摘要:

We cloned a portion of the mouse smooth muscle myosin heavy chain (SM-MHC) cDNA and analyzed its mRNA expression in adult tissues, several cell lines, and developing mouse embryos to determine the suitability of the SM-MHC promoter as a tool for identifying smooth muscle-specific transcription factors and to define the spatial and temporal pattern of smooth muscle differentiation during mouse development. RNase protection assays showed SM-MHC mRNA in adult aorta, intestine, lung, stomach, and uterus, with little or no signal in brain, heart, kidney, liver, skeletal muscle, spleen, and testes. From an analysis of 14 different cell lines, including endothelial cells, fibroblasts, and rhabdomyosarcomas, we failed to detect any SM-MHC mRNA; all of the cell lines induced to differentiate also showed no detectable SM-MHC. In situ hybridization of staged mouse embryos first revealed SM-MHC transcripts in the early developing aorta at 10.5 days post coitum (dpc). No hybridization signal was demonstrated beyond the aorta and its arches until 12.5 to 13.5 dpc, when SM-MHC mRNA appeared in smooth muscle cells (SMCs) of the developing gut and lungs as well as peripheral blood vessels. By 17.5 dpc, SM-MHC transcripts had accumulated in esophagus, bladder, and ureters. Except for blood vessels, no SM-MHC transcripts were ever observed in developing brain, heart, or skeletal muscle. These results indicate that smooth muscle myogenesis begins by 10.5 days of embryonic development in the mouse and establish SM-MHC as a highly specific marker for the SMC lineage. The SM-MHC promoter should therefore serve as a useful model for defining the mechanisms that govern SMC transcription during development and disease.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Although the heart is considered a relatively pure source of m2 muscarinic receptors, the possible expression of other muscarinic receptor genes at discrete sites within the myocardium or by intrinsic cardiac ganglia had not been evaluated. Accordingly, the present study used in situ hybridization histochemistry with35S-labeled oligonucleotide probes to address this issue. Initial experiments demonstrated that the localization of m2 mRNA was similar to that reported for muscarinic receptors labeled with the nonselective muscarinic antagonist quinuclidinyl benzilate; however, there were two important exceptions. The conducting system contained less message than expected, whereas the intrinsic cardiac ganglia contained more. The mismatch between muscarinic receptor and m2 mRNA densities in the conducting system could not be explained by the local expression of other muscarinic receptor genes, since m1, m3, and m4 mRNAs were not detected at this or any other site within the myocardium. However, the presence of a high density of prejunctional muscarinic receptors in the conducting system would be consistent with such a mismatch. Surprisingly, the intrinsic cardiac ganglia contained more than four times as much m2 mRNA as found in the atria. This level of message may be necessary for the production of prejunctional receptors on cholinergic nerve fibers within the heart and receptors localized to the ganglion cell bodies. The ganglia also contained smaller amounts of m1 and m4 mRNAs. These observations suggest that prejunctional muscarinic receptors could have a prominent role in regulating cholinergic neurotransmission in the conducting system and that multiple muscarinic receptors are present in the intrinsic cardiac ganglia.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Acetylcholine (ACh) elicits vasodilation by releasing a number of endothelium-derived relaxing factors (EDRFs). We used the isolated perfused hydronephrotic rat kidney to examine the characteristics of ACh-induced vasodilation of renal afferent arterioles during different types of underlying vasoconstriction. Basal arteriolar tone was increased by either elevating perfusion pressure to 180 mm Hg (myogenic), administering 0.3 μmol/L norepinephrine (NE), or elevating medium potassium concentration to 30 mmol/L (KCl). ACh (10 μmol/L) completely reversed myogenic and NE-induced vasoconstriction and reversed KCl-induced vasoconstriction by 80±5%. However, whereas ACh produced a sustained vasodilation during KCl- and NE-induced vasoconstriction, only a transient reversal of myogenic vasoconstriction was observed, and myogenic tone recovered within 5 to 10 minutes. ACh-induced vasodilation of arterioles preconstricted with KCl was markedly inhibited by either indomethacin (100 μmol/L) or nitro-L-arginine (100 μmol/L) and was completely abolished by pretreatment with both inhibitors. In contrast, indomethacin and nitro-L-arginine had no effect on the transient response to ACh observed during pressure-induced vasoconstriction. In vessels preconstricted with NE, nitro-L-arginine converted the normally sustained response to ACh to a transient vasodilation, which was refractory to both nitric oxide synthase and cyclooxygenase inhibition. Since this component was not observed during KCl-induced vasoconstriction, it may reflect the actions of an, as yet unidentified, endothelium-derived hyperpolarizing factor (EDHF). Our findings thus suggest that prostanoids, nitric oxide, and EDHF all contribute to ACh-induced renal afferent arteriolar vasodilation and that the relative contributions of these individual EDRFs depends on the nature of the underlying renal vascular tone.