Abstract

The present study focused on the main hypothesis that age-related changes in myofilament Ca2+ sensitivity involve simultaneous changes in the relations between cytosolic Ca2+ transients, myosin phosphorylation, and force development. To define the effects of late fetal and early postnatal maturation, parallel experiments were carried out using cerebral arteries from both term lamb fetuses (140-141 days of gestation) and nonpregnant adult sheep. Fetal and adult cerebral arteries were stimulated by either mechanically, electromechanically, and/or pharmacomechanically-induced contractions, and the relations of cytosolic Ca2+, MLC phosphorylation, and contractile tensions were measured. These measurements allowed for stimulus and age-dependent assessment of overall myofilament Ca2+ sensitivity. The study adopted the model proposed by Murphy and colleagues that states that myofilament Ca2+ sensitivity is the sum of the of the relations between cytosolic Ca2+ and myosin light chain phosphorylation, thick-filament regulation, as well the relations between MLC phosphorylation and contractile tension, thin-filament regulation. In particular, the study focused on stimulus-dependent changes in the contributions of thick-filament and thin-filament regulation to overall myofilament Ca2+ sensitivity in fetal and adult cerebral arteries.

The first part of our study focused on the mechanisms that govern myogenic tone and examined the hypothesis that myogenic tone is less dependent on Ca2+ influx and more dependent on myofilament Ca2+ sensitization in term fetal compared to adult cerebral arteries. The data demonstrated that term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses with peak tensions averaging 24% and 26% of Kmax at optimum stretch ratios (working diameter / unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic [Ca++] at stretch ratios > 2.0 in adult arteries, but increased [Ca++] only at stretch ratios > 2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %Kmax values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%). Conversely, adult %Kmax values at peak myogenic tone were significantly less at pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced MLC phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The stretch-ratios yielding half-maximal MLC phosphorylation were also similar in fetal (1.68) and adult (1.64) arteries. Overall, the data revealed that the cerebrovascular myogenic response is highly conserved during postnatal maturation and this effect my be attributable to enhanced thin-filament regulation, particularly in fetal cerebral arteries.

Our second study tested the hypothesis that age-dependent changes in patterns of agonist-induced myofilament Ca2+ sensitization involve corresponding differences in the relative contributions of thick-filament and thin-filament regulation to overall myofilament Ca2+ sensitivity. Once again, fetal and adult cerebral arteries were used in the measurements of cytosolic Ca2+, MLC phosphorylation, and contractile tensions induced by varying concentrations of K+ or serotonin (5-HT) to assess the to assess the relative contributions of thick-filament and thin-filament reactivity, to overall myofilament Ca2+ sensitivity. For K+-induced contractions, both fetal and adult arteries exhibited similar basal myofilament Ca2+ sensitivity that included a slight increase in thick-filament reactivity in fetal arteries and small increase in thin-filament reactivity in adult arteries. In contrast, 5-HT-induced contractions exhibited increased myofilament Ca2+ sensitivity compared to K+-induced contractions for both fetal and adult cerebral arteries, and the magnitude of this effect was greater in fetal compared to adult arteries. This effect was attributable to agonist-enhancement of thick-filament reactivity in fetal arteries possibly mediated by the coupling of G-protein receptor activation and PKC-independent but Rho-A dependent rho kinase and/or PAK activation. In adult arteries, agonist-enhanced thin-filament reactivity was possibly mediated thru G-protein coupled activation of Rho-A dependent activation of p21-activated protein kinase as well as the regulation of actin regulatory proteins in adult arteries. Overall, the present data demonstrated that agonist-enhanced myofilament Ca2+ sensitivity more in fetal compared to adult cerebral arteries and that this effect was mediated possibly by the coupling of G-protein activation and subsequent Rho-A activation.

Overall, the results suggest that the regulation of cerebrovascular contractility is age-dependent and differs between mechanical, electromechanical, and pharmacomechanical-induced contractions in fetal and adult cerebral arteries. In particular, the stimulus and age-dependent contribution of thick-filament and thin-filament regulation to overall myofilament Ca2+ sensitivity vary with age in ovine cerebral arteries.

LLU Discipline

Pharmacology

Department

Pharmacology

School

Graduate Studies

First Advisor

William J. Pearce

Second Advisor

John N. Buchholz

Third Advisor

William T. Gerthoffer

Fourth Advisor

Lawrence D. Longo

Fifth Advisor

Lubo Zhang

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2007

Date (Title Page)

5-2007

Language

English

Library of Congress/MESH Subject Headings

Cerebral Arteries -- pharmacology; Actin Cytoskeleton -- drug effects; Myosin Light Chains; Sarcoplasmic Reticulum Calcium-Transporting ATpases

Type

Dissertation

Page Count

xi; 12

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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