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PubMed2006

Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells.

Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP-dependent protein kinase (PKGI) in vascular smooth muscle cells (VSMC), inhibiting agonist-induced intracellular Ca2+ mobilization ([Ca2+]i). The relative roles of the two PKGI isozymes, PKGIalpha and PKGIbeta, in cyclic GMP-mediated inhibition of [Ca2+]i in VSMCs are unclear. Here we have investigated the ability of PKGI isoforms to inhibit [Ca2+]i in response to VSMC activation. Stable Chinese hamster ovary cell lines expressing PKGIalpha or PKGIbeta were created, and the ability of PKGI isoforms to inhibit [Ca2+]i in response to thrombin receptor stimulation was examined. In Chinese hamster ovary cells stably expressing PKGIalpha or PKGIbeta, 8-Br-cGMP activation suppressed [Ca2+]i by thrombin receptor activation peptide (TRAP) by 98 +/- 1 versus 42 +/- 5%, respectively (p <0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGIalpha- and PKGIbeta-specific antibodies showed PKGIalpha is the predominant VSMC PKGI isoform. [Ca2+]i following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMC cells (Co403). 8-Br-cGMP significantly inhibited TRAP-induced [Ca2+]i in Co403, causing a 4-fold increase in the EC50 for [Ca2+]i. In the absence of 8-Br-cGMP, suppression of PKGIalpha levels by RNA interference (RNAi) led to a significantly greater TRAP-stimulated rise in [Ca2+]i as compared with control RNAi-treated Co403 cells. In the presence of 8-Br-cGMP, the suppression of PKGIalpha expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca2+]i. Adenoviral overexpression of PKGIbeta in Co403 cells was unable to alter TRAP-stimulated Ca2+ mobilization either before or after suppression of PKGIalpha expression by RNAi. These results support that PKGIalpha is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Muscarinic receptor expression in the primary culture of tracheal smooth muscle cells.

Tracheal smooth muscle cells (TSMCs) were isolated from dog trachea in order to analyze the direct effects of growth factors and hormones on cell proliferation and muscarinic receptor (mAchR) expression. Dissection and dissociation of tracheal smooth muscle tissue with a collagenase I, deoxyribonuclease I and elastase IV mixture resulted in high yield and viability of TSMCs. A screen of growth factors, hormones, and serum concentration for the stimulation of cell growth, revealed that insulin-like growth factor, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, or hydrocortisone alone at the concentration used was not necessary or sufficient to stimulate growth of TSMCs in the primary culture with DMEM/F-12 containing 1% FBS. The regulation of cell surface mAchR expression in response to serum and cell growth in primary culture of TSMCs has been examined. In the presence of 1% serum, TSMCs withdraw from the cell cycle and express high levels of cell surface mAchRs. Exposure of quiescent TSMCs to 10% serum results in a loss of surface mAchRs. In addition, insulin-like growth factor, insulin or transferrin could stimulate the expression of mAchRs on TSMCs cultured in DMEM/F-12 containing 1% FBS. The results demonstrated that low serum concentration culture system may provide a useful model to elucidate the expression of mAchRs in the culture of TSMCs.

Animals↗
PubMed1993

Expression of Ca2+ binding proteins of the sarcoplasmic reticulum of striated muscle in the endoplasmic reticulum of pig smooth muscles.

The Ca2+ binding proteins in the lumen of intracellular Ca2+ stores differ between muscle and non-muscle cells, indicating a specific role of these proteins in intracellular Ca2+ regulation. Since smooth muscle cells possess both muscle and non-muscle characteristics, we have studied the presence and the differential expression of the muscle-type Ca2+ binding proteins--calsequestrin, sarcalumenin, and the histidine-rich Ca2+ binding protein (HCP)--in several smooth muscle tissues from the pig. Western blot analysis showed that among the smooth muscles studied, the cardiac isoform of calsequestrin is expressed at the highest levels in the stomach. Calsequestrin was present at lower levels in ileum and trachea, whereas this protein was undetectable in aorta and main pulmonary artery. The total amount of calsequestrin in the stomach was estimated to be 20-30-times lower than in the pig heart. Whereas calsequestrin from pig presented the same apparent M(r) in sodium dodecyl sulphate polyacrylamide gels as the well characterized protein from rabbit, the apparent M(r) of both sarcalumenin and HCP was lower in pig than in rabbit. The presence of HCP was demonstrated in pig stomach and ileum, while sarcalumenin was detected only in the stomach. These results demonstrate further biochemical differences between smooth muscle cells of large blood vessels and those of the digestive tract. The present findings on the differential distribution of muscle-type Ca2+ binding proteins are discussed in relation to biochemical and functional differences between these smooth muscle cells.