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

This study investigated the role of volume status and perfusion pressure on the hemodynamic response of cortical and medullary renal capillaries to systemic inhibition of nitric oxide.NG-Monomethyl-L-arginine (L-NMMA) was infused intravenously (15-mg/kg bolus and 500-μg · min−1· kg−1infusion), and blood flow in cortical capillaries (QCC) and in descending (QDVR) and ascending vasa recta (QAVR) was measured by fluorescence videomicroscopy in euvolemic and volume-expanded anesthetized Munich-Wistar rats. L-NMMA in euvolemic rats decreased vasa recta blood flow (ΔQDVR, 3.97 ± 0.80 nL/min [P<.01]; ΔQAVR, 1.90 ± 0.39 nL/min [P<.01]; n=6) and QCC(ΔQCC, 0.57 ± 0.15 nL/min [P<.01]; n=7) despite increases in renal perfusion pressure (RPP). Fractional excretion of sodium (FENa) remained unchanged. In volume-expanded rats, L-NMMA decreased vasa recta blood flow when RPP increased (ΔQDVR, 1.42 ± 0.79 nL/min [P= .05]; ΔQAVR, 1.95 ± 0.34 nL/min [P<.001]; n=9) or was held constant by partial aortic occlusion (ΔQDVR, 1.19 ± 0.45 nL/min [P<.05]; ΔQAVR, 1.44 ± 0.40 nL/min [P <.01]; n=8). QCCwas unchanged by L-NMMA when RPP increased (ΔQCC, 0.27 ± 0.20 nL/min; n=8) but decreased significantly by 0.61 ± 0.11 nL/min (P<.01, n=8) when increases in RPP were prevented. FENaincreased when RPP increased (ΔFENa, 2.47 ± 0.51%P<.001) and was held constant (ΔFENa, 2.64 ± 0.46%P<.001). In summary, systemic inhibition of nitric oxide increases blood pressure while decreasing QCCand medullary capillary blood flow in euvolemic rats. In volume-expanded rats, L-NMMA again increases blood pressure but selectively decreases medullary blood flow without changing QCC; QCCdecreased, however, when RPP was held constant. This study supports a role for nitric oxide synthesis in the regulation of QCCand medullary blood flow and demonstrates that volume expansion allows increases in RPP to maintain QCCbut not medullary blood flow during inhibition of nitric oxide.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Thromboxane A2(TXA2) and angiotensin II (Ang II) stimulate vascular smooth muscle hypertrophy by upregulating endogenous synthesis of basic fibroblast growth factor (bFGF). Because mitogenic phorbol esters can also stimulate bFGF formation, we investigated the role of protein kinase C (PKC) in vascular smooth muscle cell (VSMC) bFGF formation and hypertrophy. Preliminary characterization of PKC isoform expression in VSMC by use of polymerase chain reaction identified PKC α, Δ, ϵ, and ξ. Western analysis confirmed the presence of these isoforms in cultured VSMC lines and demonstrated downregulation of PKC α, Δ, and ϵ by phorbol 12-myristate 13-acetate (PMA) but not TXA2or Ang II. PKC activation with 100 nmol/L PMA stimulated VSMC mitogenesis measured as incorporation of [3H]leucine and [3H]thymidme and increased cell number. Like TXA2and Ang II, PMA increased endogenous VSMC bFGF in a time-dependent manner, whereas an inactive phorbol ester had no such effect. Addition of an antisense oligodeoxynucleotide against bFGF prevented PMA-stimulated bFGF expression and inhibited PMA-stimulated growth, suggesting that bFGF synthesis is necessary for VSMC growth stimulated by PMA. To clarify the role of PKC in vasoconstrictor-stimulated VSMC production of bFGF and hypertrophy, PKC was down-regulated by prolonged exposure to PMA or was inhibited with calphostin C or staurosporine before the addition of TXA2or Ang II. PKC inhibition prevented TXA2-stimulated and Ang II-stimulated VSMC hypertrophy without attenuating the observed increase in bFGF expression. Furthermore, PKC inhibition with calphostin C inhibited VSMC mitogenesis stimulated by exogenous bFGF. These results indicate that activation of PKC α, Δ, or ϵ is necessary to transduce TXA2-stimulated and Ang II-stimulated VSMC hypertrophy at a point in the cell-signaling pathway distal to synthesis of endogenous bFGF. In contrast, TXA2and Ang II upregulate bFGF during VSMC growth via a mechanism distinct from PKC activation. Finally, increased endogenous expression of bFGF or exposure to exogenous bFGF is not sufficient to stimulate VSMC growth in the absence of PKC activation.