Edward S. Umstot

Heme Is a Carbon Monoxide Receptor for Large-Conductance Ca2+-Activated K+ Channels

Carbon monoxide (CO) is an endogenous paracrine and autocrine gaseous messenger that regulates physiological functions in a wide variety of tissues. CO induces vasodilation by activating arterial smooth muscle large-conductance Ca2+-activated potassium (BKCa) channels. However, the mechanism by which CO activates BKCa channels remains unclear. Here, we tested the hypothesis that CO activates BKCa channels by binding to channel-bound heme, a BKCa channel inhibitor, and altering the interaction between heme and the conserved heme-binding domain (HBD) of the channel α subunit C terminus. Data obtained using thin-layer chromatography, spectrophotometry, mass spectrometry (MS), and MS-MS indicate that CO modifies the binding of reduced heme to the α subunit HBD. In contrast, CO does not alter the interaction between the HBD and oxidized heme (hemin), to which CO cannot bind. Consistent with these findings, electrophysiological measurements of native and cloned (cbv) cerebral artery smooth muscle BKCa channels show that CO reverses BKCa channel inhibition by heme but not by hemin. Site-directed mutagenesis of the cbv HBD from CKACH to CKASR abolished both heme-induced channel inhibition and CO-induced activation. Furthermore, on binding CO, heme switches from being a channel inhibitor to an activator. These findings indicate that reduced heme is a functional CO receptor for BKCa channels, introduce a unique mechanism by which CO regulates the activity of a target protein, and reveal a novel process by which a gaseous messenger regulates ion channel activity.

Postmenopausal steroid replacement with micronized dehydroepiandrosterone: Preliminary oral bioavailability and dose proportionality studies

OBJECTIVES Because dehydroepiandrosterone may protect against neoplasia, osteoporosis, and cardiac disease, we investigated the bioavailability of oral micronized dehydroepiandrosterone, anticipating its adjunctive use in postmenopausal steroid replacement. STUDY DESIGN Eight postmenopausal women randomly received either a placebo or 150 or 300 mg of oral micronized dehydroepiandrosterone in a lipid matrix. Serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, and estradiol were measured periodically over the 12 hours after each dose. All treatments, all doses, and mean serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and testosterone were compared with analysis of variance for repeated measures and Newman-Keuls a posteriori test of statistical significance. RESULTS Mean peak steroid concentrations after 150 mg (300 mg) doses were dehydroepiandrosterone 1617 (2639) ng/dl, 7 (11.5)-fold above placebo; dehydroepiandrosterone S 1185 (1688) micrograms/dl, 14 (20)-fold above placebo; and testosterone 183 (311) ng/dl, 4 (7)-fold above placebo. Estradiol concentrations remained less than 20 pg/ml, but androgen concentrations rose by 1 hour and remained elevated through the twelfth hour. Peak androgen concentrations and areas under the curves exhibited proportionality with both doses. A testosterone radioimmunoassay with celite chromatography revealed a 300% overestimation for testosterone in the direct-assay method used in this study. Thus after appropriate readjustment maximum testosterone concentrations were observed consistently within physiologic premenopausal ranges after the 150 mg dose. CONCLUSIONS Micronized dehydroepiandrosterone may provide a steroidal postmenopausal replacement that is adjunctive to estrogens and worthy of further investigation.

Hydrogen sulfide and cerebral microvascular tone in newborn pigs

Hydrogen sulfide (H2S) is a gaseous signaling molecule that appears to be involved in numerous biological processes, including regulation of blood pressure and vascular tone. The present study is designed to address the hypothesis that H2S is a functionally significant, endogenous dilator in the newborn cerebrovascular circulation. In vivo experiments were conducted using newborn pigs with surgically implanted, closed, cranial windows. Topical application of H2S concentration-dependently (10(-6) to 2×10(-4) M) dilated pial arterioles. This dilation was blocked by glibenclamide (10(-6) M). L-cysteine, the substrate of the H2S-producing enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), also dilated pial arterioles. The dilation to L-cysteine was blocked by the CSE inhibitor d,l-propargylglycine (PPG, 10 mM) but was unaffected by the CBS inhibitor amino-oxyacetate (AOA, 1 mM). Western blots detected CSE, but not CBS, in cerebral microvessels, whereas CBS is detected in brain parenchyma. Immunohistological CSE expression is predominantly vascular while CBS is expressed mainly in neurons and astrocytes. L-cysteine (5 mM) increased H2S concentration in cerebrospinal fluid (CSF), measured by GC-MS, from 561±205 to 2,783±818 nM before but not during treatment with PPG (1,030±70 to 622±78 nM). Dilation to hypercapnia was inhibited by PPG but not AOA. Hypercapnia increased CSF H2S concentration from 763±243 to 4,337±1789 nM before but not during PPG treatment (357±178 vs. 425±217 nM). These data show that H2S is a dilator of the newborn cerebral circulation and that endogenous CSE can produce sufficient H2S to decrease vascular tone. H2S appears to be a physiologically significant dilator in the cerebral circulation.

