Blood vessels express estrogen receptors, but their role in cardiovascular physiology is not well understood. We show that vascular smooth muscle cells and blood vessels from estrogen receptor beta (ERbeta)-deficient mice exhibit multiple functional abnormalities. In wild-type mouse blood vessels, estrogen attenuates vasoconstriction by an ERbeta-mediated increase in inducible nitric oxide synthase expression. In contrast, estrogen augments vasoconstriction in blood vessels from ERbeta-deficient mice. Vascular smooth muscle cells isolated from ERbeta-deficient mice show multiple abnormalities of ion channel function. Furthermore, ERbeta-deficient mice develop sustained systolic and diastolic hypertension as they age. These data support an essential role for ERbeta in the regulation of vascular function and blood pressure.

Large-conductance Ca 2 ϩ -activated K ϩ channels (BK Ca channels) are regulated by the tissue-specific expression of auxiliary ␤ subunits. ␤ 1 is predominately expressed in smooth muscle, where it greatly enhances the BK Ca channel's Ca 2 ϩ sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The ␤ 1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK Ca channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with ␤ 1 present. Furthermore, ␤ 1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by ‫ف‬ 24% (16.8 → 12.8). The effects of ␤ 1 on voltage sensing enhance the BK Ca channel's Ca 2 ϩ sensitivity by decreasing at most voltages the work that Ca 2 ϩ binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca 2 ϩ affinity brought about by ␤ 1 at negative voltages, our studies suggest that ␤ 1 also decreases the true Ca 2 ϩ affinity of the closed channel, increasing its Ca 2 ϩ dissociation constant from ‫ف‬ 3.7 M to between 4.7 and 7.1 M, depending on how many binding sites are affected.

We report here a combination of site-directed mutations that eliminate the high-affinity Ca2+ response of the large-conductance Ca2+-activated K+ channel (BKCa), leaving only a low-affinity response blocked by high concentrations of Mg2+. Mutations at two sites are required, the “Ca2+ bowl,” which has been implicated previously in Ca2+ binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BKCa channel contains three types of Ca2+ binding sites, one of low affinity that is Mg2+ sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca2+ to influence channel opening. Estimates of the binding characteristics of the BKCa channel's high-affinity Ca2+-binding sites are provided.

There is controversy over whether Ca2+ binds to the BKCa channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca2+ sensitivity act at the point of Ca2+ coordination. One region in the intracellular domain that has been implicated in Ca2+ sensing is the “Ca2+ bowl”. This region contains many acidic residues, and large Ca2+-bowl mutations eliminate Ca2+ sensing through what appears to be one type of high-affinity Ca2+-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca2+ bowl that are most important for Ca2+ sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca2+ binding to a fusion protein that contains a portion of the BKCa channel's intracellular domain. Thus, much of our data supports the conclusion that Ca2+ binds to the BKCa channel's intracellular domain, and they define the Ca2+ bowl's essential Ca2+-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca2+ sensing and those that disrupt Ca2+ binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca2+ bowl may be in a nonnative conformation.

Bystander effects induced by cytoplasmic irradiation have been reported recently. However, the mechanism(s) underlying, such as the functional role of mitochondria, is not clear. In the present study, we used either mtDNA-depleted (r 0 ) A L or normal (r þ ) A L cells as irradiated donor cells and normal human skin fibroblasts as receptor cells in a series of medium transfer experiments to investigate the mitochondria-related signal process. Our results indicated that mtDNA-depleted cells or normal A L cells treated with mitochondrial respiratory chain function inhibitors had an attenuated g-H2AX induction, which indicates that mitochondria play a functional role in bystander effects. Moreover, it was found that treatment of normal A L donor cells with specific inhibitors of NOS, or inhibitor of mitochondrial calcium uptake (ruthenium red) significantly decreased g-H2AX induction and that radiation could stimulate cellular NO and O 2 KÀ production in irradiated r þ A L cells, but not in r 0 A L cells. These observations, together with the findings that ruthenium red treatment significantly reduced the NO and O 2 KÀ levels in irradiated r þ A L cells, suggest that radiationinduced NO derived from mitochondria might be an intracellular bystander factor and calcium-dependent mitochondrial NOS might play an essential role in the process.