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B C Berk

Publications and source records attributed to B C Berk.

140 records · Page 8Linked to original sources
PubMed1988

Agonist-mediated changes in intracellular pH: role in vascular smooth muscle cell function.

Changes in intracellular pH (pHi) are likely to play an important role in regulation of vascular smooth muscle cell (VSMC) function. In most blood vessels, acidification is associated with decreased contractile tone and alkalinization with increased tone. However, the nature of agonist-mediated alterations in pHi and the role of pHi in other VSMC responses has been little studied. We have used the pH sensitive dye, BCECF, to study pHi in cultured rat aortic VSMC. Basal pHi at 37 degrees C in physiologic saline buffer (pH 7.3) was 7.08 in suspended VSMC and 7.26 in substrate-attached VSMC. An amiloride-sensitive Na+/H+ exchanger mediated pHi recovery following an acid load. Angiotensin II- and platelet-derived growth factor typified one class of VSMC agonists, causing an initial transient (less than 5 min) acidification followed by a sustained (greater than 20 min) alkalinization. The acidification phase was associated with increased Ca2+ mobilization as demonstrated by increases in intracellular Ca2+ and 45Ca2+ efflux. The alkalinization was associated with Na+ influx and H+ efflux consistent with Na+/H+ exchange. Epidermal growth factor and phorbol esters typified another class of agonists which stimulated only a sustained alkalinization. Alterations in regulation of VSMC pHi may play an important role in VSMC hypertrophy and/or proliferation as suggested by the finding of increased cell growth and Na+/H+ exchange in spontaneously hypertensive rat VSMC compared to Wistar-Kyoto VSMC. Although no functional correlate for initial acidification has been identified, cytoplasmic alkalinization appears to be required for the sustained formation of diacylglycerol following angiotensin II stimulation. These findings suggest that alterations in pHi may regulate several VSMC functions such as agonist-mediated signal transduction, excitation-response coupling, and growth.

Animals↗
PubMed1987

Protein kinase C-mediated intracellular alkalinization in rat and rabbit aortic smooth muscle cells.

The influence of protein kinase C (C-kinase) activation on intracellular pH (pHi) of cultured rat (RASM) and rabbit (RBASM) aortic smooth muscle cells was studied by employing a pH-sensitive fluorescent-dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The known C-kinase activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) and mezerine as well as the agonist angiotensin II each caused an intracellular alkalinization of approximately 0.1-0.15 pH units in RASM and RBASM cells grown in serum-free conditions. TPA-induced alkalinization was sensitive to the Na+/H+ exchange blockers amiloride and 5-N-ethylisopropyl-amiloride (EIPA). These results suggest that protein kinase C activation leads to intracellular alkalinization in vascular smooth muscle cells and the increase in pHi might play an important role in receptor-coupled arterial contraction.

Amiloride↗
PubMed1987

Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways.

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.

Angiotensin II↗
PubMed1987

Early agonist-mediated ionic events in cultured vascular smooth muscle cells. Calcium mobilization is associated with intracellular acidification.

Angiotensin II, a potent vasoconstrictor peptide, increases free cytoplasmic Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) by release of nonmitochondrial Ca2+ stores and stimulates an amiloride-sensitive Na+ influx, presumably via Na+/H+ exchange. We recently have found that the angiotensin II-mediated change in VSMC intracellular pH has two components, an early rapid acidification phase and a slower recovery phase involving Na+-dependent alkalinization. In the present study, we show that the early acidification is not mediated via Na+/H+ exchange. Instead, we propose a mechanism which involves increases in [Ca2+]i and Ca2+ efflux with a subsequent rise in intracellular H+. Agonists, in addition to angiotensin II, which increase [Ca2+]i in cultured VSMC, including platelet-derived growth factor, vasopressin, and bradykinin, induce an acidification, while agonists which fail to raise [Ca2+]i do not. The time course and magnitude of agonist-stimulated 45Ca2+ efflux correlate with the acidification response. The angiotensin II concentration-response relationship for acidification and Ca2+ mobilization are similar. Furthermore, inhibition of changes in [Ca2+]i by treatment with phorbol ester, cyclic GMP, or quin2 loading prevent agonist-mediated acidification. The effects of altering extracellular [Ca2+] and [H+] on agonist-mediated intracellular acidification and H+ efflux suggest that the acidification is due to ATP-dependent unidirectional H+ influx, perhaps via the plasma membrane Ca2+-ATPase, and not to a Ca2+/H+ antiport. This agonist-mediated acidification represents a previously undescribed ionic event in VSMC activation which may be involved in excitation-response coupling.

