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K G Morgan

Publications and source records attributed to K G Morgan.

At least 73 records · Page 4Linked to original sources
PubMed1993

Mechanisms of signal transduction during alpha 2-adrenergic receptor-mediated contraction of vascular smooth muscle.

Little is known about the signaling pathways involved in alpha 2-adrenergic receptor-mediated contraction of vascular smooth muscle. In the present study, we measured intracellular Ca2+ ([Ca2+]i), myosin light chain (MLC) phosphorylation, and myofilament Ca2+ sensitivity during stimulation with the relatively selective alpha 2-agonist UK 14304. These effects were compared and contrasted with corresponding changes during depolarization by elevation of the [K+] in the bathing medium. These studies were performed using spiral strips of the rabbit saphenous vein, a tissue with a relatively high density of postsynaptic alpha 2-receptors. UK 14304 (10(-5) M) caused parallel changes in [Ca2+]i, MLC phosphorylation, and force consisting of an initial phasic, followed by a sustained steady-state response. The steady-state increase in [Ca2+]i, MLC phosphorylation, and force caused by UK 14304 in the presence of 2.5 mM extracellular Ca2+ were indistinguishable from those during 51 mM K+ depolarization. However, when extracellular Ca2+ was removed in the presence of UK 14304, [Ca2+]i and MLC phosphorylation fell to resting levels, but force remained significantly elevated above basal levels. UK 14304 caused no change in the steady-state [Ca2+]i-MLC phosphorylation relation. Thus, the [Ca2+]i sensitization of force was not caused by a sensitization of MLC phosphorylation. These results indicate that in a 2.5-mM Ca2+ bathing medium, the dominant mechanism by which alpha 2-adrenergic receptor stimulation causes an increase in vascular tone is through a relatively large increase in [Ca2+]i and MLC phosphorylation. However, in Ca(2+)-free bathing medium, a second mechanism is unmasked which appears to involve an increased Ca2+ sensitivity and is independent of myosin phosphorylation.

Actin Cytoskeleton↗
PubMed1992

Regulation of vascular smooth muscle tone by caldesmon.

Caldesmon is an actin-binding protein present in smooth muscle cells that also inhibits actin-activated myosin ATPase activity. To assess the possible role of caldesmon in the regulation of smooth contraction, we investigated the effects of synthetic peptides on force directly recorded from single hyperpermeable smooth muscle cells of ferret aorta and portal vein. GS17C, a peptide that contains the residues from Gly651 to Ser667 of the caldesmon sequence plus an added cysteine at the C terminus, binds calmodulin in a Ca(2+)-dependent manner and also binds to F-actin but does not inhibit actomyosin ATPase activity (Zhan, Q., Wong, S.S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814). In cells in which Ca2+ was clamped at pCa 7.0, GS17C induced a dose-dependent contraction (EC50 = 0.92 microM) in aorta cells, whereas it evoked little or no contraction in portal vein cells. The GS17C-induced contraction in aorta cells was inhibited at higher Ca2+ concentrations (above pCa 6.6) and by pretreatment with calmodulin. Another peptide, C16AA, which contains the residues from Ala594 to Ala609 and does not bind actin or calmodulin, did not induce contraction. Our results strongly suggest that GS17C induces contraction by the displacement of the inhibitory region of endogenous caldesmon and, furthermore, that caldesmon present in these smooth muscle cells regulates contraction by providing a basal resting inhibition of vascular tone.

Actins↗
PubMed1992

Mechanisms of intrinsic tone in ferret vascular smooth muscle.

