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K G Morgan

Publications and source records attributed to K G Morgan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Coronary microvascular protection with mg2+: effects on intracellular calcium regulation and vascular function.

The use of Mg2+-supplemented hyperkalemic cardioplegia preserves microvascular function. However, the mechanism of this beneficial action remains to be elucidated. We investigated the effects of Mg2+ supplementation on the regulation of intracellular calcium concentration ([Ca2+]i) and vascular function using an in vitro microvascular model. Ferret coronary arterioles (80-150 micrometer in diameter) were studied in a pressurized (40 mmHg) no-flow, normothermic (37 degrees C) state. Simultaneous monitoring of internal luminal diameter and [Ca2+]i using fura 2 were made with microscopic image analysis. The microvessels (n = 6 each group) were divided into four groups according to the content of MgCl2 (nominally 0, 1.2, 5.0, and 25.0 mM) in a hyperkalemic cardioplegic solution ([K+] 25.0 mM). After baseline measurements, vessels were subjected to 60 min of hypoxia with hyperkalemic cardioplegia (equilibrated with 95% N2-5% CO2) containing each concentration of Mg2+ ([Mg2+]) and were then reoxygenated. During hyperkalemic cardioplegia, [Ca2+]i increased in a time-dependent manner in all groups. In the lower [Mg2+] cardioplegia groups, [Ca2+]i was significantly increased at the end of the 60-min cardioplegic period (247 +/- 44 nM and 236 +/- 49 nM in [Mg2+] 0 and 1.2 mM groups, respectively; both P < 0.05 vs. baseline) with 19.6-17.2% vascular contraction. Conversely, there was no significant [Ca2+]i increase in the higher [Mg2+] cardioplegia groups and less vascular contraction (5.4-4.1%, both P < 0.05 vs. [Mg2+] 1.2 mM group). After reperfusion, agonist (U-46619, thromboxane A2 analog)-induced vascular contraction was significantly enhanced in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was normalized in the higher [Mg2+] cardioplegia groups. Intrinsic myogenic contraction was significantly decreased in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was preserved in the higher [Mg2+] cardioplegia groups. These results suggest that supplementation of the solution with >5.0 mM [Mg2+] may prevent hyperkalemic cardioplegia-related intracellular Ca2+ overloading and preserve vascular contractile function in coronary microvessels.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1999

Influence of oxygenation on endothelial modulation of coronary vasomotor function during hyperkalemic cardioplegia.

BACKGROUND: The purpose of this study was to determine the influence of oxygenation of a hyperkalemic cardioplegic solution (K-CP) on endothelial modulation of vasomotor tone and to correlate these changes with the intracellular calcium concentration ([Ca++]i) in microvascular smooth muscle. METHODS: Rat coronary arterioles were studied in a pressurized, no-flow normothermic state. Simultaneous monitoring of luminal diameter and [Ca++]i (fura-2) was performed with use of microscopic image analysis. Vessels were subjected to 60 minutes of oxygenated or hypoxic K-CP (K+ = 25.0 mmol/L) and were then reperfused with oxygenated Krebs-physiologic saline solution for 60 minutes. RESULTS: In oxygenated K-CP, the K-CP-induced contraction and [Ca++]i accumulation were significantly increased in endothelium-denuded (ED) vessels compared with endothelium-intact vessels. The effect of ED in oxygenated K-CP was mimicked by administration of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine. Conversely, in hypoxic K-CP the contraction was significantly attenuated in ED vessels compared with endothelium-intact vessels, although there was no significant difference in [Ca++]i. Indomethacin did not affect the endothelium-dependent contraction during hypoxic K-CP. CONCLUSIONS: Endothelium-derived nitric oxide modulates the vascular tone during K-CP by regulating the vascular smooth muscle [Ca++]i, whereas endothelium-derived contracting factor(s), which is not predominantly a product of cyclo-oxygenase, may play a prominent role under hypoxic K-CP by increasing vascular smooth muscle Ca++ sensitivity.

Animals↗
PubMed1998

Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret.

