First-trimester serum soluble fms-like tyrosine kinase-1, free vascular endothelial growth factor, placental growth factor and uterine artery Doppler in preeclampsia.

OBJECTIVE: To compare the first-trimester serum concentrations of soluble fms-like tyrosine kinase-1 (sFlt-1), free vascular endothelial growth factor (free-VEGF), placental growth factor (PlGF), and uterine artery pulsatility index (PI) in women who later developed preeclampsia (PE). STUDY DESIGN: Prospectively collected maternal serum samples were evaluated for sFlt-1, free VEGF, and PlGF levels in 63 cases who later developed PE compared with 252 unaffected controls. Serum levels of these angiogenic factors were measured using Quantikine immunoassays. Both univariate and multivariate analyses were used to evaluate the association between angiogenic factors and PE. The relationship between the angiogenic factors and mean maternal uterine artery PI was also evaluated. RESULT: Maternal serum sFlt-1 levels were not significantly different between the cases and controls. Mean free-VEGF levels were significantly higher in women destined to develop PE compared with the controls (P=0.04), and mean PlGF levels were significantly lower in women who later developed PE (P=0.01). There was no significant correlation between maternal mean uterine artery PI and angiogenic factors evaluated. Receiver-operating characteristic curves revealed that none of the factors were clinically useful for prediction in the first trimester of PE. CONCLUSION: Despite some significant differences in the first-trimester serum levels of angiogenic factors, our models suggest that these factors are not clinically useful for prediction in women who later developed PE.

Polymorphisms in the fetal progesterone receptor and a calcium-activated potassium channel isoform are associated with preterm birth in an Argentinian population.

OBJECTIVE: To investigate genetic etiologies of preterm birth (PTB) in Argentina through evaluation of single-nucleotide polymorphisms (SNPs) in candidate genes and population genetic admixture. STUDY DESIGN: Genotyping was performed in 389 families. Maternal, paternal and fetal effects were studied separately. Mitochondrial DNA (mtDNA) was sequenced in 50 males and 50 females. Y-chromosome anthropological markers were evaluated in 50 males. RESULT: Fetal association with PTB was found in the progesterone receptor (PGR, rs1942836; P=0.004). Maternal association with PTB was found in small conductance calcium activated potassium channel isoform 3 (KCNN3, rs883319; P=0.01). Gestational age associated with PTB in PGR rs1942836 at 32-36 weeks (P=0.0004). MtDNA sequencing determined 88 individuals had Amerindian consistent haplogroups. Two individuals had Amerindian Y-chromosome consistent haplotypes. CONCLUSION: This study replicates single locus fetal associations with PTB in PGR, maternal association in KCNN3, and demonstrates possible effects for divergent racial admixture on PTB.

Characterization of a novel 132-bp exon of the human maxi-K channel.

The large-conductance Ca2+-activated voltage-dependent K+ channel (maxi-K channel) induces a significant repolarizing current that buffers cell excitability. This channel can derive its diversity by alternative splicing of its transcript-producing isoforms that differ in their sensitivity to voltage and intracellular Ca2+. We have identified a novel 132-bp exon of the maxi-K channel from human myometrial cells that encodes 44 amino acids within the first intracellular loop of the channel protein. Distribution analysis reveals that this exon is expressed predominantly in human smooth muscle tissues with the highest abundance in the uterus and aorta and resembles the previously reported distribution of the total maxi-K channel transcript. Single-channel K+ current measurements in fibroblasts transfected with the maxi-K channel containing this novel 132-bp exon demonstrate that the presence of this insert attenuates the sensitivity to voltage and intracellular Ca2+. Alternative splicing to introduce this 132-bp exon into the maxi-K channel may elicit another mode to modulate cell excitability.

Regulation of the Ca2+-sensitive domains of the maxi-K channel in the mouse myometrium during gestation.

Large conductance Ca(2+)-activated K(+) channels (maxi-K channels) are known to modulate uterine activity during gestation. Electrophysiological recordings demonstrate that myometrial maxi-K current is suppressed in term-pregnant compared to non-pregnant mice. We sought to determine whether maxi-K current suppression is due to reduction of maxi-K channel protein or differential expression of maxi-K channel isoforms that vary in their Ca(2+) and voltage sensitivities. Immunoblot analyses show an increase of maxi-K channel protein throughout gestation. Polymerase chain reaction of mouse myometrial cDNA identified four alternatively spliced sites within the maxi-K transcript and three within the Ca(2+)-sensitive "tail" domain. Ribonuclease protection analyses demonstrate that total channel transcript levels mimic protein expression; however transcript levels of alternatively spliced regions of regulatory domains that alter sensitivity to voltage and Ca(2+) differ in their gestational expression. An insert that increases the maxi-K channel sensitivity to voltage and Ca(2+) is present at steady levels throughout gestation, differing from total channel transcript regulation. The insert-less form of this transcript, which reduces the channel voltage and Ca(2+) sensitivity, is not detected until midterm pregnancy. These findings verify that multiple isoforms of the maxi-K channel are present in the mouse myometrium and are regulated differentially during gestation, which is a likely mechanism for modulation of myometrial excitability during pregnancy.

Distinct domains of the voltage-gated K+ channel Kv beta 1.3 beta-subunit affect voltage-dependent gating.

The Kvbeta1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 +/- 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvbeta1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvbeta1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvbeta1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvbeta1.3-mediated effects involve at least two distinct domains of this beta-subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvbeta1.3-induced shift in the activation midpoint is functionally distinct from inactivation.

Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv beta 2.1 subunits.

The voltage-sensitive currents observed following hKv1.5 alpha subunit expression in HEK 293 and mouse L-cells differ in the kinetics and voltage dependence of activation and slow inactivation. Molecular cloning, immunopurification, and Western blot analysis demonstrated that an endogenous L-cell Kv beta 2.1 subunit assembled with transfected hKv 1.5 protein. In contrast, both mRNA and protein analysis failed to detect a beta subunit in the HEK 293 cells, suggesting that functional differences observed between these two systems are due to endogenous L-cell Kv beta 2.1 expression. In the absence of Kv beta 2.1, midpoints for activation and inactivation of hKv1.5 in HEK 293 cells were -0.2 +/- 2.0 and -9.6 +/- 1.8 mV, respectively. In the presence of Kv beta 2.1 these values were -14.1 +/- 1.8 and -22.1 +/- 3.7 mV, respectively. The beta subunit also caused a 1.5-fold increase in the extent of slow inactivation at 50 mV, thus completely reconstituting the L-cell current phenotype in the HEK 293 cells. These results indicate that 1) the Kv beta 2.1 subunit can alter Kv1.5 alpha subunit function, 2) beta subunits are not required for alpha subunit expression, and 3) endogenous beta subunits are expressed in heterologous expression systems used to study K+ channel function.

Molecular physiology of cardiac potassium channels.

The cardiac action potential results from the complex, but precisely regulated, movement of ions across the sarcolemmal membrane. Potassium channels represent the most diverse class of ion channels in heart and are the targets of several antiarrhythmic drugs. Potassium currents in the myocardium can be classified into one of two general categories: 1) inward rectifying currents such as IK1, IKACh, and IKATP; and 2) primarily voltage-gated currents such as IKs, IKr, IKp, IKur, and Ito. The inward rectifier currents regulate the resting membrane potential, whereas the voltage-activated currents control action potential duration. The presence of these multiple, often overlapping, outward currents in native cardiac myocytes has complicated the study of individual K+ channels; however, the application of molecular cloning technology to these cardiovascular K+ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of the function and pharmacology of a single channel type. This review addresses the progress made toward understanding the complex molecular physiology of K+ channels in mammalian myocardium. An important challenge for the future is to determine the relative contribution of each of these cloned channels to cardiac function.

A novel K+ channel beta-subunit (hKv beta 1.3) is produced via alternative mRNA splicing.

Voltage-gated K+ channels can form multimeric complexes with accessory beta-subunits. We report here a novel K+ channel beta-subunit cloned from human heart, hKv beta 1.3, that has 74-83% overall identity with previously cloned beta-subunits. Comparison of hKv beta 1.3 with the previously cloned hKv beta 3 and rKv beta 1 proteins indicates that the carboxyl-terminal 328 amino acids are identical, while unique variable length amino termini exist. Analysis of human beta-subunit cDNA and genomic nucleotide sequences confirm that these three beta-subunits are alternatively spliced from a common beta-subunit gene. Co-expression of hKv beta 1.3 in Xenopus oocytes with the delayed rectifier hKv1.5 indicated that hKv beta 1.3 has unique functional effects. This novel beta-subunit induced a time-dependent inactivation during membrane voltage steps to positive potentials, induced a 13-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 13 +/- 0.5 ms versus 35 +/- 1.7 ms at -40 mV). Most notably, hKv beta 1.3 converted the Kv1.5 outwardly rectifying current voltage relationship to one showing strong inward rectification. These data suggest that Kv channel current diversity may arise from association with alternatively spliced Kv beta-subunits. A simplified nomenclature for the K+ channel beta-subunit subfamilies is suggested.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

Hypoxia increases the activity of Ca(2+)-sensitive K+ channels in cat cerebral arterial muscle cell membranes.

