摘要:

The time course of 5-bromo-2’-deoxyuridine (BrdU) uptake by medial and neointimal smooth muscle cells was examined in rat carotid arteries at periods of 1-20 days after balloon catheter injury. DNA-incorporated BrdU was determined by the indirect immunoperoxidase technique in both plastic- and paraffin-embedded specimens. The BrdU labeling index (LI) peaked at 48 h in the media (19.9% paraffin, 9.4% plastic) and at 5 days in the neointima (55.4% paraffin, 60.2% plastic). Immunohistochemistry for the endogenous marker proliferating cell nuclear antigen was variable and generally less reliable than BrdU. Using total viable medial smooth muscle cell number as an index of medial injury, the LI for BrdU was directly proportional to the extent of injury at 48 h, and correlated with loss of actin immunoreactivity. BrdU immunohistochemistry is a reliable alternative to [3H]-thymidine autoradiography for short-term labeling in this model. Proliferation of smooth muscle cells in this model is likely related to the degree of injury to the medi

ISSN:1018-1172    

DOI:10.1159/000159050

出版商:S. Karger AG    

年代:1994

数据来源: Karger

摘要:

The effects of glyburide, a purportedly selective ATP-sensitive K+ channel antagonist, were studied on dihydropyridine (DHP)-sensitive (L-type) Ca2+ channel currents in rat aortic muscle cells. Whole-cell voltage-clamp Ba2+ currents (IBa) were recorded at a series of test potentials (Vτ) from -30 to +60 mV during 300-ms voltage steps from a holding potential of –80 mV. Bay k8644 (1 µM) increased peak divalent cation currents from 47.2 ± 15.1 to 102.6 ± 13.4 pA, and the current-voltage relationship curve was shifted 10 mV to the left (n = 5). The combination of 10 µM glyburide with 1 µM Bay k8644 further increased Bay k8644-enhanced IBa in each cell (average of 223.7 ± 26.4 pA, n = 5), and caused a further 10 mV hyperpolarizing (leftward) shift of the activation curve. The kinetics of IBa were also changed (more rapid inactivation) by glyburide. These stimulatory actions of glyburide were reversed on washout. In contrast to this apparent synergism with Bay k8644, 10 µM glyburide alone inhibited (rather than potentiated) IBa by about 20% at Vτ of 0, + 10, and +30 mV. Increasing glyburide concentration to 30 µM further inhibited the IBa to about 40-50% of controls. With the pure agonist isomer, 0.5 µM Bay R5417, at theoretically the same concentration of the minus enantiomer as is present in Bay k8644, IBa increased from 137 ± 18.3 pA to 354.2 ± 12.4 pA (n = 4). However, the combination of 10 µM glyburide and 0.5 µM Bay R5417 failed to further enhance IBa (tendency to block) at any VT, implying that the glyburide-Bay k8644 interaction may depend on both the stronger agonist and weaker antagonist isomers. IBa enhanced by 10 µM glyburide seem to flow through L-type Ca2+ channels because they were significantly blocked by the DHP antagonists, nimodipine and nisoldipine. Nimodipine (3 µM)inhibited the peak IBa by 30%, and 3 µM nisoldipine blocked IBa by almost 70%. Because glyburide produced opposite effects (block) when combined with the pure agonist enantiomer of Bay k8644, similar to the glyburide alone, but opposite to enhancement of racemic Bay k8644 agonist action, it is likely that there are multiple sites for modulation of vascular muscle L-type Ca2+ channels. Possible explanations include the existence of subtypes of DHP receptors, and glyburide actions that depend on the balance of DHP agonist versus antagonist influence. Glyburide could either be weakening the Ca2+ antagonist actions or acting at a separate sulfonylurea site that inter

ISSN:1018-1172    

DOI:10.1159/000159051

出版商:S. Karger AG    

年代:1994

数据来源: Karger

摘要:

