Regional differences in endothelin converting enzyme activity in rat brain: inhibition by phosphoramidon and EDTA.

1. It has been demonstrated previously that conversion of big endothelin-1 (bET-1) to endothelin-1 (ET-1) is inhibited in vitro and in vivo by phosphoramidon. In addition, ET-1 binding sites and mRNA have been shown within the brain. Here we expand upon our previous observation that rat brain contains phosphoramidon-inhibitable endothelin converting enzyme (ECE) and show that this activity is not uniformly distributed throughout the brain. 2. ECE activity was detected by a bioassay which depended upon the 10,000 fold difference in potency between bET-1 and ET-1 as stimulants of guanosine 3':5'-cyclic monophosphate (cyclic GMP) accumulation in kidney epithelial (PK1) cells of the pig. Data were confirmed by specific enzyme-linked immunosorbent assay (ELISA) employing antibody directed against ET-1/3(17-21). 3. Following homogenization of the whole brain and ultracentrifugation the 100,000 g pellet contained greater than 4 times more ECE activity than the cytosol. Washing of the pellet with KCl (1 M) and extraction with the detergent CHAPS (20 mM) revealed a phosphoramidon-inhibitable ECE within the residual particulate fraction (nominally classified as the cytoskeletal fraction). Phosphoramidon (IC50, approx. 5 microM) or EDTA inhibited the conversion of bET-1 to ET-1 by the cytoskeletal fraction of rat brain by more than 60%.2+ 4. Following dissection of rat brain into olfactory bulb, cerebral cortex, striatum, hippocampus, cerebellum, midbrain (including thalamus), hypothalamus and medulla oblongata (including pons) the greatest ECE was detected in the hypothalamus and medulla oblongata.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium Concentration-dependent Mechanisms through which Ketamine Relaxes Canine Airway Smooth Muscle

Background: Ketamine is a potent bronchodilator that, in clinically used concentrations, relaxes airway smooth muscle in part by a direct effect. This study explored the role of calcium concentration (Ca2+) in this relaxation.

Methods: Canine trachea smooth muscle strips were loaded with the fluorescent probe fura-2 and mounted in a spectrophotometric system to measure force and intracellular calcium concentration ([Ca2+]i) simultaneously. Calcium influx was estimated using a manganese quenching technique. Cyclic nucleotides in the airway smooth muscle strips were measured by radioimmunoassay.

Results: In smooth muscle strips stimulated with submaximal (0.1 micro Meter) and maximal (10 micro Meter) concentrations of acetylcholine, ketamine caused a concentration-dependent decrease in force and [Ca2+]i. The sensitivity of the force response to ketamine significantly decreased as the intensity of muscarinic receptor stimulation increased; the median effective concentration for relaxation induced by ketamine was 59 micro Meter and 850 micro Meter for tissue contracted by 0.1 micro Meter or 10 micro Meter acetylcholine, respectively (P < 0.05). In contrast, the sensitivity of the [Ca2+] sub i response did not depend on the intensity of muscarinic receptor stimulation. Ketamine at 1 mM significantly inhibited calcium influx. Ketamine did not significantly increase cyclic nucleotide concentrations.