Amiloride↗
PubMed1987

Angiotensin II stimulation of vascular smooth muscle phosphoinositide metabolism. State of the art lecture.

Phosphoinositide hydrolysis is an integral step in the activation of vascular smooth muscle by angiotensin II. Sequential phospholipase C-mediated hydrolysis of the polyphosphoinositides and phosphatidylinositol in cultured vascular smooth muscle cells stimulated with angiotensin II results in a coordinated series of biochemical events: a transient formation of inositol trisphosphate associated with calcium mobilization, and a biphasic, sustained formation of diacylglycerol associated with activation of protein kinase C and cytosolic alkalinization. The initial, rapid phase and the sustained phase of the angiotensin II response appear to be differentially controlled. Formation of inositol trisphosphate and mobilization of calcium are attenuated by activation of protein kinase C. Sustained diacylglycerol formation is promoted by cytosolic alkalinization, and appears to require cellular processing of the angiotensin II-receptor complex. Calcium and cyclic guanosine 3',5'-monophosphate do not appear to regulate phospholipase C-mediated phosphoinositide hydrolysis in vascular smooth muscle. Thus, regulation of angiotensin II-stimulated second messenger generation in vascular smooth muscle is complex, perhaps involving protein kinase C activation, changes in intracellular pH, and processing of the angiotensin II-receptor complex.

Angiotensin II↗
PubMed1986

Vasoconstriction: a new activity for platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells that has been implicated in the pathogenesis of atherosclerosis. The potential role of PDGF in the altered vasoreactivity of atherosclerotic vessels has been studied through an examination of its effects on contractility in the rat aorta. PDGF caused a concentration-dependent contraction of aortic strips and was significantly more potent on a molar basis than the classic vasoconstrictor peptide angiotensin II. Furthermore, PDGF increased the cytosolic free calcium concentration in cultured rat aortic smooth muscle cells. These observations suggest a new biological activity for PDGF that may contribute to the enhanced vasoreactivity of certain atherosclerotic vessels.

Aminoquinolines↗
PubMed1985

Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic contraction.

Atherosclerotic arteries have enhanced reactivity to vasoconstrictors, which suggests that features of the atherosclerotic process itself may result in this abnormal responsiveness. Since vascular smooth muscle proliferation is a prominent feature of atherosclerosis, we postulated that vasoactive agonists and smooth muscle mitogens may share certain common cellular mechanisms of action which potentially contribute to this hyperreactivity. To test this hypothesis, we studied the effects of epidermal growth factor (EGF), a well-characterized mitogen, on rat aortic vascular smooth muscle, both in intact aortic strips and in culture. EGF caused contraction (EC50 = 19 nM) of rat aortic strips which maximally was equivalent to 40% of that induced by angiotensin II, a potent vasoconstrictor. EGF increased 45Ca efflux (EC50 = 3 nM) from cultured rat aortic smooth muscle cells, which was an effect shared by angiotensin II and thought to reflect increased cytosolic-free calcium concentration. EGF (7.5 nM) also stimulated growth of these cultured cells to the same extent as 10% calf serum. These results demonstrate that EGF is both a vasoconstrictor and mitogen for rat aortic smooth muscle cells. The similarities in the effects of EGF and angiotensin II suggest that certain common intracellular mechanisms of action may exist for vasoactive agonists and growth factors which may contribute to the altered vasoreactivity of atherosclerotic vessels.

Animals↗
PubMed1981

Purification of a stimulator of microtubule assembly from bovine thyroid.