1. Circular strips from ferret aorta were used to investigate the mechanism of the intrinsic basal tone. 2. Determinations of stiffness using small sinusoidal length changes showed an abolition of both stiffness and force with cooling, but the temperature dependence of the change in active stiffness did not parallel that of force. At temperatures below 22 degrees C there appeared to be a relatively large population of attached, non-force-generating cross-bridges, indicating that separate mechanisms are involved in regulating cross-bridge attachment and the force per cross-bridge. 3. Active intrinsic tone was not affected by removal of extracellular Ca2+ or removal of endothelium. 4. Intracellular ionized Ca2+ concentrations ([Ca2+]i) as measured with the photoprotein aequorin, did not significantly change when intrinsic tone was abolished by cooling. 5. Myosin light chain phosphorylation, as measured by 2-dimensional polyacrylamide gel electrophoresis, significantly decreased on cooling, but the temperature dependence of phosphorylation did not parallel that of force. The change in phosphorylation in the absence of a change in [Ca2+]i suggests the presence of a constitutively active Ca(2+)-independent form of myosin light chain kinase. 6. Maximal concentrations of staurosporine inhibited but did not eliminate intrinsic tone. 7. Changes in myosin light chain kinase and protein kinase C activities may explain part but not all of the intrinsic tone.

Animals↗
PubMed1992

Phenylephrine-induced translocation of protein kinase C and shortening of two types of vascular cells of the ferret.

1. The relationship between phenylephrine-induced smooth muscle contraction and the subcellular distribution of protein kinase C (PKC) was investigated. 2. Cell shortening induced by phenylephrine (10(-5) M) was measured in single vascular cells freshly isolated from ferret portal vein and aorta. 3. At various time points during phenylephrine activation, single cells were fixed with paraformaldehyde and the distribution of PKC was imaged in cells labelled with the fluorescent PKC probe 12-(1,3,5,7-tetramethylBODIPY-2-propionyl)phorbol-13-acetate. 4. The PKC probe located to a perinuclear region, the cytosol and surface membrane. The amplitude and time course of the phenylephrine-induced changes in the surface membrane/cytosol fluorescence ratio were measured and compared with the amplitude and time course of phenylephrine-induced cell shortening. 5. In portal vein cells incubated in 1 mM-external Ca2+, phenylephrine caused significant shortening and time-dependent translocation of the PKC probe to the surface membrane, but cell shortening preceded PKC translocation. In Ca2+free solution both cell shortening and translocation of the probe were completely inhibited. 6. Verapamil (3 x 10(-7) M) partially, but significantly, inhibited the magnitude of cell shortening and delayed the onset and time to peak shortening. Translocation of PKC in verapamil preceded or coincided with cell shortening. 7. In aorta cells incubated in 1 mM-extracellular Ca2+, phenylephrine induced significant shortening and time-dependent translocation of the PKC probe. Cell shortening preceded PKC translocation. In Ca(2+)-free solution, shortening was only partially, but significantly, inhibited and PKC translocation preceded the fraction of the shortening response that remained. 8. These data are consistent with a role for PKC in the maintenance of the phenylephrine-induced contraction in both portal vein and aorta. The data also suggest that phenylephrine-induced contraction may involve activation of a Ca(2+)-dependent PKC isoform in ferret portal vein but a Ca(2+)-independent isoform in ferret aorta.

Animals↗
PubMed1992

Ca(2+)-independent isoforms of protein kinase C differentially translocate in smooth muscle.

It is generally assumed that smooth muscle contraction is dependent on changes in intracellular Ca2+ concentration ([Ca2+]i); however, we have previously reported that alpha-agonist-induced contraction of aorta smooth muscle cells can occur in the absence of changes in [Ca2+]i [Collins, E. M., M. P. Walsh, and K. G. Morgan. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H754-H762, 1992]. The mechanism of this [Ca2+]i-independent contraction is controversial. We have now identified the Ca(2+)-independent protein kinase C (PKC) isoforms epsilon and zeta in ferret aorta and have used digital imaging microscopy to determine their subcellular distribution. At rest, epsilon-PKC is diffusely distributed in the cytosol, whereas zeta-PKC is concentrated in the perinuclear region; both isoforms are excluded from the nuclear space. Agonist stimulation causes a [Ca2+]i-independent translocation of epsilon-PKC to the surface membrane and of zeta-PKC to the intranuclear compartment. In comparison, ferret portal vein cells, which display a totally Ca(2+)-dependent agonist contraction, are lacking in epsilon-PKC but display perinuclear zeta-PKC, which translocates intranuclearly on activation. Thus the Ca(2+)-independent vascular contraction appears to be associated with plasmalemmal translocation of epsilon-PKC; in contrast, the intranuclear translocation of zeta-PKC may function in control of gene expression.