Animals↗
PubMed1996

Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle.

1. In smooth muscle, both Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx across the plasma membrane are responsible for the increase in the cytosolic Ca2+ level ([Ca2+]i). To understand further the role of SR on smooth muscle contraction, the effects of an inhibitor of the SR Ca2+ pump, cyclopiazonic acid (CPA 10 microM), an inhibitor of the Ca(2+) -induced Ca2+ release, ryanodine, (10 microM), and an activator of the Ca(2+) -induced Ca2+ release, caffeine (20 mM), on [Ca2+]i and contractile force were examined in the ferret portal vein loaded with a photoprotein, aequorin. 2. CPA induced a small increase in the aequorin signal reaching a maximum at 7 min. Several minutes after the increase in the aequorin signal, muscle tension increased reaching a maximum at 21.5 min. In contrast, ryanodine changed neither the aequorin signal nor contraction. In the presence of ryanodine, caffeine induced a sustained increase in the aequorin signal and transient contraction. After washing ryanodine and caffeine, the aequorin signal and muscle tone returned to their respective control levels. After treatment with ryanodine and caffeine, the second addition of caffeine was almost ineffective whereas CPA still increased the aequorin signal and muscle tension. 3. In the presence of external Ca2+, noradrenaline (NA, 10 microM) induced a transient increase followed by a sustained increase in the aequorin signal and sustained contraction. In contrast, KCl (70 mM) induced sustained increases in the aequorin signal and sustained contraction. In Ca(2+) -free solution, NA induced a small transient increase in the aequorin signal and a small transient contraction. These changes were inhibited in the presence of CPA or on pretreatment of the muscle with ryanodine and caffeine. These results suggest that CPA or ryanodine and caffeine depleted Ca2+ in SR. High K+ was ineffective in the absence of external Ca2+. 4. In the presence of external Ca2+ and CPA, NA and high K+ induced larger aequorin signals than in the absence of CPA, whereas the magnitude and shape of the contractions did not change. In contrast, pretreatment with ryanodine and caffeine did not have such an effect. In the muscle pretreated with ryanodine and caffeine, CPA changed the responses to high K+ and NA in a similar manner to that in the muscle without the pretreatment with ryanodine and caffeine. 5. Dissociation of contraction from [Ca2+]i as measured with aequorin suggests that NA and high K+ increase Ca2+ in two compartments: a compartment containing contractile elements (contractile compartment) and another compartment unrelated to contractile elements (non-contractile compartment). Because CPA augmented the stimulant-induced increase in aequorin signal without changing contraction, the non-contractile compartment may be located near the SR and the CPA-sensitive SR Ca2+ pump may regulate the Ca2+ level in this compartment. However, because CPA changed neither the magnitude nor shape of the contractions in the presence of external Ca2+, the SR Ca2+ pump may have little effect on regulation of Ca2+ level in the contractile compartment. Furthermore, the release of Ca2+ from SR seems to have little effect on the increase in the contractile Ca2+ because ryanodine and caffeine changed neither the aequorin signals nor contractions induced by NA and high K+ in the presence of external Ca2+ in the ferret portal vein.

Adrenergic alpha-Agonists↗
PubMedDate not supplied

Effect of normolipemic and hyperlipemic serum on biosynthetic response to cyclic stretching of aortic smooth muscle cells.

