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Biomedical subjects

K G Morgan

Publications and source records attributed to K G Morgan.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effects of calponin on force generation by single smooth muscle cells.

Although the actin-binding and actomyosin adenosinetriphosphatase (ATPase) inhibitory properties of calponin are well documented in vitro, its function in the smooth muscle cell has not been elucidated. To address this question, we utilized the ferret aortic smooth muscle cell, which shows a protein kinase C-dependent contraction even at pCa (-log [Ca2+]) 9.0 in the absence of a change in myosin light chain phosphorylation. Force was recorded from single, briefly permeabilized cells stimulated via a Ca(2+)-independent pathway by either phenylephrine or the epsilon isoenzyme of protein kinase C. Treatment of stimulated cells with wild-type recombinant calponin reduced steady-state contractile force by 45-60%. When calponin application preceded protein kinase C epsilon treatment, contraction was completely suppressed. On the other hand, calponin phosphorylated at Ser175 or mutant calponin with a Ser175 --> Ala replacement had no effect on contractile force. A peptide corresponding to Leu166-Gly194 of calponin, which included an actin-binding domain but excluded the actomyosin ATPase inhibitory region, was synthesized. Treatment of aortic smooth muscle cells with this peptide triggered a concentration-dependent contraction, presumably by alleviating the inhibitory effect of endogenous calponin. A control peptide with a scrambled sequence of the same residues produced no detectable contractile response. Although other interpretations are possible, these results are consistent with the view that calponin participates in thin filament-mediated regulation of smooth muscle contraction and that it may be part of a Ca(2+)-independent pathway downstream of protein kinase C epsilon.

Animals↗
PubMed1996

Mechanisms of smooth muscle contraction.

Work performed with differentiated contractile smooth muscle tissue over the last two decades has made clear that covalent modification of myosin by phosphorylation of the 20-kDa myosin light chains is a significant mode of regulation of contractile activity in smooth muscle, particularly in regard to the generation of phasic contractions and the initial development of tonic contractions. This regulatory mechanism appears to be of unique importance in smooth muscle compared with striated muscle. It is equally clear, however, that there is an important role for protein kinase C in the regulation of smooth muscle tone maintenance, particularly in vascular smooth muscle. Several possible signal transduction cascades involving protein kinase C are outlined. Increasing evidence suggests a link between protein kinase C and actin-based regulatory mechanisms. This review places emphasis on relating up-to-date biochemical facts to the physiological realities of the smooth muscle cell.

Animals↗
PubMed1996

Intracellular calcium, myosin light chain phosphorylation, and contractile force in experimental cerebral vasospasm.

It remains unknown what proportion of delayed arterial narrowing after subarachnoid hemorrhage depends on ongoing metabolic activity within arterial smooth muscle cells versus changes in the passive structural properties of the arterial wall. To determine this, vasospasm was induced by the double subarachnoid hemorrhage model. Anterior spinal artery segments were harvested from control dogs and from dogs with vasospasm. The segments were suspended in a force transducer and stretched to an optimal length for contraction. The difference in tension between 37 and 0 degrees C was defined as the intrinsic tone, and the residual tension at 0 degrees C was defined as the passive tension. The segments taken from dogs with vasospasm had increased intrinsic tone and passive tension (the differences were 3.8 kN/m2 [P < 0.05] and 14.8 kN/m2 [P < 0.025], respectively). Hence, the passive component accounted for 79.6% of the increased tension in vasospastic arterial segments. The intracellular calcium concentration was measured in these segments, using the luminescent calcium indicator, aequorin. The vasospastic segments had increased basal intracellular calcium concentration (398 versus 258 nmol/L, P < 0.025). In parallel experiments, control and vasospastic vessels were immediately excised when the animals were killed, and the vessels were quick-frozen. Subsequently, using two-dimensional gel electrophoresis to measure percent myosin light chain phosphorylation, vasospastic vessels were found to have increased myosin light chain phosphorylation (37 versus 2%, P < 0.05). The increased intracellular calcium concentration and increased percent myosin light chain phosphorylation in vasospastic segments implicate a role for the Ca(2+)-dependent pathway of smooth muscle cell contraction in vasospasm.

Animals↗
PubMed1996

Conditional differentiation of heart- and smooth muscle-derived cells transformed by a temperature-sensitive mutant of SV40 T antigen.