A protein from bovine thyroid which stimulated the assembly of microtubules from phosphocellulose-purified tubulin was isolated. The purification scheme involved fractionation of bovine thyroid homogenates successively on phosphocellulose, DEAE-cellulose, and Sephadex G-200. Activity was determined as ability to stimulate phosphocellulose-purified tubulin assembly measured turbidimetrically. Purification of stimulatory activity resulted in the selective enrichment to near homogeneity of a 40,000-45,000-dalton protein. The molecular weight was estimated by Sephadex G-200 gel filtration and polyacrylamide gel electrophoresis. This protein stimulated both the rate and the extent of assembly of thyroid and brain tubulin. In the turbidity assay which contained 4 M glycerol, the threshold for assembly of brain tubulin (at 1.7 mg/ml) was 0.01 mg/ml for the thyroid stimulator. Brain and thyroid microtubules assembled with thyroid stimulator appeared normal in diameter and shape by electron microscopy. Thyroid stimulator activity was destroyed by trypsinization or boiling. Activity was maximal at 1 mM GTP and 4 mM MgCl2 and was blocked by colchicine. No stimulatory protein of similar molecular weight could be isolated from bovine brain by similar techniques.

Animals↗
PubMedDate not supplied

Pulsatile and steady flow-induced calcium oscillations in single cultured endothelial cells.

The influence of flow-imposed shear stress on the intracellular calcium concentration ([Ca2+]i) of cultured endothelial cells (ECs) remains incompletely understood. In the present study, we measured [Ca2+]i in single bovine aortic ECs, using fluorescence ratiometric image analysis. The effects of several flow patterns were analysed: steady shear stress (5-70 dyn/cm2), 1-Hz pulsatile shear stress (nonreversing 40 +/- 20 dyn/cm2, reversing 20 +/- 40 dyn/cm2, or purely oscillatory 0 +/- 20 dyn/cm2), or changing shear stress levels. Under all flow conditions, single-cell analyses revealed flow-induced asynchronous [Ca2+]i oscillations, which occurred randomly over the monolayer and which were not seen in the average [Ca2+]i signal corresponding to the monolayer response. The number of single-cell [Ca2+]i oscillations and the corresponding oscillation frequency rose as the shear stress associated with the steady flow increased: 0.06 +/- 0.02 min-1 at 5 dyn/cm2, 0.19 +/- 0.03 min-1 at 20 dyn/cm2, and 0.28 +/- 0.02 min-1 at 70 dyn/cm2 (means +/- SD). Also, the number of oscillations was greater for any type of pulsatile flow (0.53 +/- 0.07 min-1 at 40 +/- 20 dyn/cm2, 0.54 +/- 0.08 min-1 at 20 +/- 40 dyn/cm2, and 0.39 +/- 0.07 min-1 at 0 +/- 20 dyn/cm2), as compared to any level of steady flow. The most dramatic finding was that purely oscillatory flow induced numerous single-cell [Ca2+]i oscillations, yet the average [Ca2+]i response for the monolayer did not change. Furthermore, an EC monolayer switched from low to high (or from high to low) steady flow consistently showed an increase (or a decrease) in the number of single-cell [Ca2+]i oscillations. These experiments show that ECs respond to different flow conditions by varying single-cell [Ca2+]i oscillatory activity. This may have important implications in the endothelium-dependent control of vascular physiology, such as the release of vasoactive substances.

Animals↗
PubMedDate not supplied

Mechanotransduction in endothelial cells: temporal signaling events in response to shear stress.

Fluid shear stress is one of the most important mechanical forces acting upon vascular endothelium, because of its location at the interface between the bloodstream and vascular wall. Recent evidence indicates that several intracellular signaling events are stimulated in endothelial cells in response to shear stress. Through these events, shear stress modulates endothelial cell function and vascular structure, but the molecular basis of shear stress mechanotransduction remains to be elucidated. In our research we have focused on three temporal signal responses to shear stress: (1) production of nitric oxide (NO) as an immediate response; (2) activation of extracellular-regulated kinases (ERK1/2; p44/p42 mitogen-activated protein (MAP) kinases) as a rapid response, and (3) tyrosine phosphorylation of focal adhesion kinase (FAK) as a sustained response. In terms of vessel biology, NO production, and ERK1/2 and FAK activation seem to be correlated with vascular homeostasis, gene expression and cytoskeletal rearrangement, respectively. In this review, we discuss the mechanisms that establish the temporal order of shear stress-stimulated responses based on a hierarchy for assembly of signal transduction molecules at the cell plasma membrane.

Animals↗
PubMedDate not supplied

Activation of extracellular signal-regulated kinases (ERK1/2) by angiotensin II is dependent on c-Src in vascular smooth muscle cells.