Arterial smooth muscle cells synthesize matrix macromolecules in response to mechanical stimulation. Exposure to serum lipids also stimulates connective tissue fiber accumulation. To assess the effect of serum lipids on the biosynthetic response to tensile stress, we subjected rabbit aortic smooth muscle cells that were cultured on purified elastin membranes to cyclic stretching and relaxation 50 times per minute in the presence of serum-free medium (SFM), normolipemic serum (NLS), or hyperlipemic serum (HLS). Incorporation of 14C-proline into proline and into hydroxyproline was taken as a measure of protein and collagen synthesis. When cells were grown in plastic Petri dishes, exposure to NLS or HLS increased both protein and collagen production to the same extent compared to synthesis in SFM (1.7 times for NLS and 1.6 times for HLS; p less than 0.001 compared to SFM). For cells grown on stationary elastin membranes, NLS and HLS also increased protein and collagen synthesis compared to SFM. The effect of NLS was 1.35 times that of HLS for protein and 1.43 times greater for collagen (p less than 0.03). Cyclic stretching in SFM doubled synthesis for both protein (p less than 0.002) and collagen (p less than 0.002) compared to stationary controls, but had no effect on synthesis in NLS. In HLS, however, cyclic stretching elevated synthesis to the same level as was found in NLS (p less than 0.003). We conclude that the relative inhibition of synthesis on stationary membranes by HLS was not due to a toxic effect, since HLS increased synthesis both in Petri dishes and on elastin membranes, and the amplifying effect of cyclic stretching in HLS was similar to that seen in SFM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Jak2 tyrosine kinase mediates oxidative stress-induced apoptosis in vascular smooth muscle cells.

In vascular smooth muscle cells, Jak2 tyrosine kinase becomes activated in response to oxidative stress in the form of hydrogen peroxide. Although it has been postulated that hydrogen peroxide-induced Jak2 activation promotes cell survival, this has never been tested. We therefore examined the role that Jak2 plays in vascular smooth muscle cell apoptosis following hydrogen peroxide treatment. Here, we report that Jak2 tyrosine kinase activation by hydrogen peroxide is required for apoptosis of vascular smooth muscle cells. Upon treatment of primary rat aortic smooth muscle cells with hydrogen peroxide, we observed laddering of genomic DNA and nuclear condensation, both hallmarks of apoptotic cells. However, apoptosis was prevented by either the expression of a dominant negative Jak2 protein or by the Jak2 pharmacological inhibitor AG490. Moreover, expression of the proapoptotic Bax protein was induced following hydrogen peroxide treatment. Again, expression of a dominant negative Jak2 protein or treatment of cells with AG490 prevented this Bax induction. Following Bax induction by hydrogen peroxide, mitochondrial membrane integrity was compromised, and caspase-9 became activated. In contrast, in cells expressing a Jak2 dominant negative we observed that mitochondrial membrane integrity was preserved, and no caspase-9 activation occurred. These data demonstrate that the activation of Jak2 tyrosine kinase by hydrogen peroxide is essential for apoptosis of vascular smooth muscle cells. Furthermore, this report identifies Jak2 as a potential therapeutic target in vascular diseases in which vascular smooth muscle cell apoptosis contributes to pathological progression.

Animals↗
PubMed1997

Effects of licorice derivatives on vascular smooth muscle function.

Vascular smooth muscle contains a bidirectional form of the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) which can inactivate (dehydrogenase) endogenous circulating glucocorticoids (GCs) or activate (oxo-reductase) 11-dehydro-metabolites by their conversion back to the parent steroid. Enzyme direction in vascular smooth muscle (VSM) has potential physiological consequences since GCs may enhance the response to known vasoconstrictors. We determined that carbenoxolone is a competitive inhibitor of 11beta-HSD contained in VSM cells with a lower Ki for forward dehydrogenase reaction (0.02 microM) compared to the oxo-reductase reaction (0.41 microM). To test whether changes in enzyme directional activity can affect the contractile response, aortae from adrenally intact Sprague Dawley rats were removed and sectioned into 2.5 mm rings. Rings were incubated with corticosterone 10 nM plus carbenoxolone (CBX) 10 microM (a concentration well above the Ki for both the dehydrogenase and oxo-reductase reaction) for 24 hrs. These rings showed an enhanced dose dependent contractile response to phenylephrine (PE) 0.01 microM(-1) microM and to angiotensin II 1 microM compared to rings incubated with corticosterone alone, CBX alone, or controls: [e.g. response to PE 1 microM in mg of contractile force, mean +/- SE: corticosterone plus CBX 1495 +/- 162 (n=10) vs corticosterone 1039 +/- 64 (n=9), p<0.05]. Aortic rings preincubated with 11-dehydrocorticosterone 10(-7)M and CBX 10 microM displayed a decreased contractile response compared to 11-dehydrocorticosterone alone. Thus in situ glucocorticoid metabolism is important in mediating the constrictor responses of vascular tissue.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Levosimendan, a calcium sensitizer in cardiac muscle, induces relaxation in coronary smooth muscle through calcium desensitization.