To create muscle cell lines that conditionally differentiate in vitro we introduced a temperature-sensitive SV40 T antigen by retroviral infection into rat aortic smooth muscle cells (SMCs) and neonatal heart-derived cells. After G418 selection cell lines isolated were characterized at permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. [3H]Thymidine uptake showed tht progression through the cell cycle is greatly reduced at 39 degrees C. Cytoskeletal proteins, such as actins and vimentin did not change significantly after temperature shift, while the number of desmin-positive SMCs significantly increased when cells were switched to 39 degrees C. Heart-derived muscle cells showed sarcomeric myosin heavy chain reactivity only when grown at 39 degrees C. After thrombin stimulation intracellular calcium in both cell types increased severalfold in 39 degrees C-cells but not in 33 degrees C-cells. Whole cell patch-clamp recordings of SMCs and heart-derived cells revealed a strong increase in nicardipine-sensitive Ca2+ current when cells were switched to 39 degrees C. Nicardipine-insensitive Ca2+ current also increased in both cell types at the non-permissive temperature. Na+ current in SMCs was large at 33 degrees C and small or not detectable at 39 degrees C and absent in heart-derived cells. Using a cDNA probe specific for the alpha 1 subunit of the dihydropyridine-sensitive Ca2+ channel we demonstrate a temperature-sensitive expression of the dihydropyridine receptor mRNA in smooth muscle-derived cells but not in heart-derived H10 cells. Our results suggest that upon downregulation of SV40 T antigen these cells become quiescent and exhibit a more differentiated phenotype. These cell lines may provide a useful tool to investigate ion channel- and receptor signal transduction, as well as cell cycle control in smooth and possibly cardiac muscle cell differentiation.

Animals↗
PubMed1995

Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells.

Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes.

Animals↗
PubMed1994

Smooth muscle protein kinase C.

Protein kinase C (PKC) was first implicated in the regulation of smooth muscle contraction with the observation that phorbol esters induce slowly developing, sustained contractions. In some vascular smooth muscles, e.g., ferret aorta, phorbol ester induced contractions occur without an increase in sarcoplasmic free-Ca2+ concentration ([Ca]i) or myosin light chain phosphorylation. This response appears to be mediated by a Ca(2+)-independent isoenzyme of PKC (probably PKC epsilon), since saponin-permeabilized single ferret aortic smooth muscle cells, which retain receptor coupling, developed force in response to phenylephrine at low free [Ca2+] (pCa 7.0-8.6) and the constitutively active proteolytic fragment of PKC (PKM) elicited a contraction at pCa 7 comparable with the phenylephrine-induced contraction. Both contractions were reversed by a pseudo-substrate peptide inhibitor of PKC. These observations suggest a mechanism whereby alpha-adrenergic agonists may elicit a contractile response without a Ca2+ signal: alpha-adrenergic stimulation of phosphatidylcholine-specific phospholipase C or D (the latter in conjunction with phosphatidate phosphohydrolase) generates diacylglycerol. In the absence of an increase in [Ca2+]i, diacylglycerol specifically activates so-called novel PKCs, of which epsilon is the only isoenzyme known to be expressed in vascular smooth muscle. Recent evidence suggests that PKC may trigger a cascade of phosphorylation reactions, resulting in activation of mitogen-activated protein kinase and phosphorylation of the thin filament associated protein caldesmon. Alternatively, or additionally, PKC may directly phosphorylate calponin, another thin filament associated protein. These phosphorylations are predicted to alleviate inhibition of the cross-bridge cycling rate by these thin-filament proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform.

Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.

Animals↗
PubMed1994

Redistribution of protein kinase C isoforms in association with vascular hypertrophy of rat aorta.

Freshly enzymatically isolated cells from the aorta of a rat model of vascular hypertrophy were used to investigate the role of protein kinase C (PKC) isoforms during a physiologically relevant growth response. With the combination of immunofluorescence with digital imaging microscopy, PKC isoforms alpha, delta, and zeta were found to be present in single cells from control and hypertrophied rat aortas. The alpha- and zeta-isoforms were distributed in the cytoplasm of control cells; however, in hypertrophied cells, sustained translocations of alpha-PKC to the surface membrane and zeta-PKC to the intranuclear area were seen. delta-PKC was concentrated in the perinuclear area in control cells but appeared to translocate to a more diffuse localization in the cytosol of hypertrophied cells. Staining of mitochondria with rhodamine 123 indicated some similarity in the spatial distribution compared with that of delta-PKC. In control cells, translocation of isoforms alpha and delta was activated by phenylephrine or 12-deoxyphorbol 13-isobutyrate 20-acetate. Agonist stimulation produced translocation of no isoforms in the hypertrophied cells. These results indicate that isoform-specific spatial distribution and translocation of PKC occur in association with the growth response of vascular hypertrophy.