Animals↗
PubMed1992

Contraction of single vascular smooth muscle cells by phenylephrine at constant [Ca2+]i.

The mechanism of alpha-adrenergic agonist-mediated force generation was investigated in single hyperpermeable vascular smooth muscle cells. By use of a previously described method, force was recorded from individual ferret aortic cells made hyperpermeable by exposure to saponin. When the cells were clamped at pCa 7, addition of protein kinase M (PKM), the constitutively active form of protein kinase C (PKC), caused a sustained increase in force, which was reversible upon addition of the PKC pseudosubstrate inhibitor peptide (PSSI) corresponding to residues 19-31 of PKC. Phenylephrine at pCa 7 caused a dose-dependent increase in contractile force of the hyperpermeable cells, which was reversible on addition of phentolamine. The phenylephrine contraction could also be inhibited by the same concentration of PSSI that was effective toward the PKM-induced contraction. The response of the cells to a constant [phenylephrine] in different Ca buffers showed a lack of Ca dependence between pCa 8.6 and 7.0. The addition of PSSI to unstimulated cells caused a small but significant drop in basal tone. Taken together, these results suggest that a fraction of the basal tone, as well as the phenylephrine contraction that occurs in the skinned cells at constant intracellular free Ca2+ concentration, is the result of activation of a Ca-independent isozyme of PKC.

Animals↗
PubMed1991

The cellular basis of contraction and relaxation in cardiac and vascular smooth muscle.

Changes in cytoplasmic levels of free-ionized calcium regulate the contraction and relaxation of cardiac and vascular smooth muscle. Aside from changing the intrinsic rate of energy use within the cell, an intervention that alters the strength of contraction of cardiac or vascular smooth muscle must in general alter either the intracellular calcium level, change the calcium requirements of the contractile apparatus, or exert both effects. Intracellular calcium handling is modulated by at least three other important second messengers: cyclic adenosine monophosphate, cyclic guanosine monophosphate, and inositol 1,4,5 triphosphate. These substances modulate intracellular calcium handling at multiple steps in the excitation-contraction coupling scheme. In vascular smooth muscle, diacylglycerol appears to play an important role in the regulation of myofilament calcium requirements. Intracellular calcium homeostasis is maintained through the action of sarcolemmal mechanisms that extrude calcium into the extracellular space; these mechanisms include a sodium-calcium exchange mechanism and an energy-dependent calcium pump. Abnormalities in electrophysiologic properties, sarcoplasmic reticulum function, energy use and supply, and contractile element interaction have been identified in experimental studies of animals and patients with various cardiovascular diseases. These abnormalities may be differentially affected by therapeutic agents that act on the heart or vasculature; therefore it is important to understand the underlying cellular abnormalities and subcellular actions of inotropic, vasoconstrictor, and vasodilatory drugs to apply rational therapeutics in the clinical setting.

Aequorin↗
PubMed1991

Flosequinan, a vasodilator with a novel mechanism of action.