1. Biochemical and quantitative image analysis methods were used to investigate the anatomical basis for the previously described agonist-induced redistribution of calponin. 2. At 140 nm resolution, the quantitative distribution of calponin in resting cells was statistically indistinguishable from that of filament bundles containing alpha-smooth muscle actin and myosin, but was significantly different from that of filaments containing beta-non-muscle actin. Conversely, in stimulated cells, the distribution of calponin was not significantly different from that of beta-actin filaments in the subplasmalemmal cell cortex but was significantly different from the distribution of alpha-actin- and myosin-containing filamentous bundles. 3. The distribution of calponin significantly differed from that of the intermediate filament proteins vimentin and desmin as well as that of the dense body protein alpha-actinin either by ratio analysis of the subcellular distribution or by colocalization analysis. 4. The imaging results, although limited to 140 nm spatial resolution, suggested the hypothesis that the agonist-induced redistribution involves the binding of calponin to isoform-specific actin filaments. This hypothesis was tested by quantifying the relative affinity of calponin for purified alpha- and beta-actin. Light scattering measurements showed that calponin induces bundle formation with beta-actin more readily than alpha-actin, indicating that calponin may be preferentially sequestered by beta-actin under appropriate conditions. 5. These results are consistent with a model whereby agonist activation decreases calponin's binding to filaments, but the tighter binding to beta-actin filaments results in a spatial redistribution of calponin to the submembranous cortex.

Actins↗
PubMed1998

PKC-dependent signalling mechanisms in differentiated smooth muscle.

Protein kinase C (PKC) is now known to play an important physiological role in essentially all cell types. This review will focus on what is known about the kinase in contractile differentiated smooth muscle. Current knowledge on the molecular structure of PKC isoforms will be discussed as they relate to mechanisms of translocation and targeting of the kinase within smooth muscle cells. Studies performed on PKC-dependent signalling pathways in differentiated smooth muscle cells will be discussed with emphasis on studies form our laboratory, especially discussing thin filament linked pathways. Thick filament linked PKC-dependent pathways will be described in more detail elsewhere in this monograph.

Animals↗
PubMed1998

A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha-adrenoceptor stimulation.

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in smooth muscle contraction by monitoring MAP kinase activation, caldesmon phosphorylation, and contractile force during agonist stimulation. Isometric tension in response to KCl and phenylephrine (PE) was measured from strips of ferret aorta. MAP kinase activation was monitored by Western blot using a phosphospecific p44/p42 MAP kinase antibody. Caldesmon phosphorylation was assessed using specific phosphocaldesmon antibodies. We report here that treatment of smooth muscle strips with PD-098059, a specific inhibitor of MAP kinase kinase, did not detectably modify the KCl-evoked contraction but significantly inhibited the contraction to PE in the absence of extracellular Ca2+. In this experimental condition, where the contraction occurs in the absence of increases in 20-kDa myosin light chain phosphorylation, PD-098059 also inhibited significantly MAP kinase and caldesmon phosphorylation. Collectively, these results demonstrate a direct cause-and-effect relationship between MAP kinase activation and Ca2+-independent smooth muscle contraction and support the concept of caldesmon phosphorylation as the missing link between both events.

Adrenergic alpha-Agonists↗
PubMed1997

Requirement for protein kinase C theta for cell cycle progression and formation of actin stress fibers and filopodia in vascular endothelial cells.

Activation of the protein kinase C (PKC) family with phorbol esters induces endothelial proliferation and angiogenesis, but which of the events that constitute angiogenesis are affected by individual members of the PKC family is unknown. In rat capillary endothelial (RCE) cells, serum stimulation increased expression of a single PKC isoenzyme, PKCtheta, and its translocation to the periphery. Conditional overexpression of a dominant-negative mutant of PKCtheta markedly inhibited RCE proliferation, as well as closure of a "wound" by RCE migration and formation of capillary rings and tubules in vitro. PKCtheta inhibition delayed the endothelial cell cycle at the G2/M phase and prevented formation of actin stress fibers and filopodia but not lamellipodia. The defect in cell morphology and wound closure in PKCtheta-kn cells was reversed by overexpressing kinase-active PKCtheta, indicating that these RCE functions depend upon PKCtheta substrates. Thus, PKCtheta is required for multiple processes essential for angiogenesis and wound repair, including endothelial mitosis, maintenance of a normal actin cytoskeleton, and formation of an enclosed tube.