Animals↗
PubMed1994

Agonist-induced redistribution of calponin in contractile vascular smooth muscle cells.

Calponin is a thin filament-associated protein that has been implicated in playing an auxiliary regulatory role in smooth muscle contraction. We have used immunofluorescence and digital imaging microscopy to determine the cellular distribution of calponin in single cells freshly isolated from the ferret portal vein. In resting cells calponin is distributed throughout the cytosol, associated with filamentous structures, and is excluded from the nuclear area of the cell. The ratio of surface cortex-associated calponin to cytosol-associated calponin (R) was found to be 0.639 +/- 0.021. Upon depolarization of the cell with physiological saline solution containing 96 mM K+, the distribution of calponin did not change from that of a resting cell (R = 0.678 +/- 0.025, P = 0.369). Upon stimulation with an agonist (10 microM phenylephrine) that is known to activate protein kinase C (PKC) in these cells, the cellular distribution of calponin changed from primarily cytosolic to primarily surface cortex associated (R = 1.24 +/- 0.085, P < 0.001). This agonist-induced redistribution of calponin was partially inhibited by the PKC inhibitor calphostin, overlapped in time with PKC translocation, and preceded contraction of these cells. These results suggest that the physiological function of calponin may be to mediate agonist-activated contraction via a PKC-dependent pathway.

Actins↗
PubMed1994

Mechanosensitive modulation of myosin phosphorylation and phosphatidylinositol turnover in smooth muscle.

Myosin light chain phosphorylation and phosphatidylinositol turnover were measured at different muscle lengths in bovine tracheal smooth muscle. The relationship between myosin phosphorylation and muscle length was linear between optimal length (Lo) and 0.1 Lo in both unstimulated and carbachol-activated tissues. However, myosin phosphorylation in carbachol-activated tissues was more sensitive to changes in muscle length. As a result, suprabasal myosin phosphorylation induced by carbachol was significant at Lo but became insignificant at 0.1 Lo. Phosphatidylinositol turnover was assayed by measuring the formation of myo-[3H]inositol phosphates in unstimulated and carbachol-activated tissues using the Li+ method. Pairs of time-matched and length-matched muscle strips were used for control (unstimulated) and activation by carbachol. Phosphatidylinositol turnover in carbachol-activated tissues was more sensitive than that in unstimulated tissues to changing length. As a result, suprabasal phosphatidylinositol turnover induced by carbachol was significant at Lo but became insignificant at 0.1 Lo. These results indicated that myosin phosphorylation and phosphatidylinositol turnover mediated by muscarinic receptor activation were modulated by the mechanical state of smooth muscle.

Animals↗
PubMed1994

[Ca2+]i signaling in pregnant human myometrium.

The purpose of the present study was to determine the changes in intracellular ionized calcium concentration ([Ca2+]i) or [Ca2+]i sensitivity accompanying spontaneous and agonist-induced contraction of human myometrium at term pregnancy, as well as to quantify the response to three prototypical agonists: 1) oxytocin, 2) vasopressin, and 3) phenylephrine. Uterine biopsies were obtained at the time of cesarean section from patients who delivered at or near full-term gestation. These preparations were used to measure isometric force development and [Ca2+]i levels with the luminescent calcium indicator aequorin. Concentration-response relationships were determined with respect to isometric force development in the presence of the agonist. [Ca2+]i-force relationships were determined with respect to spontaneous phasic contractions, as well as agonist-induced phasic and tonic contractions. The results provide evidence that the phasic nature of term human myometrium is due to 1) the resting [Ca2+]i level being less than the calcium threshold for contractions and 2) the inability of the tissue to maintain high [Ca2+]i levels for prolonged periods of time. In addition, calcium-independent mechanisms of regulation were suggested by the relatively minor calcium sensitizing action of oxytocin and the observation that relaxation of tonic contractions preceded the fall in [Ca2+]i levels.

Calcium↗
PubMed1994

Factors involved in delaying the rise in peripheral resistance in developing heart failure.