1. The mechanism of action of flosequinan was investigated in ferret aortic smooth muscle by the simultaneous measurement of aequorin luminescence and isometric force. 2. The control calcium-force curve was obtained by plotting the calibrated aequorin luminescence against the force from potassium-depolarized muscles. Flosequinan relaxed potassium-depolarized muscles by causing parallel changes in [Ca2+]i and force with no shift in the control [Ca2+]i-force relationship. 3. The [Ca2+]i-force relationship in the presence of a maximally effective concentration of phenylephrine was significantly shifted to the left of that for the control, potassium-depolarized muscle. Flosequinan relaxed the phenylephrine-contracted muscle by causing a large decrease in force with only a minimal decrease in [Ca2+]i, resulting in an apparent rightward shift of the [Ca2+]i-force relationship, toward the control curve. 4. In comparison, sodium nitroprusside caused relaxation of either the potassium- or phenylephrine-induced contraction solely by a decrease in [Ca2+]i with no shift in either calcium-force relationship. 5. Milrinone caused no significant rightward shift of the calcium-force relationship during phenylephrine- or potassium-induced contractions, but when milrinone was added in the absence of vasoconstrictors, relaxation was obtained with no significant decrease in [Ca2+]i. 6. Flosequinan appears to differ in mechanism of action from both nitroprusside and milrinone. It relaxes depolarization-mediated contractions solely by decreasing [Ca2+]i but also appears to be capable of reversing the apparent calcium sensitizing action of phenylephrine.

Aequorin↗
PubMed1991

Ca(2+)-independent change in phosphorylation of the myosin light chain during relaxation of ferret aorta by vasodilators.

1. The effects of the vasodilators atrial natriuretic peptide (ANP) and forskolin were determined on isometric force, intracellular ionized Ca2+ concentration ([Ca2+]i) as indicated by aequorin, and myosin light chain (MLC) phosphorylation in ferret aorta. 2. Atrial natriuretic peptide (10(-7) M) inhibited intrinsic tone with an associated significant decrease in [Ca2+]i. ANP also inhibited the contraction induced by KCl with a significant decrease in [Ca2+]i. MLC phosphorylation induced by KCl was inhibited by ANP. 3. Forskolin (10(-6) M) decreased the intrinsic tone without significantly decreasing [Ca2+]i, although MLC phosphorylation was significantly decreased. 4. A calcium-force curve was constructed by plotting the calibrated aequorin light signal against the resulting force. The control (potassium-generated) calcium-force curve was not shifted by ANP, but was significantly shifted to the right by forskolin. Forskolin also shifted the phosphorylation-calcium curve to the right without changing the phosphorylation-force curve. 5. We conclude that the vasodilatory effect of ANP on vascular smooth muscle is mainly due to a decrease in [Ca2+]i. On the other hand, the effect of forskolin is via both a decrease in [Ca2+]i and a change in the Ca2+ requirement for MLC phosphorylation.

Animals↗
PubMed1991

Change of Ca2+ requirement for myosin phosphorylation by prostaglandin F2 alpha.

The mechanism of contraction of vascular smooth muscle by prostaglandin F2 alpha (PGF2 alpha) was examined by simultaneous measurement of the intracellular Ca2+ concentration [( Ca2+]i), force, and myosin light-chain (MLC) phosphorylation in ferret aorta. In the presence of 2.5 mM extracellular Ca2+, PGF2 alpha (10(-5)M) produced a tonic contraction with a transient spike in [Ca2+]i, followed by a relatively small sustained increase in [Ca2+]i (from a basal level of 2.32 +/- 0.07 x 10(-7) to 2.72 +/- 0.05 x 10(-7) M). In Ca(2+)-free bathing media, PGF2 alpha also produced a tonic contraction with a small spike in [Ca2+]i, indicating a release of Ca2+ from intracellular store sites, followed by no significant increase in [Ca2+]i. Ca(2+)-force curves were constructed by plotting the calibrated steady-state aequorin light signal against the resulting steady-state force. The curve was significantly shifted to the left by PGF2 alpha. PGF2 alpha also shifted the Ca(2+)-phosphorylation curve to the left. These results suggest that PGF2 alpha causes contraction by both elevating [Ca2+]i and decreasing the Ca2+ requirement for MLC phosphorylation. The data are consistent with a mechanism where there is either an increase in activity of MLC kinase or a decrease in phosphatase activity. Additionally, there was a smaller, but statistically significant, effect to increase force at any one phosphorylation level, pointing to the possibility of regulation of contractile force separate from MLC phosphorylation.

Animals↗
PubMed1991

Intracellular free Ca2+ is elevated in hypertrophic aortic muscle from hypertensive rats.