Actins↗
PubMed1997

Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle.

Contraction of smooth muscle cells is generally assumed to require Ca2+/calmodulin-dependent phosphorylation of the 20-kDa myosin light chains. However, we report here that in the absence of extracellular calcium, phenylephrine induces a contraction of freshly isolated ferret aorta cells in the absence of increases in intracellular ionized calcium or light chain phosphorylation levels but in the presence of activation of mitogen-activated protein kinase. A protein at 36 kDa co-immunoprecipitated with the mitogen-activated protein kinase and was identified as the actin-binding protein, calponin, by immunoblot. An overlay assay further confirmed an interaction between the kinase and calponin, even though the kinase did not phosphorylate calponin in vitro. Calponin also co-immunoprecipitated from smooth muscle cells with protein kinase C-epsilon. High resolution digital confocal studies indicated that calponin redistributes to the cell membrane during phenylephrine stimulation at a time when mitogen-activated protein kinase and protein kinase C-epsilon are targeted to the plasmalemma. These results suggest a role for calponin as a signaling molecule, possibly an adapter protein, linking the targeting of mitogen-activated protein kinase and protein kinase C-epsilon to the surface membrane.

Animals↗
PubMed1997

Effect of metabolic inhibition on intracellular Ca2+, phosphorylation of myosin regulatory light chain and force in rat smooth muscle.

1. The effect of the inhibition of oxidative phosphorylation on intracellular calcium concentration ([Ca2+]i), phosphorylation of the 20 kDa regulatory light chain of myosin (MLC20) and contractility was investigated in isolated longitudinal smooth muscle from rat uteri. 2. Cyanide (2 mM) application to normally polarized preparations resulted in an elevation of basal [Ca2+]i but an inhibition of [Ca2+]i transients and the accompanying contractions. 3. Depolarization with high-K+ solution (40 mM KCI) resulted in elevation of [Ca2+]i and maintained force production. Phosphorylation of MLC20 was transiently increased followed by a steady-state augmentation above resting levels. 4. Carbachol (100 microM) produced a transient elevation of [Ca2+]i and force of depolarized tissues followed by a steady-state augmentation of both parameters. PGF2 alpha (1 microM) did not significantly potentiate [Ca2+]i or force in depolarized preparations. Both carbachol and PGF2 alpha potentiated phosphorylation of MLC20 in depolarized tissues. 5. Addition of cyanide to depolarized preparations, in the presence or absence of carbachol or PGF2 alpha, resulted in significant attenuation of force under each condition. The magnitude and normalized rates of force inhibition by cyanide were not significantly different for each stimulus condition. MLC20 phosphorylation levels were unaltered by cyanide treatment. However, cyanide increased the maintained level of [Ca2+]i under each experimental protocol. 6. It is concluded that the inhibition of oxidative phosphorylation with cyanide results in dissociation of both the [Ca2+]i-force and MLC20 phosphorylation-force relationships in rat uterine smooth muscle.

Animals↗
PubMed1997

F-actin disruption attenuates agonist-induced [Ca2+], myosin phosphorylation, and force in smooth muscle.

Cytochalasins B and D (at 10 microM) inhibited stress development induced by 1 microM carbachol in bovine tracheal smooth muscle by 55% and 90%, respectively. Glucose depletion was ineffective in inhibiting carbachol-induced contraction, indicating that inhibition of glucose transport was not the cause. Cytochalasin D-treated smooth muscle cells appeared collapsed, with spiky protrusions from the cell membrane. Deconvolution of fluorescent images of fluorescein isothiocyanate-phalloidin-labeled smooth muscle cells revealed concentrations of actin filaments near the cell periphery, including near the spiky protrusions. Cytochalasin B attenuated carbachol-induced intracellular Ca2+ concentration ([Ca2+]), especially the initial peak intracellular [Ca2+]. Cytochalasin B also attenuated carbachol-induced myosin light chain phosphorylation. However, when the myosin phosphorylation data were plotted against time-matched intracellular [Ca2+] data, the two relationships in control and cytochalasin B-treated smooth muscle were similar, suggesting that the changes in myosin phosphorylation could be explained by the changes in intracellular [Ca2+]. These results suggest that actin filaments in smooth muscle cells are dynamic and may be an integral component of Ca2+ regulation and/or signal transduction in receptor-coupled mechanisms.