Astrocyte-Derived CO Is a Diffusible Messenger That Mediates Glutamate-Induced Cerebral Arteriolar Dilation by Activating Smooth Muscle Cell KCa Channels

Astrocyte signals can modulate arteriolar tone, contributing to regulation of cerebral blood flow, but specific intercellular communication mechanisms are unclear. Here we used isolated cerebral arteriole myocytes, astrocytes, and brain slices to investigate whether carbon monoxide (CO) generated by the enzyme heme oxygenase (HO) acts as an astrocyte-to-myocyte gasotransmitter in the brain. Glutamate stimulated CO production by astrocytes with intact HO-2, but not those genetically deficient in HO-2. Glutamate activated transient KCa currents and single KCa channels in myocytes that were in contact with astrocytes, but did not affect KCa channel activity in myocytes that were alone. Pretreatment of astrocytes with chromium mesoporphyrin (CrMP), a HO inhibitor, or genetic ablation of HO-2 prevented glutamate-induced activation of myocyte transient KCa currents and KCa channels. Glutamate decreased arteriole myocyte intracellular Ca2+ concentration and dilated brain slice arterioles and this decrease and dilation were blocked by CrMP. Brain slice arteriole dilation to glutamate was also blocked by L-2-alpha aminoadipic acid, a selective astrocyte toxin, and paxilline, a KCa channel blocker. These data indicate that an astrocytic signal, notably HO-2–derived CO, is used by glutamate to stimulate arteriole myocyte KCa channels and dilate cerebral arterioles. Our study explains the astrocyte and HO dependence of glutamatergic functional hyperemia observed in the newborn cerebrovascular circulation in vivo.

Delivery of dehydroepiandrosterone to premenopausal women: effects of micronization and nonoral administration.

OBJECTIVES This single-dose study compares three dehydroepiandrosterone delivery methods (oral crystalline steroid, micronized steroid, and vaginal administration) to ascertain whether physiologic levels of circulating dehydroepiandrosterone and dehydroepiandrosterone sulfate can be obtained while increases in testosterone are minimized. STUDY DESIGN Two randomized, double-blind, placebo-controlled single-dose comparisons were made. For oral micronized versus crystalline dehydroepiandrosterone 300 mg doses of micronized or crystalline dehydroepiandrosterone were administered, followed by 6 hours of blood sampling (n=7). Serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and testosterone levels were measured; areas under the curve and mean peak values were analyzed by Student-Newman-Keuls tests. For oral versus vaginal micronized dehydroepiandrosterone 150 mg oral or vaginal doses of micronized dehydroepiandrosterone were administered, followed by blood sampling over 12 hours (n=5). Data analysis was as described. RESULTS Oral micronized and unmicronized dehydroepiandrosterone resulted in increases in serum dehydroepiandrosterone, dehydroepiandrosterone sulfate, and testosterone. Micronization increased the area-under-the-curve ratios for dehydroepiandrosterone sulfate/dehydroepiandrosterone and dehydroepiandrosterone sulfate/testosterone. Vaginal administration provided equivalent serum dehydroepiandrosterone; however, it failed to increase dehydroepiandrosterone sulfate or testosterone over placebo. CONCLUSION Micronization of oral dehydroepiandrosterone diminishes bioconversion to testosterone. Vaginal dehydroepiandrosterone delivers equivalent dehydroepiandrosterone but substantially diminishes dehydroepiandrosterone bioconversion.

Matrix-assisted laser desorption/ionization mass spectrometric quantification of the mu opioid receptor agonist DAMGO in ovine plasma

The synthetic opioid peptide analog Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO), which is a mu opioid receptor-selective agonist, was quantified in ovine plasma samples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), using delayed extraction and a reflectron. The internal standard was pentadeuterated DAMGO. Timed-ion selection was used to select the precursor ion. The analysis of the post-source decay fragments improved the detection sensitivity, and the use of the precursor-product ion relationship optimized the specificity. For plasma samples, the inter-assay variability of this method was 6.4% (n = 79) and the intra-assay variability was 6.0% (n = 10). The variability for controls was 3.4% (n = 43). The profile of DAMGO amount versus time was determined in sheep plasma, and the corresponding pharmacokinetic data were calculated.

Dehydroepiandrosterone Attenuates Study‐Induced Declines in Insulin Sensitivity in Postmenopausal Womena

Dehydroepiandrosterone (DHEA) and its sulfated congener dehydroepiandrosterone sulfate (DHEAS) may play a rolc in the regulation of immune competence’ and insulin sensitivity2 and also may be cardioprotective.2 Age-related declines in adrenocortical androgen secretion, independent of cortisol, may thus contribute to the increased prevalence of insulin resistance in the elderly. In rodents, DHEA decreases the severity of genetic and induced diabetes2 In humans, amelioration of diabetes in a DHEA-supplemented patient has been described.3 Additionally, 17-hour DHEA infusions enhance postreceptor insulin a ~ t i o n , ~ and 3 weeks of oral DHEA increases T-lymphocyte insulin binding and degradation.z We have defined a low dosc preparation of DHEA that recreates the reproductive age adrenal androgen milieu in elderly subjects.? We hypothesize that with this system, DHEA supplementation will decrease insulin resistance in an androgen-deficient population of postmenopausal women.