Vessels from hypertensive animals have been shown to have increased medial thickness and also to exhibit increased sensitivity to agonists. We tested the hypothesis that changes in intracellular Ca2+ concentration [( Ca2+]i) may be associated with the growth response and the altered contractility of the aortic smooth muscle of modified coarctation-hypertensive rats. Freshly isolated single cells, loaded with fura-2, showed significantly higher [Ca2+]i both at rest and after depolarization compared with normal rat aortic cells. These cells also exhibited at rest a significant nuclear-cytoplasmic [Ca2+] gradient. Furthermore, the increased [Ca2+]i was associated with increased nuclear volumes, suggesting the presence of polyploid nuclei, as determined by ethidium dimer fluorescence imaging. Intact vascular rings from hypertrophic rat aortas, loaded with aequorin, also showed significantly higher [Ca2+]i at rest and after depolarization compared with normal rat aortas. In addition, the maximal force per cross-sectional area generated by the hypertrophic muscle was less than normal, consistent with a change in the contractile phenotype of the growing smooth muscle cells. This is the first report to directly indicate an increase in [Ca2+]i in hypertrophic vascular smooth muscle in hypertensive animals.

Aequorin↗
PubMed1991

Increased Ca2+ signaling after alpha-adrenoceptor activation in vascular hypertrophy.

In an effort to explain the increased sensitivity to agonists of hypertrophic vascular muscle, intracellular Ca2+ concentration ([Ca2+]i)-signaling mechanisms were studied in normal and hypertrophic rat aortas from normotensive and coarctation-hypertensive rats. Based on both fura 2 fluorescence and aequorin luminescence measurements, qualitatively different patterns of Ca2+ mobilization occur in normal and hypertrophic rat aortic muscle. Normal rat aortic muscle contracts to phenylephrine with little or no increase in [Ca2+]i, whereas the angiotensin II-induced contraction is accompanied by a marked [Ca2+]i transient. In contrast, hypertrophic rat aortic muscle shows a dramatic increase in Ca2+ signaling after phenylephrine stimulation. Moreover, both the amplitude of the angiotensin-induced [Ca2+]i transient and the contractile sensitivity to this agonist are decreased in the hypertrophic muscle. Our results strongly suggest that the amplitude of the [Ca2+]i transient after agonist stimulation determines the contractile sensitivity and that there is an altered coupling of the alpha-adrenoceptor in the hypertrophic vascular muscle.

Angiotensin II↗
PubMed1991

Imaging of protein kinase C distribution and translocation in living vascular smooth muscle cells.

The subcellular distribution of protein kinase C was directly imaged in single living smooth muscle cells using a new fluorescent protein kinase C probe. The probe localized prominently to perinuclear organelles and, to a lesser extent, to the cytosol and surface membrane. The perinuclear signal did not detectably change over the time observed. The ratio between the surface membrane intensity and that in the cytosol (R) was measured in parallel with cell shortening. In 1 mM extracellular Ca2+, the time to peak R and time to peak shortening were not significantly different. In Ca(2+)-free solution, no significant increase in the surface membrane/cytosol fluorescence ratio was observed with time, and shortening was inhibited. These results provide a new method for monitoring protein kinase C localization in living cells and directly demonstrate that protein kinase C moves in a Ca(2+)-dependent fashion from the cytosol to the surface membrane simultaneously with contraction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Effects of cocaine on excitation-contraction coupling of aortic smooth muscle from the ferret.