The development of heart failure (HF) on peripheral vascular control was studied in 10 conscious dogs with measurements of cardiac output (CO) and left ventricular (LV), arterial, and right atrial pressures. At 3 wk after pacing-induced HF, CO was not decreased from 2.5 +/- 0.2 l/min, whereas LV dP/dt fell (from 2,858 +/- 71 to 1,409 +/- 69 mmHg/s) and LV end-diastolic pressure increased (from 4.8 +/- 0.4 to 27.3 +/- 1.1 mmHg) (P < 0.05). At 4-7 wk after pacing, CO was significantly decreased (to 1.6 +/- 0.1 l/min; P < 0.05), but total peripheral resistance (TPR) did not rise, despite increases in plasma norepinephrine and renin activity (P < 0.05). In the presence of ganglionic blockade, TPR was still not increased in HF. In vitro studies in isolated femoral artery segments demonstrated reduced intrinsic tone (0.028 +/- 0.007 g/mg; P < 0.05) as compared with vessels from sham-operated controls (0.124 +/- 0.023 g/mg), whereas the intracellular calcium level was not altered in HF. Thus, during the development of HF, severe contractile dysfunction precedes the fall in CO, which, in turn, precedes the rise in TPR. The delayed rise in TPR appears to involve a reduction in intrinsic peripheral vascular tone, despite neurohumoral activation.

Animals↗
PubMed1994

The bradykinin analog RMP-7 increases intracellular free calcium levels in rat brain microvascular endothelial cells.

The vasoactive peptide bradykinin is believed to cause increased vascular permeability by the activation of B2 receptors on the vascular endothelium. A bradykinin analog, H-Arg-Pro-Hyp-Gly-Thi-Ser-Pro-4-Me-Tyr(psi CH2NH)-Arg-OH (RMP-7), was designed and it was proposed that it might increase cerebrovascular permeability by activating B2 receptors on brain microvasculature. In this report, the effects of RMP-7 and related peptides on bradykinin receptor-induced calcium signaling were examined in rat brain microvascular endothelial (RBME) cultures. RBME cells are responsive to bradykinin and exhibit specific [3H]-bradykinin binding, with Scatchard analysis indicating a major class of sites with a Kd of 3.9 +/- 1.4 nM and a minor class of higher affinity sites (Kd approximately 33 pM). RMP-7 displaces a significant component of specific [3H]-bradykinin binding from RBME cells; RMP-10, and RMP-7 diastereomer with a D-amino acid substitution in the number 9 position, does not. [3H]-bradykinin binding to RBME cells is not displaced by desArg9-bradykinin, which indicates that neither class of sites is a B1 bradykinin receptor. RMP-7 induces an increase in intracellular free calcium levels in RBME cells. The time course, magnitude and concentration dependence of RMP-7-induced calcium signaling is similar to that induced by bradykinin in RBME and other endothelial cells. Compared with RMP-7, RMP-10 is at least 1000 times less potent. However, the level of contamination by RMP-7 in the RMP-12 preparation accounts for its activity, an indication that both the RMP-10 and RMP-12 diastereomers are essentially inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The proto-oncogene c-myb mediates an intracellular calcium rise during the late G1 phase of the cell cycle.

The intracellular concentration of ionized calcium is involved in regulating mitosis. However, little is known about intracellular levels of calcium during G1. We have demonstrated in vascular smooth muscle cells a mid-G1 decrease in ionized calcium concentration followed by a 2-fold rise at the G1/S interface (44 nM +/- 0.6 nM versus 98 nM +/- 1.1 nM, p < 0.01). The elevation of intracellular calcium is preceded by an increase in c-myb mRNA levels and is abolished with antisense but not missense c-myb oligonucleotides. Furthermore, cells stably transfected with c-myb show a similar 2-fold augmentation in intracellular calcium concentrations, as compared with untransfected cells, which is also abolished by antisense c-myb oligonucleotides. The c-myb-induced rise in intracellular calcium is dependent upon the presence of extracellular calcium and is not suppressed by L type calcium channel blockers. We conclude that c-myb induces an elevation in intracellular calcium levels of vascular smooth muscle cells at the G1/S interface which provides a novel role for this proto-oncogene as well as a potentially important control point for cell cycle regulation.

Animals↗
PubMed1993

Negative inotropic and relaxant effects of cocaine on myopathic human ventricular myocardium and epicardial coronary arteries in vitro.