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Increased glomerular capillary hydrostatic pressure (PGC) is an important hemodynamic determinant of glomerular injury, but the molecular events responsible for this association are poorly understood. PGC is normal in spontaneously hypertensive rats (SHR), but uninephrectomy leads to an increase in PGC and accelerated glomerulosclerosis. Since recent studies have implicated transforming growth factor-β1(TGF-β1) and platelet-derived growth factor (PDGF) in the pathogenesis of glomerulosclerosis, we first sought to determine if uninephrectomy increased mRNA levels for TGF-β1and PDGF in glomeruli of SHR. Since treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril lowers PGC and prevents glomerulosclerosis in uninephrectomized SHR, we also sought to determine if ACE inhibitor lowered mRNA levels for TGF-β1and PDGF in the glomeruli of uninephrectomized SHR. PGC increased from 53 ± 1 to 64 ± 1 mm Hg 1 week after uninephrectomy in SHR (P<.05). The increase in PGC was associated with a sixfold rise in mRNA levels for TGF-β1and a twofold rise in mRNA levels for PDGF in glomeruli. mRNA levels for PDGF returned to normal 2 weeks after nephrectomy, but the increase in mRNA levels for TGF-β1was sustained. An increase in TGF-β1immunostaining was detectable in glomeruli 4 weeks after nephrectomy. Treatment with ACE inhibator normalized PGC (51 ± 1 mm Hg) and prevented the rise in glomerular mRNA levels for TGF-β1and PDGF. We conclude that an acute increase in PGC leads to increased TGF-β1and PDGF expression in the glomerulus, thus linking changes in PGC to cytokine gene expression.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The intracellular mechanism underlying the Ca2+-induced enhancement of the L-type Ca2+current (1Ca) was examined in adult rabbit cardiac ventricular myocytes by using patch-clamp methodology. Internal Ca2+was elevated by flash photolysis of the Ca2+chelator Nitr 5, and intracellular Ca2+levels were simultaneously monitored by Fluo 3 fluorescence. Flash photolysis of Nitr 5 produced a rapid (<l-second) elevation of internal Ca2+, which led to enhancement (39% to 51% above control) of the peak inward Ca2+current after a delay of 20 to 120 seconds. Internal dialysis of myocytes with synthetic inhibitory peptides derived from the pseudosubstrate (peptide 273-302) and calmodulin binding (peptide 291-317) regions within the regulatory domain of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) blocked enhancement of ICaproduced by elevation of internal Ca2+but not that produced by β-adrenergic stimulation. These inhibitory peptides also had no effect on the elevation of internal Ca2+produced by flash photolysis of Nitr 5. A pseudosubstrate inhibitory peptide derived from protein kinase C had no significant effect on Ca2+-dependent enhancement of ICa. We conclude that CaM kinase mediates the Ca2+-induced enhancement of ICain mammalian cardiac myocytes by a mechanism likely involving direct phosphorylation of the L-type Ca2+channel complex or an associated regulatory protein.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The effects of β-adrenergic agonists on pHiwere studied on single ventricular myocytes isolated from adult rat heart and loaded with the acetoxymethyl ester (AM) form of the pH indicator SNARF-1. In modified Krebs' solution containing 20 mmol/L HEPES and 4.4 mmol/L HCO3−, isoproterenol (1 μmol/L) caused a significant decrease of steady-state pHifrom 7.20 ± 0.02 to 7.13 ± 0.02 (mean ± SEM) within 2 minutes. This acidification, which was also observed in myocytes that were preloaded with the Ca2+chelator BAPTA and supervised with nominally Ca2+-free solution, was blocked by propranolol as well as by the specific β1-antagonist CGP 20712 A but not by the β2-antagonist ICI 118,551. Forskolin (10 μmol/L) induced a similar reversible decrease of pHi(average decrease, 0.11 ± 0.02 pH unit). Furthermore, adenosine (100 μmol/L) substantially attenuated the isoproterenol-induced decrease of pHi. The effect of isoproterenol was not prevented by inhibitors of the Na+-H+antiport, amiloride (1 mmol/L) and 2-N,N-hexamethylene amiloride (20 μmol/L). On the other hand, blockers of Cl−transport mechanisms, DIDS (200 μmol/L) and probenecid (100 μmol/L), inhibited this acidification. Isoproterenol also failed to induce a decrease of steady-state pHiin myocytes incubated in Cl−-free medium. Rather, the initial rate of rise of pHiobserved on removal of external Cl−ions was significantly increased in the presence of isoproterenol or dibutyryl cAMP. Because the alkalinization induced by removal of Cl−ions is mainly due to reversal of the Cl−-HCO3−exchanger, the augmentation of this initial rate of pHirise directly points to a β-adrenergic stimulation of the exchanger. Furthermore, the pHirecovery following NH4Cl exposure was accelerated by isoproterenol in the presence of probenecid, indicating that the Na+-HCO3−cotransport and/or the Na+-H+antiport also could be activated. In conclusion, the present results demonstrate that β-adrenergic agonist-induced acidification of rat ventricular myocytes occurs mainly through alteration of Cl−transport systems, most likely via a cAMP-dependent stimulation of the Cl−-HCO3−exchanger. Since the alkalinizing mechanisms are also stimulated, an increased apparent cellular buffering capacity is expected.