Peripheral venous congestion causes time- and dose-dependent release of endothelin-1 in humans.

Endothelin-1 (ET-1) is a pivotal mediator of vasoconstriction and inflammation in congestive states such as heart failure (HF) and chronic kidney disease (CKD). Whether peripheral venous congestion (VC) increases plasma ET-1 at pressures commonly seen in HF and CKD patients is unknown. We seek to characterize whether peripheral VC promotes time- and dose-dependent increases in plasma ET-1 and whether these changes are sustained after decongestion. We used a randomized, cross-over design in 20 healthy subjects (age 30 ± 7 years). To experimentally model VC, venous pressure was increased to either 15 or 30 mmHg (randomized at first visit) above baseline by inflating a cuff around the subject's dominant arm; the nondominant arm served as a noncongested control. We measured plasma ET-1 at baseline, after 20, 60 and 120 min of VC, and finally at 180 min (60 min after cuff release and decongestion). Plasma ET-1 progressively and significantly increased over 120 min in the congested arm relative to the control arm and to baseline values. This effect was dose-dependent: ET-1 increased by 45% and 100% at VC doses of 15 and 30 mmHg, respectively (P < 0.05), and declined after 60 min of decongestion though remaining significantly elevated compared to baseline. In summary, peripheral VC causes time- and dose-dependent increases in plasma ET-1. Of note, the lower dose of 15 mmHg (more clinically relevant to HF and CKD patients) was sufficient to raise ET-1. These findings support the potentially contributory, not merely consequential, role of VC in the pathophysiology of HF and CKD.

Orientations and proximities of the extracellular ends of transmembrane helices S0 and S4 in open and closed BK potassium channels.

The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory ß1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in ß1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the ß1 subunit than in its absence, consistent with ß1 acting through S0 to stabilize its immobilization relative to α S4.

Positions of ß2 and ß3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.

Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory ß subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in ß2 and ß3, as in ß1 and ß4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse ß3 (mß3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mß3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in ß1. These are conserved in ß2-ß4, which have four additional Cys (eight in total), except that mß3 has one fewer. In ß1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mß3, the free Cys is at position 7; position 2 lacks a Cys present in all other ß2-ß4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other ß2-ß4. Cross-linking of mß3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mß3 perturbed. Although the mß3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.

Reduced vascular smooth muscle BK channel current underlies heart failure-induced vasoconstriction in mice.

Excessively increased peripheral vasoconstriction is a hallmark of heart failure (HF). Here, we show that in mice with systolic HF post-myocardial infarction, the myogenic tone of third-order mesenteric resistance vessels is increased, the vascular smooth muscle (VSM) membrane potential is depolarized by ~20 mV, and vessel wall intracellular [Ca(2+)] is elevated relative to that in sham-operated control mice. Despite the increased [Ca(2+)], the frequency and amplitude of spontaneous transient outward currents (STOCs), mediated by large conductance, Ca(2+)-activated BK channels, were reduced by nearly 80% (P<0.01) and 25% (P<0.05), respectively, in HF. The expression of the BK α and ß1 subunits was reduced in HF mice compared to controls (65 and 82% lower, respectively, P<0.01). Consistent with the importance of a reduction in BK channel expression and function in mediating the HF-induced increase in myogenic tone are two further findings: a blunting of paxilline-induced increase in myogenic tone in HF mice compared to controls (0.9 vs. 10.9%, respectively), and that HF does not alter the increased myogenic tone of BK ß1-null mice. These findings identify electrical dysregulation within VSM, specifically the reduction of BK currents, as a key molecular mechanism sensitizing resistance vessels to pressure-induced vasoconstriction in systolic HF.

Echocardiographic changes following hemodialysis initiation in patients with advanced chronic kidney disease and symptomatic heart failure with reduced ejection fraction.

BACKGROUND: In patients without overt cardiac disease, the degree of left ventricular hypertrophy (LVH) gets worse following hemodialysis (HD) initiation; however, in patients with both advanced chronic kidney disease (CKD) and symptomatic heart failure (HF) with reduced ejection fraction (EF), the short-term effect of HD on LVH and LV geometry has not been examined. We hypothesized that left ventricular mass index (LVMI) would decrease following HD initiation in CKD patients with symptomatic HF. METHODS: We retrospectively evaluated changes in LVMI, LV geometry, and LV fractional shortening (LVFS), assessed by 2D transthoracic echocardiography (TTE), in 41 patients with HF initiating HD while hospitalized from 1995 to 2006. HF was defined by LVEF ≤ 45% or dyspnea plus two of the following: raised jugular venous pressure, bibasilar crackles, pulmonary venous hypertension, interstitial edema on chest X-ray, or both. TTE was performed within 3 months prior to first HD and repeated 8.6 ± 5.2 months after start of HD. TTE recordings were obtained from storage and analyzed by a cardiologist blinded to patient clinical characteristics. RESULTS: Before initiation of HD, LVMI in 39 patients was 167.9 ± 53.1 g/m2 and it decreased by -24.3 ± 35.4 g/m2 by follow-up, p < 0.001. 26% of patients with concentric LVH at baseline had concentric remodeling or eccentric LVH at follow-up. LVFS did not significantly change over time in all 41 patients with HF (25.7 ± 8.7% vs. 26.4 ± 8.7%, p = 0.66). However, in an expanded analysis of all 69 patients with serial TTEs, a 1% increase in LVFS after starting HD was associated with a 16% reduction in risk of cardiovascular hospitalization at follow-up (HR 0.84, 95% CI 0.73 - 0.96, p = 0.01). CONCLUSIONS: LVMI decreases following HD initiation in CKD patients with symptomatic HF and reduced LVEF, possibly due to relief of venous congestion. Increase in LVFS following HD initiation predicts improved cardiac outcome.

Location of modulatory beta subunits in BK potassium channels.

Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming alpha subunits and four modulatory beta subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK alpha, and of the voltage-sensing domain TM helices S1-S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two beta1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of alpha with two substituted Cys's, one in S0 and one in S2, and beta1 also with two substituted Cys's, one in TM1 and one in TM2, resulted in two alphas cross-linked by one beta. Thus, each beta lies between and can interact with the voltage-sensing domains of two adjacent alpha subunits.

Location of the beta 4 transmembrane helices in the BK potassium channel.

Large-conductance, voltage- and Ca(2+)-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of alpha subunits, which contain the voltage-sensor domains and the Ca(2+)- sensor domains and form the pore, and often one of four types of beta subunits, which modulate the channel in a cell-specific manner. beta 4 is expressed in neurons throughout the brain. Deletion of beta 4 in mice causes temporal lobe epilepsy. Compared with channels composed of alpha alone, channels composed of alpha and beta 4 activate and deactivate more slowly. We inferred the locations of the two beta 4 transmembrane (TM) helices TM1 and TM2 relative to the seven alpha TM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that beta 4 TM2 is close to alpha S0 and that beta 4 TM1 is close to both alpha S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3-S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V(50) and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states.