Levosimendan is a pyridazinone-dinitrile derivative belonging to a new class of cardiac inotropic drugs, Ca++ sensitizers. Levosimendan is also a vasodilator both in vitro and in vivo, but its mechanism is not well understood. The cardiac target protein of levosimendan, troponin C, is a Ca++-binding EF-hand protein. This raises the possibility that levosimendan may also interact with smooth muscle EF-hand proteins, such as, calmodulin, the regulatory myosin light chains, or S100 proteins. We investigated the effects of levosimendan on [Ca++]i, and force in porcine coronary arteries, with receptor-mediated (U46619) or KCl stimulation. At high levels of stimulation, levosimendan decreased force without changing or increasing [Ca++]i, measured with the Ca++-sensitive fluorescent probe fura-2 in the intact artery. With lower levels of U46619, levosimendan (1 microM) lowered force by 70% and reduced [Ca++]i by 38%. The relationship between force and [Ca++]i for KCl stimulation are significantly rightward shifted, indicating Ca++ desensitization by levosimendan. In contrast, the phosphodiesterase III inhibitor, milrinone, does not shift the force-Ca++ relations but elicits relaxation via lowering [Ca++]i. There was little change in pHi, indicating that the Ca++ desensitization by levosimendan was not attributable to decreasing pHi. Levosimendan relaxes coronary arteries and lowers [Ca++]i by mechanisms different than milrinone. Our results indicate a lowering of [Ca++]i by levosimendan consistent with opening of potassium channels and a relaxation that is independent of [Ca++]i. Our evidence points to a novel mechanism that might involve the direct effect of levosimendan on the smooth muscle contractile or regulatory proteins themselves.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Gene structure and chromosomal mapping of the rat smooth muscle calponin gene.

Smooth muscle cells (SMC) express a battery of lineage-restricted genes whose encoded proteins impart the unique contractile phenotype that characterizes this muscle type. While the encoded function of many SMC-restricted genes has been extensively analyzed, less is known about their position within the genome and the regulatory factors governing their transcription. In this report, we define the gene structure, 5' promoter analysis, and chromosomal mapping of the rat smooth muscle calponin (CnnI) gene. The rat CnnI gene is comprised of seven exons spanning approximately 8 kb of genomic sequence. The intron-exon boundaries of the rat CnnI gene match precisely those in human and mouse. Primer extension and RNase protection assays indicate two major transcription start positions (tsp). Comparative sequence analysis of the 5' promoter region reveals several conserved cis regulatory elements, including a TA-rich element within 30 nt of the tsp that could be a recognition site for TATA-binding protein and two CCAAT boxes. Transient and stable transfection studies support the hypothesis that distal regulatory elements confer SMC-restricted expression of CnnI. Finally, using an F2 intercross, we have mapped the rat CnnI gene to the telomeric end of Chromosome (Chr) 8. These studies provide additional information relating to the control of CnnI gene expression and provide a platform to begin assessing the potential linkage of CnnI to spontaneous and experimental disease phenotypes in rats.

Animals↗
PubMed2002

Evidence that action potential generation is not the exclusive determinant to trigger spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle.

Urinary bladder smooth muscle (UBSM) exhibits spontaneous contraction. This spontaneous mechanical activity is myogenic and can be closely related to the UBSM cell action potential to facilitate Ca2+ influx through voltage-gated Ca2+ channels. In the present study, to know whether this membrane electrical event is the exclusive mechanism to trigger spontaneous smooth muscle contraction, we compared the inhibitory effects of Ca2+ channel blockers on the spontaneous action potential and mechanical activity in the isolated guinea-pig UBSM. Both action potential and rhythmic contraction were generated spontaneously in the presence of atropine (1 microM), phentolamine (1 microM), propranolol (1 microM), suramin (10 microM) and tetrodotoxin (1 microM), which suggest that both phenomena were myogenic in origin. Nisoldipine (100 nM) and diltiazem (10 microM) completely eliminated the generation of action potential whereas its frequency was dramatically increased by a dihydropyridine Ca2+ agonist, BayK 8644 (1 microM). In contrast to disappearance of action potential in the presence of Ca2+ channel blockers, spontaneous contraction of UBSM was inhibited only partly by nisoldipine or diltiazem and most of the mechanical components persisted in these channel blockers. These results indicate that spontaneous action potential in UBSM cell is generated through the activation of L-type voltage-gated Ca2+ channels. The subsequent elevation of intracellular Ca2+ concentrations during a burst of action potentials can be partly responsible for the induction of UBSM mechanical activity. In addition, the present study provides evidence that UBSM spontaneous mechanical activity is also attributable to the mechanism(s) other than the generation of Ca2+ spike.