A comparison of the bioavailability of oral and intramuscular dexamethasone in women in late pregnancy

Objective To compare the bioavilability of oral and intramuscular (IM) dexamthasonei in third-trimester pregnant women. Methods Oral and IM dexamethasone levels were compared in a randomized, parallel, crossover bioavailability study involving 11 gravid women in the third trimester of pregnancy. Subjects were randomized to receive either 6 mg of IM or 8 mg of oral dexamethasone. The following week, the alternative regimen was administered. Serial blood samples were obtained after drug administration. Dexamethasone concentrations were measured by radioimmunoassay. Total area under the curve was compared for the oral and IM groups using a paired t test. Results Eight of the 11 women completed the study through 12 hours; all 11 women completed the study through 6 hours. Among the 11 women, peak levels of dexamethasone occurred 30 minutes after IM injection (mean ± standardf deviation, 101.7 ± 19.2 ng/mL) and 120 minutes after oral administration (65.9 ± 20.5 ng/mL). Area under the curve did not differ significantly between those receiving IM dexamethasone (258.3 ± 50.0 ng/minu/mL) when measured 6 hours after administration of the drug. Terminal half-lives were similar in the IM and oral groups. Similar findings were noted among the eight women who were studied through 12 hours. This study had a power of 87% to detect a 20% difference in area under the curve between the two groups. Conclusion The bioavailability of 8 mg of oral dexamethasone is similar to that of a 6-mg IM dose, as determined by the area under the curve.

Downregulation of a unique photoreceptor protein correlates with improper outer segment assembly

A unique photoreceptor protein has been characterized. This protein, termed XAP‐1 antigen, is expressed by photoreceptors exclusively under conditions in which the outer segment membranes are properly assembled. When the retinal pigment epithelium is adherent to the underlying neural retina, the XAP‐1 antigen is localized to the plasma membrane that surrounds the inner and outer segments in the areas juxtaposed to the subretinal space. A similar labeling pattern is detected in retinal pigment epithelium‐deprived retinas in which assembly of nascent outer segments is supported by lactose. In retinas that undergo degeneration subsequent to the removal of the retinal pigment epithelium, the expression of this protein is completely downregulated. Immunohistochemical analyses and subcellular fractionation along with Western blot analysis, indicate that the XAP‐1 antigen is a membrane‐associated soluble protein. Mass spectrometric analysis indicates that the XAP‐1 antigen shares homology via 12 tryptic peptide masses with the gamma‐crystallin (lens structural protein) subclasses, although it does not immunolocalize to the same ocular structures as reported for the gamma‐crystallins. We propose that XAP‐1 antigen is a unique protein that is expressed extensively by healthy photoreceptor cells; the expression of the XAP‐1 antigen exclusively by photoreceptors with organized outer segments suggests that this protein may play a critical role in outer segment assembly.

Glutamate regulates Ca2+ signals in smooth muscle cells of newborn piglet brain slice arterioles through astrocyte- and heme oxygenase-dependent mechanisms

Glutamate is the principal cerebral excitatory neurotransmitter and dilates cerebral arterioles to match blood flow to neural activity. Arterial contractility is regulated by local and global Ca(2+) signals that occur in smooth muscle cells, but modulation of these signals by glutamate is poorly understood. Here, using high-speed confocal imaging, we measured the Ca(2+) signals that occur in arteriole smooth muscle cells of newborn piglet tangential brain slices, studied signal regulation by glutamate, and investigated the physiological function of heme oxygenase (HO) and carbon monoxide (CO) in these responses. Glutamate elevated Ca(2+) spark frequency by approximately 188% and reduced global intracellular Ca(2+) concentration ([Ca(2+)](i)) to approximately 76% of control but did not alter Ca(2+) wave frequency in brain arteriole smooth muscle cells. Isolation of cerebral arterioles from brain slices abolished glutamate-induced Ca(2+) signal modulation. In slices treated with l-2-alpha-aminoadipic acid, a glial toxin, glutamate did not alter Ca(2+) sparks or global [Ca(2+)](i) but did activate Ca(2+) waves. This shift in Ca(2+) signal modulation by glutamate did not occur in slices treated with d-2-alpha-aminoadipic acid, an inactive isomer of l-2-alpha-aminoadipic acid. In the presence of chromium mesoporphyrin, a HO blocker, glutamate inhibited Ca(2+) sparks and Ca(2+) waves and did not alter global [Ca(2+)](i). In isolated arterioles, CORM-3 [tricarbonylchloro(glycinato)ruthenium(II)], a CO donor, activated Ca(2+) sparks and reduced global [Ca(2+)](i). These effects were blocked by 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one, a soluble guanylyl cyclase inhibitor. Collectively, these data indicate that glutamate can modulate Ca(2+) sparks, Ca(2+) waves, and global [Ca(2+)](i) in arteriole smooth muscle cells via mechanisms that require astrocytes and HO. These data also indicate that soluble guanylyl cyclase is involved in CO activation of Ca(2+) sparks in arteriole smooth muscle cells.