In many different species, fetal and newborn cerebral arteries are more reactive to contractile amines than are adult cerebral arteries, a fact which may have direct bearing on the high incidence of hypoxic/ischemic encephalopathy following intracranial hemorrhage in neonates. In ovine cerebral arteries, for example, the ED50 for serotonin is more than an order of magnitude lower in newborns than adults. The purpose of the present application is to identify the mechanisms responsible for this age-related difference in reactivity. In these studies, we will systematically quantitate each of the four main factors which together determine the dose-response relation for, serotonin including: 1) receptor affinity; 2) receptor density; 3) receptor gain; and 4) second messenger sensitivity. Receptor affinity will be measured in intact arteries using the method of partial irreversible blockade. Receptor density will be measured in freshly dispersed whole smooth muscle cells by saturation binding of 3H-Ketanserin. Receptor gain will be measured for the first time in any tissue using a novel approach based on timed measurements of the inositol 1,4,5 trisphosphate (IP3) mass mobilized in response to varying concentrations of serotonin. IP3 mass will be measured using a highly sensitive protein binding assay recently developed in the applicant's laboratory, and the total IP3 mobilized will be quantitated by integrating the area beneath the curve relating IP3 mass to the time of exposure to serotonin. The resulting values of IP3 area will then be plotted against their corresponding serotonin concentrations which, in turn, will be converted into values of %occupancy using the Furchgott equation and the previous measures of receptor affinity. The values of %occupancy will then be further converted into values of fmol receptor bound by multiplying %occupancy times the corresponding Bmax values obtained from the receptor density measurements. This final calculation will yield a plot relating IP3 area to fmol receptors bound, and the slope of this curve at any point will be defined as receptor gain, in units of IP3 area per fmol receptor bound. Finally, second messenger sensitivity will be quantitated by normalizing the IP3 mass values relative to intracellular water, measured using a recently developed 57Co-EDTA method. These values of IP3 values in units of pmol/liter cell water will then be plotted against corresponding values of contractile tension, which will be measured simultaneously during IP3 mobilization studies. This IP3-tension dose-response curve will then be used to determine values of ED5O and maximum response for IP3. Parallel measurements of receptor affinity, receptor density; receptor gain; and second messenger sensitivity will be made in common carotid and middle cerebral arteries from both newborn (3- 7d) and adult non-pregnant sheep, and values of these variables will be compared across age and artery type to determine how each factor is modulated by maturation.
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