Carbon monoxide (CO) is produced by catabolism of heme via heme oxygenase (HO). CO dilates cerebral arterioles by activating Ca activated K (Kca) channels. The constitutive HO isoform, HO-2, is highly expressed in astrocytes that coat cerebral arterioles and have glutamate receptors. This is important because CO is a mediator of newborn cerebral vasodilatation in response to glutamatergic neuronal activity. Although microvascular cells can produce CO, the physiological origin of CO that regulates cerebral vascular tone is unknown. The proposed research will pursue the hypothesis that perivascular astrocytes HO-2 makes CO to control newborn cerebrovascular circulation during glutamatergic stimulation.
Three specific aims will be addressed: 1. Compare, in vivo, contributions of astrocytes, neurons, and endothelium to cerebral CO production and pial arteriolar dilation in response to glutamate, 2. Determine, in vitro, causal relationships among glutamate, cystolic Ca transients, global Ca, and astrocytes production of CO, 3. Investigate the ability of CO produced by astrocytes to activate vascular smooth muscle KCa channels. Techniques allowing investigation of intact cerebral microcirculation, intact neurovascular units in brain slices, freshly isolated astrocytes, astrocytes in primary culture, and freshly isolated cerebral arteriolar myocytes alone and in combination with astrocytes will be employed. Such research is unique by studying intact cerebral circulation and investigating, at the cellular level, the mechanisms regulating astrocytes production of a gasotransmitter, and how such astrocytes production can affect adjacent vascular smooth muscle to alter vascular tone. Cranial windows allow investigation of intact cerebral circulation, monitoring of brain surface CO production, and topical application of agonists and inhibitors. Freshly isolated astrocytes and astrocytes grown on microcarrier beads allow study of the mechanisms involved in glutamate stimulation of CO production. Global cystolic Ca and Ca transients in brain slices, astrocytes, cerebrovascular myocytes, and reunited astrocytes and myocytes will be studied using fluorescent indicator technology with a dual excitation, single emission system and laser scanning confocal microscopy, respectively. Patch clamp techniques will be used to examine KCa channel activity. Cerebral circulatory disorders in newborns are major causes of morbidity and mortality and can result in life long disabilities. Control of cerebrovascular circulation is impaired by pathological conditions. Better understanding of mechanisms of cerebromicrovascular humoral communication is needed. ? ? ?
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