Actins↗
PubMed1997

Inhibition of maxi-K currents in ferret portal vein smooth muscle cells by the antifungal clotrimazole.

The antifungal agent clotrimazole (CLT) is a potent small-molecule inhibitor of Ca-activated K (KCa) currents of intermediate conductance in murine erythroleukemia cells. This study demonstrates that CLT also inhibits large-conductance KCa currents (maxi-K currents) in acutely dissociated vascular smooth muscle (VSM) cells of ferret portal vein. The magnitude of block of a component of the whole cell K current by CLT was sensitive to test potential. CLT inhibited unitary maxi-K currents in outside-out patches, apparently by decreasing the mean open time. A metabolite of CLT lacking an imidazole ring also inhibited K currents. In contrast, the antifungal drug ketoconazole increased these same currents. Thus the inhibitory action of CLT appears to be due to a direct interaction with the channel protein rather than to imidazole block of cytochrome P-450 activity. Consistent with inhibition of maxi-K currents by CLT, superfusion of strips of portal vein VSM with CLT enhanced isometric tension and spontaneous rate of contraction, suggesting that CLT modulation of maxi-K currents may alter vasomotor functioning.

4-Aminopyridine↗
PubMed1996

Strong interaction between caldesmon and calponin.

Caldesmon was labeled at either Cys-153 in the NH2-terminal domain or Cys-580 in the COOH-terminal domain with a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan) fluorescence probe. The addition of smooth muscle calponin to Cys-580-labeled caldesmon resulted in an 18% drop in fluorescence intensity, which titrated with a stoichiometry of 0.9 and a binding constant of 9.5 x 10(7) M-1. For Cys-153-labeled caldesmon, there was no change in fluorescence upon adding calponin. These findings indicate strong binding between calponin and the COOH-domain of caldesmon. The association was sensitive to ionic strength, suggesting that ionic interactions between calponin, a basic protein, and caldesmon, an acidic protein, contribute to the stabilization of the protein complex. That non-muscle acidic calponin interacts with caldesmon with a much reduced association constant of 3.5 x 10(6) M-1 supports such a model. The binding between acidic calponin and caldesmon is strengthened to 1.8 x 10(7) M-1 in the presence of Ca2+, which might bind to acidic residues of the calponin and partially neutralize its negative charge. The strong, specific binding between calponin and caldesmon suggests that this interaction occurs within smooth muscle cells and possibly plays a role in the regulation of contraction.

Animals↗
PubMed1996

Intrinsic tone as potential vascular reserve in conductance and resistance vessels.

BACKGROUND: The purpose of this study was to define the degree of intrinsic tone in conductance and resistance vessels, to define the calcium dependency of intrinsic tone in these vascular preparations, and to investigate the efficacy of vasodilatory agents on the level of intrinsic tone in these vascular preparations. METHODS AND RESULTS: All vessels were deendothelialized. Isometric force was recorded from strips of ferret aorta, ferret pulmonary artery, and human coronary artery. Vessel diameter was recorded from the ferret epicardial coronary artery and from ferret coronary microvessel in a pressurized no-flow state. Intrinsic tone was defined as the active increase in force or decrease in diameter with warming from 6 degrees C to 37 degrees C. Changes in force or diameter with various pharmacological agents were expressed as a percentage of intrinsic tone. Our results indicate that intrinsic tone accounts for approximately 35% to 40% of total tone in all vascular preparations studied and is not dependent on extracellular calcium. Agents that increased cAMP levels (eg, forskolin, milrinone) and agents that decreased protein kinase C activity (eg, staurosporine) were partially effective in decreasing intrinsic tone. Nitroprusside, adenosine, hydralazine, and nifedipine had no significant effect. CONCLUSIONS: Our results indicate that intrinsic tone represents a significant component of vascular tone that has not been previously recognized and remains largely unexploited by current pharmacological therapies.

Animals↗
PubMed1996

Angiogenesis in normal, hyperplastic, and neoplastic endometrium.

