Purification and characterization of beta-adrenergic receptor mRNA-binding proteins.

Beta-adrenergic receptors (beta-ARs), like other G-protein-coupled receptors, can undergo post-transciptional regulation at the level of mRNA stability. In particular, the human beta(1)- and beta(2)-ARs and the hamster beta(2)-AR mRNA undergo beta-agonist-mediated destabilization. By UV cross-linking, we have previously described an approximately M(r) 36,000 mRNA-binding protein, betaARB, that binds to A/C+U-rich nucleotide regions within 3'-untranslated regions. Further, we have demonstrated previously that betaARB is immunologically distinct from AUF1/heterogeneous nuclear ribonucleoprotein (hnRNP) D, another mRNA-binding protein associated with destabilization of A+U-rich mRNAs (Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B. C., Minobe, W., Sherman, J. A., Bisognano, J., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501). In this report, we describe the peptide composition of betaARB. Mass spectrometric analysis of an approximately M(r) 36,000 band isolated from ribosomal salt wash proteins revealed the presence of two mRNA-binding proteins, hnRNP A1, and the elav-like protein, HuR, both of which are known to bind to A+U-rich nucleotide regions. By immunoprecipitation, HuR appears to be the biologically dominant RNA binding component of betaARB. Although hnRNP A1 and HuR can both be immunoprecipitated from ribosomal salt wash proteins, the composition of betaARB (HuR alone versus HuR and hnRNP A1) appears to be dependent on the mRNA probe used. The exact role of HuR and hnRNP A1 in the regulation of beta-AR mRNA stability remains to be determined.

A comparison of pulmonary arterial occlusion algorithms for estimation of pulmonary capillary pressure.

Using the arterial occlusion method, we compared five literature-based estimates of pulmonary capillary pressure (Ppc) with the corresponding double occlusion pressures (Pdo) in anesthetized dogs whose chests had been closed after sternotomy for instrumentation. Arterial occlusions were performed with a balloon-tipped pulmonary artery catheter that housed pressure transducers immediately proximal and distal to the balloon. Separation of the proximal and distal pressure waveforms during balloon inflation allowed us to precisely define the moment of occlusion. We fit a monoexponential curve to pressure data beginning 200 ms after the onset of occlusion and a biexponential curve to data beginning at the instant of occlusion, with data obtained over a range of vascular states (control, serotonin infusion, histamine infusion). In addition, we investigated the use of sampling of the raw data to estimate capillary pressure. Three of the five literature-based estimates of Ppc yielded values similar to Pdo. The optimal (least average difference from Pdo) interpolation/extrapolation time points of the curve fits varied, depending on the type of curve fitting and the state of the pulmonary vasculature. We also determined that a close approximation of Pdo may be derived from the raw data, as an alternative to exponential curve fitting.

Hypoxemia and hypoxic pulmonary vasoconstriction: autonomic nervous system versus mixed venous PO2.

Hypoxemia interferes with hypoxic pulmonary vasoconstriction (HPV). We investigated the respective roles of the autonomic nervous system and the mixed venous PO2 (PVO2) in the attenuation of HPV by hypoxemia. Pentobarbital-anesthetized dogs had their lungs separately ventilated with a dual-lumen endotracheal tube. Left (Ql) and total (Qt) pulmonary blood flows were determined using electromagnetic flow probes. HPV was initiated by ventilating the left lung with nitrogen for 5-10 min while the right lung received 100% oxygen. The animals were subsequently made hypoxemic by switching the right lung to room air ventilation (5-10 min). Two different protocol groups received either intravenous atropine during hypoxemia (group I) or intravenous propranolol prior to protocol initiation (group II). A third group of dogs (group III) had their mixed venous PO2S maintained above 30 torr during hypoxemia. In response to left lung hypoxia, Ql/Qt decreased from 44 +/- 5, 48 +/- 3 and 46 +/- 2% to 25 +/- 4, 28 +/- 2 and 26 +/- 3% in the three groups, respectively. During hypoxemia Ql/Qt increased to 50 +/- 7 and 47 +/- 3% in groups I and II. In group III dogs, Ql/Qt remained significantly decreased at 31 +/- 3%. Subsequent administration of atropine in group I had no effect on Ql/Qt. We conclude that the loss of flow diversion from a hypoxic lung during hypoxemia may be mediated primarily by a decreased in mixed venous PO2 when PVO2 is allowed to decrease to 15-20 torr.

Endothelin produces systemic vasodilation independent of the state of consciousness.

The effects of endothelin, ET-1, on pulmonary and systemic hemodynamics were studied in the open chest dog and changes in systemic arterial pressure in dogs under conscious and anesthetized states were compared. Rapid intravenous (IV) bolus injections of ET-1, 100-1,000 nanograms/kg, significantly decreased systemic arterial pressure, and significantly decreased systemic vascular resistance whereas left atrial pressure and pulmonary vascular resistance were not altered. Reductions in systemic arterial pressure in response to bolus injection of ET-1, 100 and 300 nanograms/kg IV, during conscious state and during anesthesia were similar, respectively. The present data suggest that ET-1 dilates the systemic vascular bed independent of the animal's state of consciousness. The present data also suggest that when compared to the systemic vascular bed, the pulmonary vascular bed is less responsive to bolus administration of ET-1.