Biomedical subjects

M Keith Wilkerson

Publications and source records attributed to M Keith Wilkerson.

7 recordsLinked to original sources

PubMed2006

Endothelium-dependent vasodilation of cerebral arteries is altered with simulated microgravity through nitric oxide synthase and EDHF mechanisms.

Cephalic elevations in arterial pressure associated with microgravity and prolonged bed rest alter cerebrovascular autoregulation in humans. Using the head-down tail-suspended (HDT) rat to chronically induce headward fluid shifts and elevate cerebral artery pressure, previous work has likewise shown cerebral perfusion to be diminished. The purpose of this study was to test the hypothesis that 2 wk of HDT reduces cerebral artery vasodilation. To test this hypothesis, dose-response relations for endothelium-dependent (2-methylthioadenosine triphosphate and bradykinin) and endothelium-independent (nitroprusside) vasodilation were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (10(-5) M) and cyclooxygenase inhibitor indomethacin (10(-5) M). MCA caveolin-1 protein content was measured by immunoblot analysis. Endothelium-dependent vasodilation to 2-methylthioadenosine triphosphate and bradykinin were both lower in MCAs from HDT rats. These lower vasodilator responses were abolished with N(G)-nitro-L-arginine methyl ester but were unaffected by indomethacin. In addition, HDT was associated with lower levels of MCA caveolin-1 protein. Endothelium-independent vasodilation was not altered by HDT. These results indicate that chronic cephalic fluid shifts diminish endothelium-dependent vasodilation through alterations in the endothelial nitric oxide synthase signaling mechanism. Such decrements in endothelium-dependent vasodilation of cerebral arteries could contribute to the elevations in cerebral vascular resistance and reductions in cerebral perfusion that occur after conditions of simulated microgravity in HDT rats.

Adenosine Triphosphate↗

PubMed2006

Local potassium signaling couples neuronal activity to vasodilation in the brain.

The mechanisms by which active neurons, via astrocytes, rapidly signal intracerebral arterioles to dilate remain obscure. Here we show that modest elevation of extracellular potassium (K+) activated inward rectifier K+ (Kir) channels and caused membrane potential hyperpolarization in smooth muscle cells (SMCs) of intracerebral arterioles and, in cortical brain slices, induced Kir-dependent vasodilation and suppression of SMC intracellular calcium (Ca2+) oscillations. Neuronal activation induced a rapid (<2 s latency) vasodilation that was greatly reduced by Kir channel blockade and completely abrogated by concurrent cyclooxygenase inhibition. Astrocytic endfeet exhibited large-conductance, Ca2+-sensitive K+ (BK) channel currents that could be activated by neuronal stimulation. Blocking BK channels or ablating the gene encoding these channels prevented neuronally induced vasodilation and suppression of arteriolar SMC Ca2+, without affecting the astrocytic Ca2+ elevation. These results support the concept of intercellular K+ channel-to-K+ channel signaling, through which neuronal activity in the form of an astrocytic Ca2+ signal is decoded by astrocytic BK channels, which locally release K+ into the perivascular space to activate SMC Kir channels and cause vasodilation.

Animals↗

PubMed2006

Dynamic inositol trisphosphate-mediated calcium signals within astrocytic endfeet underlie vasodilation of cerebral arterioles.

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca(2+)](i) increases in astrocytic processes ("endfeet"), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca(2+)](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca(2+)](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca(2+) indicator and confocal microscopy. Increases in endfoot [Ca(2+)](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca(2+)](i) and vascular diameter. Neuronal activity-evoked elevation of endfoot [Ca(2+)](i) was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP(3)) receptor Ca(2+) release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca(2+) uptake. To probe the Ca(2+) release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP(3) was utilized to liberate InsP(3) directly within endfeet. This maneuver generated large amplitude [Ca(2+)](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)-induced [Ca(2+)](i) increases were sensitive to depletion of the intracellular Ca(2+) store, but not to ryanodine, suggesting that Ca(2+)-induced Ca(2+) release from ryanodine receptors does not contribute to the generation of endfoot [Ca(2+)](i) signals. Neuronally evoked increases in astrocytic [Ca(2+)](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca(2+)](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca(2+) release processes within the endfeet were evident, as were localized regions of Ca(2+) release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca(2+)](i) increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP(3) within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local "vasoregulatory units" by translating information from active neurons into complex InsP(3)-mediated Ca(2+) release signals that modulate arteriolar diameter.

Animals↗

PubMed2005

Inositol trisphosphate receptor calcium release is required for cerebral artery smooth muscle cell proliferation.

Vascular damage signals smooth muscle cells to proliferate, often exacerbating existing pathologies. Although the role of changes in "global" Ca2+ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role of specific Ca2+ signals in determining VSM phenotype remains relatively unexplored. Earlier work with cultured VSM cells suggests that inositol 1,4,5-trisphosphate receptor (IP3R) expression and sarcoplasmic reticulum (SR) Ca2+ release may be linked to VSM cell proliferation in native tissue. Thus we hypothesized that SR Ca2+ release through IP3Rs in the form of discrete transient signals is necessary for VSM cell proliferation. To investigate this hypothesis, we used mouse cerebral arteries to design an organ culture system that permitted examination of Ca2+ dynamics in native tissue. Explanted arteries were cultured in normal medium with 10% FBS, and appearance of individual VSM cells migrating from explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca2+ waves in myocytes in the arteries that were blocked by the IP3R antagonist 2-aminoethoxydiphenylborate (2-APB). Inhibition of IP3R opening (via 100 microM 2-APB, 10 microM xestospongin C, or 25 microM U-73122) dramatically reduced outgrowth cell number compared with untreated or ryanodine-treated (10 microM) arteries. Consistent with this finding, 2-APB inhibited cell proliferation, as measured by reduced proliferating cell nuclear antigen immunostaining within 48 h of culture but did not inhibit cell migration. These results indicate that activation of IP3R Ca2+ release is required for VSM cell proliferation in these arteries.