The purpose of this study was to quantify vascular density in the stroma of normal, hyperplastic, and neoplastic endometria and to explore its relationship to other prognostic features of endometrioid adenocarcinoma. Curettage specimens of proliferative and mid-secretory endometrium; simple, complex, and atypical endometrial hyperplasia; and grade I endometrioid adenocarcinoma were stained with Factor VIII-related antigen. The number of vessels per mm2 of stroma was calculated for each case. The stroma of mid-secretory and hyperplastic endometrium was more vascular than that of proliferative endometrium. The stroma of adenocarcinoma, though reduced in proportion to the epithelium, was significantly more vascular than that of normal or hyperplastic endometrium. Stromal vascular density was not related to the depth of invasion, the presence of lymphovascular space permeation, or the state of the adjacent endometrium, whether atrophic or hyperplastic.

Adenocarcinoma↗
PubMed1996

Protein kinase C mediation of Ca(2+)-independent contractions of vascular smooth muscle.

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca(2+)-independent isoenzyme of PKC. Of the two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, identified in ferret aorta, PKC epsilon is more likely to mediate the contractile response because (i) PKC epsilon, but not PKC zeta, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKC epsilon translocates from the sarcoplasm to the sarcolemma, whereas PKC zeta, translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKC epsilon, but not PKC zeta, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKC epsilon is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of alpha, beta, and gamma isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKC epsilon but is a very poor substrate of PKC zeta. A signal transduction pathway is proposed to explain Ca(2+)-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKC epsilon would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (approximately 0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKC epsilon is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to alpha 1-adrenergic agonists than to K+.

Amino Acid Sequence↗
PubMed1996

Epsilon-isoenzyme of protein kinase C induces a Ca(2+)-independent contraction in vascular smooth muscle.

We provide here the first direct evidence for in situ functional specificity of protein kinase C (PKC)-epsilon as a regulator of smooth muscle contractility. PKC is known to cause a Ca(2+)-independent contraction of ferret aortic smooth muscle, and the expression of two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, has been demonstrated in this tissue. To test directly the hypothesis that one of these isoenzymes regulates contractility, constitutively active forms of PKC-epsilon and PKC-zeta were applied to saponin-permeabilized single ferret aortic smooth muscle cells. PKC-zeta caused no significant force response, but PKC-epsilon induced contraction of a magnitude (105 +/- 8 micrograms) similar to that produced by phenylephrine (110 +/- 10 micrograms), a relatively selective alpha 1-adrenergic agonist that triggers a PKC-dependent contraction. The PKC-epsilon-induced contraction was reversed by the PKC pseudosubstrate inhibitory peptide, PKC19-31. The myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) did not affect the force response of PKC-epsilon-activated cells, suggesting that PKC-epsilon may induce this contraction solely via thin filament disinhibition. In support of this conclusion, calponin and caldesmon were shown to be good in vitro substrates of PKC-epsilon but not of PKC-zeta.

Animals↗
PubMed1996

Mitogen-activated protein kinase and proliferation of human vascular smooth muscle cells.

The intracellular messenger mitogen-activated protein kinase (MAPK) is activated in vascular smooth muscle cells (SMC) by various growth factors as well as by agonists that have no proliferative effect. We explored the hypotheses that SMC proliferation is associated with a specific pattern of MAPK activation by evaluating the kinetics of MAPK activation and tyrosine phosphorylation and the intracellular location of MAPK in SMC following addition of agonists of varying mitogenic potential. A peak in MAPK activation and tyrosine phosphorylation occurred 3-10 min after the addition of agonists to SMC derived from human saphenous vein (early phase), followed by a plateau of activity, which was variable in duration (late phase). A correlation was not found between mitogenicity and the degree to which MAPK became activated or tyrosine phosphorylated in the early phase. However, the duration of MAPK activation and tyrosine phosphorylation correlated strongly with the ability of agonists to stimulate SMC proliferation. Nuclear translocation of MAPK was associated with SMC proliferation, although the degree to which each agonist induced nuclear translocation did not parallel its mitogenic potential. The relative dependency of all three events on protein kinase C differed for each agonist and was greater in the late versus the early phase. Thus, in human SMC, nuclear translocation of MAPK and prolonged activation and tyrosine phosphorylation of MAPK are associated with growth factor-induced mitogenesis.

Calcium-Calmodulin-Dependent Protein Kinases↗