 (6)