The mechanism by which cocaine alters vascular tone is not fully understood. We determined the effects of cocaine on excitation-contraction coupling of isolated ferret aorta. Cocaine in concentrations less than or equal to 10(-4) M caused a contractile response in a dose-dependent manner. The response of control muscle was significantly larger than that in muscle from ferrets pretreated with reserpine. Cocaine-induced contraction was not affected by endothelial factors, but was significantly inhibited by prazosin 10(-7) M pretreatment. The intracellular calcium [( Ca++]i), as measured with aequorin, rose in conjunction with cocaine-induced contraction. The degree of contraction generated by 10(-4) M cocaine decreased after higher concentrations of cocaine greater than or equal to 10(-3) M, while aequorin luminescence remained elevated above the levels before 10(-6) M cocaine. The dose-response relationships of norepinephrine and sympathetic nerve stimulation were enhanced by 10(-6) M cocaine in control muscles; this did not occur in muscles from reserpine pretreated ferrets. In conclusion, (a) cocaine in concentrations less than or equal to 10(-4) M caused vascular contraction presumably by its presynaptic action with consequent alpha-1 adrenoceptor activation and consequent [Ca++]i rise; (b) high concentrations of cocaine greater than or equal to 10(-3) M reduced muscle tone by decreasing the Ca++ sensitivity of the contractile proteins; and (c) supersensitivity to norepinephrine was mediated by cocaine's action on adrenergic nerve endings.

Adrenergic Fibers↗
PubMed1991

Effects of cocaine on intracellular calcium handling in cardiac and vascular smooth muscle.

The inotropic and lusitropic (i.e., relaxant) actions of cocaine on the heart appear to be caused primarily by changes in intracellular calcium handling. The positive inotropic and lusitropic effects of low and moderate concentrations (i.e., less than or equal to 10(-5)M) are mediated by catecholamines; the negative inotropic effects of higher concentrations appear to be due to the direct local anesthetic effects of cocaine on excitation-contraction coupling mechanisms. The relevance of these findings to humans is suggested by the fact that blood levels of cocaine in excess of 10(-5) M have been described in patients abusing this drug (Van Dyke et al. 1976; Paly et al. 1982). Blood vessels from certain vascular beds, including the epicardial coronary arteries of humans and swine, show little or no constrictor response to low concentrations of cocaine and relax at higher concentrations. In contrast to the effects on the heart, the relaxant effects of cocaine on vascular smooth muscle appear to be related to marked changes in myofilament calcium responsiveness, which may be mediated by the protein kinase-C system. These results at least indicate that the depressant effects of cocaine on cardiac vs. vascular smooth muscle occur by different mechanisms and suggest the need for specific therapeutic approaches to managing cardiac depression vs. vasodilatation when they occur in cocaine-intoxicated individuals. Moreover, these data provide evidence supporting the hypothesis that the net effects of cocaine in the intact organism are highly dependent on the underlying level of sympathetic adrenergic activity.

Actin Cytoskeleton↗
PubMed1990

Regulation of force in skinned, single cells of ferret aortic smooth muscle.

An isolation technique was developed for single cells from the ferret aorta, which resulted in the isolation of long (87 +/- 27 microns; x +/- SD, n = 62), relaxed, pharmacologically active smooth muscle cells. These cells were attached to microtools, one of which was connected to a force transducer. Force in maximally phenylephrine-stimulated contractions of the intact cells averaged 2.3 +/- 1.4 microN (n = 17). After cell skinning with saponin, the threshold for force development was 0.05 microM [Ca2+], and force reached a maximum of 4.4 +/- 1.6 microN (n = 36) at 0.5 microM [Ca2+]. Plots of relative steady-state force vs pCa (-log10[Ca2+]) were fit to the Hill equation, which yielded a pCa at half-maximal force of 6.87 +/- 0.30 and a Hill coefficient of 2.3 +/- 1.4 (n = 29). When 2.5 microM calmodulin was added to the solutions, the calcium sensitivity of force was significantly increased (P less than 0.05) without changing the maximal force (P greater than 0.05). In a solution of pCa 7, the skinned cells developed 2.5 +/- 0.5 microN (n = 5) of force when stimulated with a phorbol ester. The addition of a specific inhibitor (17 kDa) of protein kinase C to the calcium buffers depressed (P less than 0.05) the maximally Ca2(+)-activated force without a change in the calcium sensitivity of force (P greater than 0.05). These data strongly suggest that in vascular smooth muscle, protein kinase C may be involved in a physiological, regulatory system for force.

Animals↗