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Atrial tumor myocytes derived from transgenic mice (AT-1 cells) maintain a well-differentiated cardiac biochemical and histological phenotype. In addition, they beat spontaneously in culture and exhibit long action potentials whose repolarization resembles that observed in native mammalian myocytes. In this study, we identified the major depolarization-activated outward currents in AT-1 cells; also, the presence of mRNAs that encode outwardly conducting ion channels was determined by cloning from an AT-1 cDNA library or by Northern hybridization. Among K+channel isoforms, Kv2.1, minK, and Kvl.4 were readily detected in tumors and at 1 day in culture. Their abundance remained relatively stable (twofold or less change) after 14 days. The major outward current in AT-1 cells is a delayed rectifier that displays prominent inward rectification, activates rapidly (eg, 182 ± 27 milliseconds [mean ± SEM] at + 20 mV, n=12), exhibits biexponential deactivation kinetics, and is extremely sensitive to the methanesulfonanilide dofetilide (IC50, 12 nmol/L). These characteristics identify this current as IKr, a delayed rectifier observed only in cardiac cells. IKrin AT-1 cells displayed slow inactivation: dofetilide-sensitive deactivating tails were greater after 1-second than after 5-second pulses. When IKrwas blocked by ≤0.5 μmol/L dofetilide, time-independent current was usually recorded (50 of 65 experiments); rapidly inactivating (6 of 65) or slowly inactivating (9 of 65) outward currents were occasionally observed. We conclude that AT-1 cells express mRNAs encoding cardiac K+channels and display a cardiac electrophysiological phenotype. This system may therefore be a useful tool in studies of regulation of cardiac ion channel gene expression and its functional consequences. The presence of a readily recorded IKrwill facilitate study of the physiology and pharmacology of this important repolarizing current.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

There is a high incidence of diuretic use among patients who develop exaggerated QT prolongation and polymorphic ventricular tachycardia (torsade de pointes) during treatment with action potential-prolonging agents. Diuretic-induced hypokalemia is thought to be the usual mechanism, but a direct effect of diuretic drugs on repolarizing currents is an additional possibility. Therefore, in this study, we examined the effects of the diuretic agents chlorthalidone and indapamide on the cardiac delayed rectifier current. In guinea pig ventricular myocytes, this current is made up of two components: IKr, a rapidly activating, inwardly rectifying current blocked by most action potential-prolonging antiarrhythmics, and Iks, a slowly activating component. In this preparation, indapamide blocked outward current in a time-, voltage- and concentration-dependent fashion, whereas chlorthalidone (1 mmol/L) was without effect. The following features of the effect of indapamide strongly suggest selective block of Iks: (1) Indapamide block was significantly greater with 5000-millisecond activating pulses (−43±5% at +50 mV [100 μmol/L indapamide]) than with 225-millisecond ones (−20±5%n=5, P<.01), and the signature of the indapamide-sensitive current was a slowly activating delayed rectifier current. (2) The voltage dependence of indapamide block (EC50,101 μmol/L at +50 mV and 196 μmol/L at +10 mV) was consistent with preferential block of Iksrelative to IKr(3) In the presence of indapamide, an envelope-of-tails test for IKrwas satisfied. The drug-insensitive current had rectifying properties similar to those described for IKrin these cells. In addition, under experimental conditions where only IKrwas present, indapamide was without effect. WhenXenopusoocytes were injected with minK, a cRNA encoding an Iks-like current, induced current was blocked by indapamide. These studies demonstrate that indapamide is a K+channel blocker but, unlike most antiarrhythmics, targets Iks

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID