Rhythmic supplementation of 17beta-estradiol according to the physiological estrous cycle: effect on blood pressure in female ovariectomized rats.

Female Wistar-Kyoto rats (WKY) show a 4-day estrous cycle. The aim of this study was to examine the impact of 17beta-estradiol supplementation every fourth day to ovariectomized rats - mimicking the physiological estrous cycle - on regulation of blood pressure. We monitored blood pressure telemetrically in intact females, ovariectomized (OVX), and ovariectomized WKY injected subcutaneously with 17beta-estradiol (OVX (E2)) in a 4-day rhythm for 24 weeks. Blood pressure decreased both in intact females and OVX (E2), whereas that of OVX persisted at constant levels. The underlying mechanisms studied include the nitric oxide pathway, the rennin-angiotensin system as well as the endothelin system. Serum and urinary nitrate/nitrite (NOx) as well as aortic eNOS decreased in OVX and were restored to normal in OVX (E2). Conversely, caveolin-1 was higher in OVX than in intact females and OVX (E2) while Hsp90 did not differ among groups. Plasma angiotensin II and aortic AT (1) receptor expression increased in OVX and were normalized in OVX (E2). AT (2) receptor expression was regulated reciprocally. Serum endothelin-1 was significantly elevated in OVX and OVX (E2). There was no difference in aortic ET (A) receptor expression between groups whereas ET (B) receptor expression was higher in intact females and OVX (E2) than in OVX. The study suggests that supplementation of 17beta-estradiol in female WKY according to the natural estrous cycle maintains the physiological blood pressure encompassing vasorelaxing and vasoconstricting pathways. The physiological estrous cycle should be kept in mind when cardiovascular data are to be collected/interpreted under estrogen supplementation.

A fluorescence-based microplate assay to quantify DOM-induced catabolic activity.

This note describes a novel method to quickly quantify the dissolved organic matter (DOM)-induced catabolic activity from low-volume samples. The concept is based on the catabolic response profiles (CRP) assay and is described as an inverse CRP, where the reactivity of a complex and diverse mixture of organic compounds towards single strains of bacteria is quantified. A strain of Pseudomonas fluorescens was grown and then transferred to an organic carbon-free mineral salt medium. 90 microL of a fluorogenic redox indicator was added to 90 microL of the bacterial suspension in a well on a 96-well microplate. The DOM sample (90 microL) was added to the well and the fluorescence emitted by the reduced indicator was read over the period of incubation. Only 0.8 mL of the DOM sample, including controls and replicates, was required to quantify the activity of each sample. Results are presented for a surface soil DOM sample and they were compared to glucose samples of various concentrations. The detection limit was reached for samples containing as little as 55 microM of glucose (0.3 mg C L(-1)). The assay showed that only 9% of the total carbon of the soil surface DOM sample was readily biodegradable.

Molecular biology of the voltage-gated potassium channels of the cardiovascular system.

K+ channels represent the most diverse class of voltage-gated ion channels in terms of function and structure. Voltage-gated K+ channels in the heart establish the resting membrane K+ permeability, modulate the frequency and duration of action potentials, and are targets of several antiarrhythmic drugs. Consequently, an understanding of K+ channel structure-function relationships and pharmacology is of great practical interest. However, the presence of multiple overlapping currents in native cardiac myocytes complicates the study of basic K+ channel function and drug-channel interactions in these cells. The application of molecular cloning technology to cardiovascular K+ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of channel function and pharmacology without contaminating currents. To date six different K+ channels have been cloned from rat and human heart, and all have been functionally characterized in either Xenopus oocytes or mammalian tissue culture systems. This initial research is an important step toward understanding the molecular basis of the action potential in the heart. An important challenge for the future is to determine the cell-specific expression and relative contribution of these cloned channels to cardiac excitability.

Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel.

The interaction of quinidine with a cloned human cardiac potassium channel (HK2) expressed in a stable mouse L cell line was studied using the whole-cell tight-seal voltage-clamp technique. Quinidine (20 microM) did not affect the initial sigmoidal activation time course of the current. However, it reduced the peak current and induced a subsequent decline, with a time constant of 8.2 +/- 0.8 msec, to 28 +/- 6% of control (at +60 mV). The concentration dependence of HK2 block at +60 mV yielded an apparent KD of 6 microM and a Hill coefficient of 0.9. The degree of block was voltage dependent. Block increased from 0.60 +/- 0.09 at 0 mV to 0.72 +/- 0.06 at +60 mV with 20 microM quinidine and from 0.39 +/- 0.20 to 0.48 +/- 0.16 with 6 microM. Paired analysis in seven experiments with 20 microM quinidine indicated that the voltage-dependent increase in block was significant (difference, 12 +/- 4%; p less than 0.001). This voltage dependence was described by an equivalent electrical distance delta of 0.19 +/- 0.02, which suggested that at the binding site quinidine experienced 19% of the applied transmembrane electrical field, referenced to the inner surface. Quinidine reduced the tail current amplitude and slowed the time course relative to control, resulting in a "crossover" phenomenon. These data indicate that 1) the charged form of quinidine blocks the HK2 channel after it opens, 2) binding occurs within the transmembrane electrical field (probably in or near the ion permeation pathway), and 3) unbinding is required before the channel can close.