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O2, PO2, on the predominant K+ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K(+)-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NPo) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]i) between 0 and 100 microM, but was significantly increased upon elevation of intracellular free Ca2+ concentration ([Ca2+]i). Low concentrations of external tetraethylammonium (0.1-3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O2 from 21% to < 2% reversibly increased the NPo, mean open time, and event frequency of the Ca(2+)-sensitive, high-conductance single-channel K+ current recorded at a patch potential of +20 mV. A similar reduction in PO2 also produced a transient increase in the activity of the 215-pS K+ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca2+]i or intracellular pH (pHi) of cat cerebral arterial muscle cells, as measured using Ca(2+)- or pH-sensitive fluorescent probes. Reduced PO2 caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pretreatment with 1 mM tetraethylammonium. These results suggest that reduced PO2 increases the activity of a high-conductance, Ca(2+)-sensitive K+ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca2+]i and pHi.

Enhanced single-channel K+ current in arterial membranes from genetically hypertensive rats.

Arterial smooth muscle from hypertensive rats shows an increased membrane permeability to K+ that depends on Ca2+ influx. To define the mechanism of this membrane alteration, we tested the hypothesis that Ca(2+)-dependent K+ current (IK(Ca)) is increased in arterial muscle membranes from genetically hypertensive rats. Single-channel K+ currents measured in cell-attached and inside-out aortic membrane patches from spontaneously hypertensive rats (SHR) were compared with those from normotensive Wistar-Kyoto rats (WKY). Inside-out patches from both rat strains showed a predominant 225 pS, Ca(2+)- and voltage-dependent K+ channel in symmetrical 145 mM KCl solutions, which was blocked by tetraethylammonium [concentration for half-maximal block (IC50) < or = 0.3 mM]. In cell-attached patches of aortic muscle cells bathed in physiological salt solution, this channel [IK(Ca) channel] showed a fivefold higher open-state probability (NPo) in SHR as compared with WKY. This increased NPo of SHR IK(Ca) channels in membranes of intact aortic muscle cells was not correlated with an altered membrane potential in current-clamped SHR myocytes or with changes in cytosolic free Ca2+ concentration in fura-2-loaded aortic muscle cells. However, inside-out aortic membrane patches from SHR showed more detected IK(Ca) channels per patch, a higher IK(Ca) channel NPo, and a greater total patch current than their WKY counterparts. Further analysis revealed a greater Ca2+ sensitivity of SHR than WKY IK(Ca) channels. These results suggest that IK(Ca) channel function is altered in isolated membrane patches of arterial muscle from genetically hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A Ca(2+)-dependent K+ current is enhanced in arterial membranes of hypertensive rats.

This study was designed to investigate the role and regulation of arterial membrane K+ channels in hypertension. Aortic segments from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were suspended for isometric tension recording. In other experiments, proximal aortic segments (PS) (exposed to high pressure) and distal aortic segments (DS) (exposed to lower pressure) were removed from surgically coarcted Sprague-Dawley rats and similarly prepared. Aortas from SHR and PS dose-dependently contracted to the K+ channel blocker tetraethylammonium (TEA) (0.1-10 mM), and this contraction was abolished by preincubation with 0.1 microM nifedipine. In contrast, the same concentrations of TEA did not contract either WKY or DS aortas. Since block of K+ channels by TEA had a different effect on aortic segments exposed to high versus low blood pressure, we compared whole-cell K+ currents in isolated vascular cells from the same aortas. The reversal potentials of depolarization-induced outward currents in WKY, SHR, DS, and PS aortic cells showed a Nernst relation to external K+ concentration indicative of selective K+ permeability. TEA (1 and 10 mM) was equipotent in blocking these K+ currents in patch-clamped cells from all aortic preparations, suggesting that the lack of TEA-induced contractions in WKY and DS aortas was not due to an absence of TEA-sensitive K+ channels in these arterial membranes. However, when the Ca2+ ionophore A23187 (10 microM) was used to increase the level of cytosolic Ca2+ in patch-clamped cells, the K+ current density in SHR and PS aortic cells was twofold or more higher than in WKY and DS cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Normalization of arterial pressure after barodenervation: role of pressure natriuresis.

Studies in several species have demonstrated that mean arterial pressure (MAP) is normal or only slightly elevated after chronic arterial baroreceptor denervation. We hypothesized that the absence of sustained hypertension after barodenervation was the result of a pressure natriuresis response, secondary to sympathetic vasoconstriction of nonrenal vasculature. To test this hypothesis, MAP, sodium balance (NaBal), and water balance were measured before and after aortic baroreceptor denervation (ABD), sinoaortic denervation (SAD), or sham surgery in conscious rats. MAP was increased 20.0 +/- 3.7 mmHg 1 day after ABD but returned to control by day 3. ABD had no significant effect on daily NaBal or water balance. The responses to SAD were similar to those after ABD, with the exception that a significant natriuresis was observed the first day after SAD. However, this was followed by a significant antinatriuresis on day 2, when MAP was still elevated. By day 3 after SAD, MAP, NaBal, and water balance were not significantly different from control. These results suggest that the normalization of MAP after ABD or SAD is not the result of pressure natriuresis but rather failure to maintain a chronic elevation of sympathetic activity after barodenervation.