Animals↗

PubMed2004

Simulated microgravity enhances cerebral artery vasoconstriction and vascular resistance through endothelial nitric oxide mechanism.

Elevations in arterial pressure associated with hypertension, microgravity, and prolonged bed rest alter cerebrovascular autoregulation in humans. Using head-down tail suspension (HDT) in rats to induce cephalic fluid shifts and elevate arterial pressure, this study tested the hypothesis that 2-wk HDT enhances cerebral artery vasoconstriction and that an enhanced vasoconstriction described in vitro will alter regional cerebral blood flow (CBF) and vascular resistance (CVR) during standing and head-up tilt. To test this hypothesis, basal tone and vasoconstrictor responses to increases in transmural pressure, shear stress, and K(+) were determined in vitro in middle cerebral arteries (MCAs) from HDT and control rats. All in vitro measurements were done in the presence and absence of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-5) M) and with endothelium removal. Endothelial NOS (eNOS) mRNA and protein expression levels were measured by RT-PCR and immunoblot, respectively. Regional CBF and CVR were determined with a radiolabeled tracer technique and quantitative autoradiography. Basal tone and all vasoconstrictor responses were greater in MCAs from HDT rats. L-NAME and endothelium removal abolished these differences between groups, and HDT was associated with lower levels of MCA eNOS protein. CBF in select regions was lower and CVR higher during standing and head-up tilt in HDT rats. These results indicate that chronic cephalic fluid shifts enhanced basal tone and vasoconstriction through alterations in the eNOS signaling mechanism. The functional consequence of these vascular alterations with HDT is regional elevations in CVR and corresponding reductions in cerebral perfusion.

Animals↗

PubMed2003

Constitutively elevated nuclear export activity opposes Ca2+-dependent NFATc3 nuclear accumulation in vascular smooth muscle: role of JNK2 and Crm-1.

The transcription factor NFAT (nuclear factor of activated T-cells) is a cytosolic phosphoprotein that accumulates in the nucleus following dephosphorylation by the calcium (Ca2+)/calmodulin-dependent phosphatase, calcineurin. A defining feature of stimuli that induce NFAT nuclear accumulation/activation is a sustained increase in global intracellular Ca2+. Contrary to expectations, we have found that a sustained elevation of intracellular Ca2+, induced by membrane potential depolarization and mediated by voltage-dependent Ca2+ channels, does not result in nuclear localization of the NFATc3 isoform in smooth muscle. However, vasoconstrictors (e.g. uridine triphosphate (UTP)) and growth factors, which elevate intracellular Ca2+ and engage multiple intracellular signaling pathways, induce a robust increase in smooth muscle nuclear NFATc3. Here we show that depolarizing stimuli that normally fail to induce NFATc3 nuclear accumulation in arterial smooth muscle effectively induce nuclear accumulation under conditions in which Crm-1-dependent or JNK2-mediated nuclear export processes are disrupted. Consistent with an important regulatory role for JNK, UTP exerts a suppressive effect on JNK activity in smooth muscle. Export of nuclear NFATc3 following UTP-induced nuclear accumulation is dramatically slowed in cerebral arteries from JNK2-/- animals. These data indicate that in smooth muscle, stimulation of Ca2+-dependent, calcineurin-mediated nuclear import and suppression of Crm-1/JNK-dependent nuclear export are both required for induction of NFATc3 nuclear accumulation. These results highlight the dynamic interplay between influences that promote and oppose NFAT nuclear accumulation and suggest that in arterial smooth muscle suppression of constitutive nuclear export activity is an important property of NFAT-activating stimuli.

Active Transport, Cell Nucleus↗

PubMed2002

Acute and chronic head-down tail suspension diminishes cerebral perfusion in rats.

The purpose of this study was to test the hypothesis that regional brain blood flow and vascular resistance are altered by acute and chronic head-down tail suspension (HDT). Regional cerebral blood flow, arterial pressure, heart rate, and vascular resistance were measured in a group of control rats during normal standing and following 10 min of HDT and in two other groups of rats after 7 and 28 days of HDT. Heart rate was not different among conditions, whereas mean arterial pressure was elevated at 10 min of HDT relative to the other conditions. Total brain blood flow was reduced from that during standing by 48, 24, and 27% following 10 min and 7 and 28 days of HDT, respectively. Regional blood flows to all cerebral tissues and the eyes were reduced with 10 min of HDT and remained lower in the eye, olfactory bulbs, left and right cerebrum, thalamic region, and the midbrain with 7 and 28 days of HDT. Total brain vascular resistance was 116, 44, and 38% greater following 10 min and 7 and 28 days of HDT, respectively, relative to that during control standing. Vascular resistance was elevated in all cerebral regions with 10 min of HDT and remained higher than control levels in most brain regions. These results demonstrate that HDT results in chronic elevations in total and regional cerebral vascular resistance, and this may be the underlying stimulus for the HDT-induced smooth muscle hypertrophy of cerebral resistance arteries.

Animals↗