Molecular cloning and characterization of two voltage-gated K+ channel cDNAs from human ventricle.

K+ channels represent the most complex class of voltage-gated ion channels from both functional and structural standpoints. In the heart these channels are responsible for the rapid repolarizing phases of the action potential and are the targets of several antiarrhythmic drugs. Full-length cDNA clones were isolated from human ventricular libraries that encode two voltage-gated K+ channels. These two cDNAs, designated HK1 and HK2, encode proteins of 653 and 605 amino acids, respectively. HK1 is the human equivalent (98% identity) of an inactivating K+ channel previously described in rat heart (RHK1) whereas the HK2 channel is 86% identical to a cloned rat brain K+ channel (Kv1). The only amino acid sequence identity (72%) between HK1 and HK2 is within the central region containing the membrane spanning domains. Northern blot analysis of human mRNA indicated that HK1 is slightly more abundant in ventricle than atrium whereas HK2 is much more abundant in atrium relative to ventricle. Both channel transcripts are present in ventricle at levels equivalent to voltage-gated Na+ channels. Analysis of the gene encoding HK1 suggests the coding sequence is intronless and is represented once in the human genome.

Sertraline-induced desensitization of the serotonin 5HT-2 receptor transmembrane signaling system.

Sertraline is a new, selective serotonin (5HT) uptake inhibitor with antidepressant activity. The effect of chronic administration of sertraline on 5HT-2 receptors in rat cortex was compared with that of the tricyclic antidepressant, amitriptyline. 5HT-2 receptors were evaluated in binding assays using [3H]-ketanserin and in functional assays of transmembrane signaling, hydrolysis of phosphoinositides. The daily injection of 17 mg/kg sertraline induced a desensitization of 5HT-2-mediated phosphoinositide hydrolysis after 28, but not 21, days. The administration of 1.2 mg/kg/day via continuous release pumps caused a more rapid desensitization. Amitriptyline administered chronically also produced a desensitization of the 5HT-2-mediated phosphoinositide hydrolysis response. A decrease in the density of 5HT-2 binding sites accompanies the functional desensitization after amitriptyline, but changes in 5HT-2 binding sites were not detected after chronic sertraline administration. Studies of the mechanism of action of sertraline show that the desensitization of the phosphoinositide hydrolysis response is homologous in nature, and that it is not secondary to changes in the synthesis of precursor lipids. Other possibilities such as alterations in coupling efficiency or in the activity of effector enzymes are currently being considered. The present results suggest a new postsynaptic action of antidepressant drugs at central 5HT-2 receptors (i.e., changes in 5HT-2 signal transduction at a site distal to the cell surface binding site) and illustrate the importance of studies of receptor signaling pathways to complement radioligand binding.

Lysergic acid diethylamide and 2,5-dimethoxy-4-methylamphetamine are partial agonists at serotonin receptors linked to phosphoinositide hydrolysis.

Based on electrophysiological, radioligand binding, and behavioral studies in laboratory animals, it is generally believed that the psychotomimetic effects of the phenethylamine and indolealkylamine hallucinogens are mediated by central serotonin (5-HT) receptors, in particular the 5-HT-2 subtype. Agonist-stimulated phosphoinositide hydrolysis was utilized to determine the potency and efficacy of racemic 1-(2,5)-dimethoxy-4-methyl-phenyl)-2-aminopropane (DOM), and d-lysergic acid diethylamide (LSD) at the 5-HT-2 receptor in rat cerebral cortex and the 5-HT-1c receptor in rat choroid plexus. Both DOM and LSD stimulated phosphoinositide hydrolysis in cerebral cortex. These effects were blocked by the 5-HT-2 antagonists, ketanserin and spiperone, but not by antagonists of muscarinic, alpha-1 adrenergic or histaminergic receptors. The maximum responses of DOM and LSD, respectively, were 76% and 25% of the maximum response to 5-HT. However, LSD was 500 times more potent than was racemic DOM. Consistent with a partial agonist effect, LSD partially blocked the effect of 5-HT, with a maximal inhibition equivalent to the intrinsic activity of LSD alone. In choroid plexus, DOM and LSD stimulated phosphoinositide hydrolysis and both responses were blocked by mianserin and less effectively by spiperone. The maximum effect of DOM was 67% of that of 5-HT, whereas the maximum effect of LSD was only 34% of the maximum response of 5-HT. LSD was 50 times more potent than was racemic DOM. LSD partially antagonized the effect of 5-HT in the choroid plexus, consistent with a partial agonist effect at the 5-HT-1c receptor in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)