The caldesmon content of vertebrate smooth muscle.

Caldesmon and tropomyosin can be selectively and quantitatively extracted from vascular and visceral smooth muscle following heat treatment; all other smooth muscle proteins are precipitated by this procedure. Estimates of the caldesmon/tropomyosin molar ratio in heat-extracts determined by SDS-PAGE densitometry are 1 caldesmon:5.1-5.3 tropomyosin for rabbit and sheep aorta, and 1 caldesmon:5.9 tropomyosin for rabbit stomach and chicken gizzard. If the assumption is made that tropomyosin serves as a true reference of thin-filament content in intact muscle, it follows that the relative caldesmon contents in the above tissues are similar to each other. Caldesmon in heat extracts was identified by Western blotting, by its anomalous migration on several different SDS-PAGE systems and by its position on two-dimensional PAGE. Values of caldesmon contents in unfractionated total tissue homogenates were found to be similar to those cited above. Smooth muscles contain different thin-filament classes and only one type appears to possess caldesmon. By comparing values for the molar composition of caldesmon-specific filaments (1 caldesmon:2 tropomyosin:14 actin) with the values above determined for intact tissue, we conclude that the caldesmon filaments account for approx. 35-45% of the total thin-filament pool in arterial smooth muscle and slightly less in visceral muscles.

WC-2 mediates WC-1-FRQ interaction within the PAS protein-linked circadian feedback loop of Neurospora.

Eukaryotic circadian clocks comprise feedback loops where PAS domain-containing transcriptional activators drive gene expression of negative elements. In NEUROSPORA:, clock models posit a White Collar complex (WCC) containing WC-1 and WC-2 that activates expression of the central clock gene frequency (frq); FRQ protein is hypothesized to feed back to block the activity of the WCC. We have characterized the WC-2 protein and its role in this complex: WC-2 is an abundant constitutive nuclear protein, in contrast to rhythmically expressed FRQ and WC-1. WC-2 interacts with WC-1 and FRQ but, significantly, WC-1 and FRQ do not interact in the absence of WC-2. By quantifying the relative numbers of WC-2, FRQ and WC-1 proteins and complexes in cell extracts, both the numbers and types of complexes at different circadian times were estimated, yielding results consistent with the model. Constitutive and abundant WC-2 appears to provide a scaffold allowing for the interaction of two limiting and rhythmically out-of-phase proteins, FRQ and WC-1, and this temporal and physical relationship may be responsible for rhythmic expression of frq.

Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells.

Specific regulatory mechanisms of aldosterone-stimulated Na+ reabsorption through the apical amiloride-sensitive channel are unknown. In this study, we examined the effects of aldosterone on Na+ channel gamma-subunit mRNA levels in cultured rabbit cortical collecting duct cells. With the use of reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from aldosterone-treated cells and degenerate primers, a 446-base pair (bp) PCR product was amplified and further characterized by nested PCR and sequencing. The nested PCR yielded a predicted 164-bp product. Sequencing of the 446-bp PCR product revealed 83% nucleotide and 91% amino acid identity to the rat colonic Na+ channel gamma-subunit. The relative abundance of Na+ channel mRNA was determined by quantitative PCR after a 24-h aldosterone treatment. The results demonstrate that Na+ channel gamma-subunit mRNA levels were significantly higher (2.6 +/- 0.42) in aldosterone-treated cultures vs. the controls. This increase, however, is less than the aldosterone-induced increase (3.2 +/- 2.0) in the amiloride-sensitive short-circuit current. These results indicate that Na+ channel gamma-subunit mRNA levels are increased by aldosterone and that this increase is likely to be responsible, at least in part, for the aldosterone-induced Na+ current in the kidney.

Expression and characterization of a new species of 11 beta-hydroxysteroid dehydrogenase in Xenopus oocytes.

11 beta-Hydroxysteroid dehydrogenase (11-OHSD) plays a critical role in conferring aldosterone specificity to mineralocorticoid target cells. We have recently described a novel isoform of 11-OHSD in the collecting duct (11-OHSD/CD), which differs from the previously characterized isoform (11-OHSD-1) in kinetic parameters, cofactor dependency, and reversibility of the reaction. Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation of endogenous glucocorticoids, has a very high affinity for its substrate, and is located in mineralocorticoid target cells; it thus appears well suited to "protect" the mineralocorticoid receptors from occupancy by glucocorticoids. As a first step in attempting to isolate the cDNA for the 11-OHSD/CD isoform, we isolated mRNA from immunodissected cortical collecting duct (CCD) cells and characterized the 11-OHSD in oocytes injected with this mRNA. Water-injected oocytes had no measurable 11-OHSD activity. In contrast, oocytes injected with as little as 1 ng CCD mRNA expressed detectable 11-OHSD activity. Expression of 11-OHSD activity was dependent on the amount of mRNA injected and was maximal with 30 ng mRNA. Similar to the findings in CCD cells, the expressed enzyme preferred NAD over NADP (activity was 0.46 +/- 0.04 and 0.011 +/- 0.01 fmol.min-1.oocyte-1 with 0.1 mM NAD and NADP, respectively). The Michaelis constant (Km) for corticosterone was 11.5 +/- 3.7 nM. Similar to the findings in CCD cells, the expressed enzyme worked predominantly in the oxidative direction, as back-conversion of [3H]dehydrocorticosterone to corticosterone was negligible. (ABSTRACT TRUNCATED AT 250 WORDS)