Action Potentials↗
PubMed2000

[Special characteristics of bronchial smooth muscle cells].

Airway smooth muscle is one of the main effectors of bronchial reactivity. Our understanding of the cellular mechanisms involved in the contraction of this muscle has advanced in the recent past since isolated cells in culture can now be studied. Extracellular messengers (neurotransmitters and mediators) as well as their specific membrane receptors have been analyzed in some details. Membrane transduction of extracellular messengers brings about the formation (or the increase in the concentration) of the intracellular second messenger which, in airway smooth muscle, is cytosolic calcium (Ca2+i) via activation of calcium channels which depend on surface membrane potential changes (electromechanical coupling) on one hand and mainly via mechanisms independent of surface membrane potential changes, the so called--pharmacomechanical coupling--which involves membrane phosphoinositide metabolism. Changes in Ca2+i activate contractile proteins leading the muscle to shorten and to develop force via several controlled steps such as phosphorylation of myosin or changes in the sensitivity to Ca2+ of the contractile elements. Information about the cellular physiology and pathophysiology of this muscle is of value to design new drugs for the treatment of bronchoconstriction.

Bronchi↗
PubMed1990

Competence growth factors evoke the phenotypic transition of arterial smooth muscle cells.

Vascular smooth muscle cells (V-SMC) alter their functions from contraction to production of extracellular matrices during the response to injury of the arterial wall. Accumulation of these matrices characterize the chronic structural change of arteriosclerosis. In this paper, we reported as follows. (1) V-SMC freshly isolated from human aortic media had strong reactivity with anti-smooth muscle specific alpha-actin (alpha-SM actin) antibody but not with anti-prolyl 4-hydroxylase (ProHy) antibody. (2) The exponentially growing cells in serum-containing culture media reduced alpha-SM actin and induced ProHy distinctly. (3) Growth-arrested cells with serum starvation rearranged numerous alpha-SM actin fibers in their cytoplasm. And the rearrangement of contractile filaments inhibited by the addition of single competence growth factor, such as platelet derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). These results suggest that V-SMC change their phenotype in a competence-factor-dependent manner.

Actins↗
PubMed1999

Phosphorylation-dependent structural changes in the regulatory light chain domain of smooth muscle heavy meromyosin.

Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 A) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin-heavy meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.

Amino Acid Sequence↗
PubMed2006

Upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle.

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.

Actins↗
PubMed1999

Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

Phospholipase C inhibitors suppress spontaneous mechanical activity of guinea pig urinary bladder smooth muscle.

Urinary bladder smooth muscle (UBSM) exhibits spontaneous rhythmic contraction. This spontaneous mechanical activity is generated in the presence of neuronal blockade and thus is myogenic in origin. The spontaneous myogenic contraction of UBSM may be the fundamental determinant of the physiological functions of the urinary bladder to store and excrete urine. Although the mechanisms by which UBSM generates spontaneous contraction have not been completely ascertained, its induction has been suggested to be intimately associated with smooth muscle cell action potentials to enhance extracellular Ca(2+) influx through voltage-gated L-type Ca(2+) channels. However, the alteration of membrane electrical activity does not seem to be the exclusive trigger mechanism for the generation of the spontaneous contraction. In the present study, we show that spontaneous mechanical activity of guinea pig UBSM is substantially diminished by an inhibitor of phospholipase C (PLC), U-73122, but is not affected by its inactive form, U-73343. Significant attenuation of the mechanical activity can be also obtained with another PLC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate. Our present findings suggest a significant role for the activation of PLC and subsequent inositol 1,4,5-trisphosphate-induced Ca(2+) release mechanism as an alternative triggering system for inducing spontaneous mechanical activity of UBSM. The present results support the idea that the action potential is not the sole pacemaker mechanism by which spontaneous contraction is induced in UBSM.

Animals↗