OBJECTIVE: Cocaine produces both vascular and myocardial effects that can lead to serious cardiovascular complications in man. Tissue catecholamine stores are known to be depleted in the advanced stages of heart failure. The effects of cocaine on cardiac and coronary smooth muscle isolated from patients with end stage heart failure was tested in order to evaluate the direct actions of this drug on human tissue. METHODS: Effects of cocaine HCl were studied on cardiac ventricular trabeculae carneae and epicardial coronary artery segments obtained at heart transplantation from patients with end stage heart failure. Muscles were placed in organ baths under physiological conditions for recording isometric tension; a subset of muscles was loaded intracellularly with the bioluminescent calcium indicator, aequorin. Cardiac muscles were stimulated with threshold pulses delivered via a punctate electrode at 0.33 Hz. Coronary segments were studied under basal conditions and after contraction with 60 mM KCl. RESULTS: In both cardiac muscle and vascular smooth muscle preparations, cocaine (10(-6)-10(-3) M) produced dose related negative inotropic and relaxant effects, respectively; positive inotropic actions and vasoconstriction were not seen. In cardiac muscle, the negative inotropic actions were associated with a simultaneous decrease in peak intracellular calcium levels. In contrast, cocaine induced relaxation of potassium contracted vascular smooth muscle was not associated with a fall in peak intracellular calcium levels, a result consistent with decreased myofilament calcium responsiveness. CONCLUSIONS: These results indicate that the depressant effects of cocaine on cardiac versus vascular smooth muscle occur by different mechanisms and suggest the need for specific therapeutic approaches to managing cardiac depression versus vasodilation when these occur in cocaine intoxicated patients.

Adult↗
PubMed1993

PKC-mediated redistribution of mitogen-activated protein kinase during smooth muscle cell activation.

Protein kinase C (PKC) translocates from the cytosol to the surface membrane at the time it mediates agonist-induced contraction of ferret vascular smooth muscle cells (R. A. Khalil and K. G. Morgan. J. Physiol. Lond. 455: 585-599, 1992). However, no direct communication between membrane-associated PKC and the contractile filaments has been identified. Mitogen-activated protein (MAP) kinase is a substrate for PKC and is also capable of phosphorylating the actin-binding protein caldesmon at sites phosphorylated during smooth muscle contraction in vivo (L. P. Adam, C. J. Gapinski, and D. R. Hathaway. FEBS Lett. 302: 223-226, 1992). In the present study, the hypothesis that PKC and MAP kinase are involved in a signal-transduction cascade leading to smooth muscle contraction was tested. Immunofluorescence and digital-imaging microscopy were used to localize the epsilon-PKC isoform and MAP kinase during phenylephrine-induced Ca(2+)-independent activation of ferret aorta cells. We report that maintained phenylephrine-induced translocation of cytosolic PKC to the surface membrane is associated with transient redistribution of cytosolic MAP kinase to the surface membrane before cell contraction. Coincident with cell contraction, MAP kinase undergoes a second redistribution away from the plasmalemma and toward the vicinity of contractile filaments. Redistribution of MAP kinase is not stimulated by Ca2+ but is completely prevented by PKC inhibitors. The transient Ca(2+)-independent but PKC-dependent redistribution of MAP kinase points to MAP kinase as a missing link in the signal-transduction cascade between membrane-bound PKC and smooth muscle activation.

Animals↗
PubMed1993

Mechanisms of Ca(2+)-independent contraction in single permeabilized ferret aorta cells.

The mechanisms by which prostaglandin F2 alpha (PGF2 alpha) can cause contractions at constant intracellular Ca2+ were investigated by the direct measurement of force from single saponin-permeabilized smooth muscle cells from the ferret aorta. The size of PGF2 alpha contractions did not change between pCa 9.0 and pCa 6.6. The remainder of the experiments were carried out at pCa 7.0. At pCa 7.0, PGF2 alpha (0.1-100 microM) induced sustained force in a dose-dependent manner, reaching a maximum (2.61 +/- 0.20 microN, n = 14) in 10 minutes. Both protein kinase C pseudosubstrate inhibitor (3 microM) and staurosporine (1 microM) significantly inhibited PGF2 alpha (100 microM)-induced contractions, but staurosporine was more effective. Staurosporine caused 88.8 +/- 13.3% inhibition, whereas protein kinase C pseudosubstrate inhibitor inhibited 62.3 +/- 9.6% of the PGF2 alpha-induced contraction. An inhibitor of type-1 and type-2A protein phosphatases, microcystin-LR, at a concentration of 1 microM induced a gradual and sustained contraction (1.53 +/- 0.21 microN). A lower concentration of microcystin-LR (100 nM) also induced a small but significant contraction (0.36 +/- 0.26 microN). Pretreatment with both 1 microM and 100 nM microcystin-LR caused significant inhibition of the PGF2 alpha-induced contraction from 2.61 +/- 0.20 microN (n = 14) to 0.32 +/- 0.20 microN (n = 6) (p < 0.01) and 1.52 +/- 0.21 microN (n = 6) (p < 0.01), respectively. These results indicate that the part of the PGF2 alpha-induced contraction that occurs at a constant, low intracellular Ca2+ is the combined result of activation of protein kinase C and phosphatase inhibition.

Alkaloids↗