To evaluate the direct effects of the angiotensin-converting enzyme (ACE) inhibitors, captopril, enalaprilat, enalapril (a prodrug without therapeutically significant ACE inhibitory effect) and ramiprilat, on cellular calcium metabolism, the cytosolic free calcium concentration was measured in cultured rat vascular smooth muscle cells using the fluorescent dye, fura-2. Preincubation with captopril, enalaprilat, enalapril, or ramiprilat for 40 min significantly reduced the angiotensin II-induced transplasma membrane calcium influx but did not influence the angiotensin II-induced calcium release from internal stores. Captopril and ramiprilat also inhibited arginine vasopressin, but not the thapsigargin-, norepinephrine-, or the BayK 8644-induced changes in cytosolic calcium. Phorbol 12-myristate 13-acetate pretreatment for 30 s caused an increase in the angiotensin II-induced rise in cytosolic calcium. Although both captopril and verapamil reduced responses to angiotensin II to similar extents, only verapamil blocked the ability of phorbol 12-myristate 13-acetate to enhance responses to angiotensin II. It is concluded that ACE inhibitors modulate the effects of some but not all agonist-induced transplasma membrane calcium influx.

ISSN:1018-1172    

DOI:10.1159/000159052

出版商:S. Karger AG    

年代:1994

数据来源: Karger

摘要:

In a previous study, we demonstrated that a high concentration (≧1 µM)of isoproterenol (ISO) produced a dual effect on L-type Ca2+ current (Ica(L)) in vascular smooth muscle (VSM) cells from the portal vein: an initial stimulatory action followed by a sustained inhibition. The first stimulatory phase was fast (presumably more direct) and may reflect G-protein gating of the Ca2+ channels. The second inhibitory phase was slower (presumably more indirect) and may be mediated by the adenylate cyclase/cAMP pathway. In order to define further the mechanism for the ISO inhibition of Ica(L), the effects of cyclic nucleotides and their related protein kinases were examined in freshly isolated single smooth muscle cells from the rabbit portal vein using the whole-cell voltage clamp technique. To isolate Ica(L), the pipette solution contained high Cs+ (to block K+ outward current), and the bath contained physiological salt solution. Upon extracellular application of membrane-permeable cAMP and cGMP analogs (8-Br-cAMP and 8-Br-cGMP, 3 mM), ICa(L) was significantly inhibited by 27.9 ± 5.0 and 33.5 ± 4.8%, respectively. Forskolin (100 µM) also depressed Ica(L)· The protein kinase inhibitor, H-7, prevented the inhibitory effects of both cyclic nucleotides and forskolin. In addition, intracellular application (via the patch pipettes) of cAMP-dependent protein kinase (PK-A, catalytic subunit; 1.76 µM)and cGMP-dependent protein kinase (PK-G, 50 nM, pre-activated by 10 µM cGMP) significantly inhibited the peak amplitude of Ica(L) by 45.5 ± 10 and 43.2 ± 6.2%, respectively. These results indicate that, in portal vein VSM cells, phosphorylation of the Ca2+ channel protein, or of an associated regulatory protein, by PK-A and PK-G depresses Ica(L)· The inhibition of Ica(L) by cyclic nucleotides may decrease the intracellular Ca2+ concentration and contractility, and therefore contributes to their vasodilatory effects. Thus, Ca2+ channel phosphorylation may provide an important mechanism for the cyclic nucleotide-dependent actions of some v

ISSN:1018-1172    

DOI:10.1159/000159053

出版商:S. Karger AG    

年代:1994

数据来源: Karger

摘要:

We have used a double-immunolabelling technique on human carotid atherosclerotic plaques to measure cell proliferation and type-I collagen gene expression, using antibodies to proliferating cell nuclear antigen (PCNA) and type-I procollagen protein, respectively. Although cell proliferative activity and type-I collagen gene expression can occur simultaneously in the same cell, this is a rare event, and the vast majority of collagen-producing cells do not show proliferative activity. These two processes also tend to occur in separate locations, although they can coexist in certain regions of the plaque. This disparate location of these two important modes of plaque growth suggests that cell proliferation and collagen gene expression may be under separate biological controls during the development and evolution of human atherosclerosis.