Among the angiotensin II (Ang II)-mediated signal events likely to be important in vascular smooth muscle cells (VSMCs) is activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). The upstream mediators by which Ang II activates ERK1/2 remain poorly defined. Recently, we showed that Ang II activated c-Src, a nonreceptor kinase, which is a candidate to mediate Ang II signal events. To determine whether c-Src is required for ERK1/2 activation by Ang II, we studied the effects of Src family-selective tyrosine kinase inhibitors on ERK1/2 activation and also studied Ang II-mediated signal events in Src-deficient and Src-overexpressing VSMCs. The tyrosine kinase inhibitors, genistein and CP-188,556, blocked Ang II-mediated ERK1/2 activation in rat VSMCs (rVSMCs). We derived Src-deficient VSMCs from the aortas of c-Src knockout mice (Src-/- mVSMCs). Basal ERK1/2 activity was lower, and activation of ERK1/2 by Ang II was significantly decreased in Src-/- mVSMCs compared with wild-type mVSMCs, whereas ERK1/2 protein expression and ERK1/2 activation by phorbol 12-myristate 13-acetate were similar. To examine the role of c-Src further, we overexpressed wild-type or dominant-negative c-Src in rVSMCs using retroviral vectors. ERK1/2 activation by Ang II was significantly increased in rVSMCs that overexpressed c-Src, whereas ERK1/2 activation by Ang II was significantly inhibited in rVSMCs that overexpressed dominant-negative c-Src compared with control rVSMCs. These findings demonstrate that c-Src activation is required for Ang II stimulation of ERK1/2 in VSMCs and suggest an important role for c-Src in Ang II-mediated signal transduction.

Angiotensin II↗
PubMedDate not supplied

Differential regulation of p90 ribosomal S6 kinase and big mitogen-activated protein kinase 1 by ischemia/reperfusion and oxidative stress in perfused guinea pig hearts.

Reactive oxygen species (ROS) activate members of the Src kinase and mitogen-activated protein kinase superfamily, including big mitogen-activated protein kinase 1 (BMK1) and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth through the activation of several transcription factors, as well as the Na(+)/H(+) exchanger. Previously, we showed that Src regulates BMK1 via a redox-sensitive signaling pathway. Because ROS are generated during ischemia and reperfusion after ischemia, we assessed the effects of these stimuli (H(2)O(2), ischemia, and reperfusion) in the activation of ERK1/2, p90RSK, Src, and BMK1 in perfused guinea pig hearts. H(2)O(2) (100 micromol/L) significantly activated all kinases. Ischemia alone stimulated p90RSK, Src, and BMK1 but not ERK1/2. These results suggest that p90RSK activation through ischemia occurs via a pathway other than ERK1/2. A role of Src in ischemia-mediated BMK1 activation was demonstrated through inhibition with the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Reperfusion after ischemia stimulated both p90RSK and ERK1/2. In contrast, although ROS increase during reperfusion after ischemia, the activities of both BMK1 and its upstream regulator, Src, were markedly attenuated by reperfusion after ischemia. The activation of C-terminal Src kinase during ischemia but not during reperfusion suggests that the attenuation of Src and BMK1 activity by reperfusion was not regulated by C-terminal Src kinase activity. The antioxidant N-2-mercaptopropionylglycine completely inhibited ERK1/2 and p90RSK activation by reperfusion but only partially inhibited ischemia-induced Src and BMK1 activation. The present study is the first to show the coregulation of Src and BMK1 by reperfusion after ischemia, which we propose to occur via a novel, ROS-independent pathway.

Animals↗
PubMedDate not supplied

Growth factors and the vessel wall.

The vascular tree is a highly specialized organ that has developed a complex and highly orchestrated mechanism of response to injury and stress. After PTCA-like injury many of these mechanisms are activated to maintain vessel integrity. Similar but clearly different growth mechanisms are activated by hypertension and hemodynamic stress. In this light, restenosis is merely a reparative response that has gone on too long or to a greater extent than necessary. The number of growth factors that affect this system is enormous, and their temporal and spatial distribution suggests that therapy targeted to any single factor is unlikely to be successful. In contrast, therapies directed at common mediators such as extracellular matrix and downstream cellular effectors may be more effective. Increasing knowledge of these interactions should point the way to new therapies based on cell-cell and cell-matrix interactions that are tissue specific and focused in time and that take advantage of the common mechanisms by which vessels normally adapt to stress.

Animals↗