ISSN:1018-1172    

DOI:10.1159/000159054

出版商:S. Karger AG    

年代:1994

数据来源: Karger

摘要:

Intravital microscopy of the hamster cheek pouch microvasculature was used for in vivo studies on the effects of buflomedil, phentolamine (α-adrenergic receptor antagonist) and norepinephrine on the mean internal arteriolar diameter and spontaneous vasomotion. All drugs were applied topically. The vasomotor activity was studied in 125 arterioles (internal diameter range 18.0–62.0 µm) of 34 preparations. Addition of buflomedil (10–9 to 10–5M) did not affect the arteriolar diameter significantly (from 100.7 ± 3.5 to 106.4 ± 1.8%, values expressed in percent of the initial diameter as mean ± SE), but increased the vasomotion frequency and amplitude by approximately 20% (from 7.5 ± 0.3 to 9.2 ± 0.2 cpm) and 30% (from 7.3 ± 0.3 to 10.0 ± 0.5 µm), respectively. Phentolamine (10–9 to 10–5M) dose-dependently increased the microvascular diameter (from 102.3 ± 1.2 to 139.1 ± 4.3%) and reduced the vasomotion frequency and amplitude (from 8.0 ± 0.3 to 1.9 ± 0.5 cpm and from 9.0 ± 2.1 to 3.1 ± 0.2 µm, respectively). Addition of buflomedil (10–7M) reduced the vasodilation evoked by phentolamine (from 103.3 ± 0.7 to 127.0 ± 1.5%) and potentiated its depressive effect on vasomotion frequency and amplitude (from 7.6 ± 0.1 to 1.0 ± 0.3 cpm and from 9.0 ± 0.3 to 1.9 ± 0.6 µm, respectively). Norepinephrine (10–9 to 10–5M) dose-dependently decreased to arteriolar diameter (from 102.3 ± 0.7 to 69.6 ± 1.6%) and the vasomotion frequency and amplitude (from 8.4 ± 0.2 to 0.4 ± 0.3 emp and from 8.7 ± 0.2 to 0.5 ± 0.4 µm, respectively). Addition of buflomedil (10–7 M) reduced the vasoconstriction elicited by norepinephrine (from 103.2 ± 0.9 to 82.9 ± 3.9%) and restored the vasomotion frequency and amplitude (from 8.4 ± 0.3 to 7.8 ± 0.4 cpm and from 8.6 ± 0.2 to 7.8 ± 0.3 µm, respectively). The effects observed with buflomedil on the hamster cheek pouch microcirculation suggest a direct action on the smooth muscle excitability and further support its properties as a competitive inhibitor of

ISSN:1018-1172    

DOI:10.1159/000159055

出版商:S. Karger AG    

年代:1994

数据来源: Karger

ISSN:1018-1172    

DOI:10.1159/000159056

出版商:S. Karger AG    

年代:1994

数据来源: Karger

摘要:

Oscillatory contractions in uterine smooth muscle are mechanistically related to gap junction complex formation. We have tested the hypothesis that agonist-induced oscillations in vascular smooth muscle are also mediated by gap junctions and that gap junctions are important for vascular smooth muscle cell communication. Total RNA from cultured Wistar-Kyoto rat (WKY) mesenteric arterial cells hybridized strongly with a cDNA probe for the message for connexin43, a monomer of the gap junction. In these same cells, the quaternary ion tetraethylammonium (TEA) (10 mM) increased Lucifer yellow dye transfer between contiguous cells, a measure of cell-to-cell communication via gap junctions, approximately 35% above basal levels. Heptanol, an established inhibitor of gap junction communication, completely blocked both basal- and TEA-stimulated dye transfer between neighboring cells. In other experiments, helical strips of superior mesenteric and tail arteries from WKY rats were mounted in tissue baths for measurement of isometric contractile force. TEA (10–3–10–1M) induced oscillatory contractions (1–5 cycle/min) in both mesenteric and tail arteries. Removal of endothelium did not affect the pattern of TEA-stimulated oscillations. Oscillations to TEA were blocked in a concentration-dependent manner in both arteries by heptanol (10–7–10–3M). Heptanol (10–3M) also significantly reduced (40%) acetylcholine-induced relaxation in the mesenteric artery (contracted with phenylephrine). Thus, these data document that: (1) the message for the gap junction protein connexin43 is located in cultured arterial cells; (2) TEA induces oscillatory contractions in vascular smooth muscle and stimulates intercellular communication in cultured cells, and (3) oscillatory contractions, vascular relaxation and intercellular communication are blocked by the gap junction inhibitor heptanol. Collectively, these data strongly support the importance of gap junctional communication in vascular smooth muscle reactivity, including both oscillatory contractions and vasc

ISSN:1018-1172    

DOI:10.1159/000159057

出版商:S. Karger AG    

年代:1994

数据来源: Karger