Defective smooth muscle regulation in cGMP kinase I-deficient mice.

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP-dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross-activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI-deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.     

Modulation of Ca2+ sensitivity by cyclic nucleotides in smooth muscle from protein kinase G-deficient mice

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. We have explored cGMP- and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity.     

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI-/-) mice. We demonstrate that murine neutrophils expressed cGKIalpha. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI-/- bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI-/- than in WT. Enhanced chemotaxis was also observed with cGKI-/- peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI-/- vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI-/- than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI-/- (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI-/- (approximately 60%) than in WT (approximately 90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIalpha down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIalpha on the secretagogue responses are complex, depending on the granule subtype.     

Nitric oxide sensitivity in pulmonary artery and airway smooth muscle: a possible role for cGMP responsiveness

We aimed to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Porcine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used in concentration-response studies to the NO donor (Z)-1-[N-2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM consistently exhibited greater relaxation at a given DETA-NO concentration (NO responsiveness) than TSM NO responsiveness, with DETA-NO log EC(50) being -6.55 +/- 0.11 and -5.37 +/- 0.13 for PASM and TSM, respectively (P < 0.01). We determined relationships between tissue cGMP concentration ([cGMP](i)) and relaxation using the particulate guanylyl cyclase agonist atrial natriuretic peptide. Atrial natriuretic peptide resulted in nearly complete relaxation, with no detectable increase in [cGMP](i) in PASM and only 20% relaxation (10-fold increase in [cGMP](i)) in TSM, indicating that TSM is less cGMP responsive than PASM. Total cGMP-dependent protein kinase I (cGKI) mRNA expression was greater in PASM than in TSM (2.23 +/- 0.36 vs. 0.93 +/- 0.31 amol mRNA/mug total RNA, respectively; P < 0.01), but total cGKI protein expression was not significantly different (0.56 +/- 0.07 and 0.49 +/- 0.04 ng cGKI/mug protein, respectively). The phosphotransferase assay for the soluble fraction of tissue homogenates demonstrated no difference in the cGMP EC(50) between PASM and TSM. The maximal phosphotransferase activity indexed to the amount of total cGKI in the homogenate differed significantly between PASM and TSM (1.61 +/- 0.15 and 1.04 +/- pmol.min(-1).ng cGKI(-1), respectively; P < 0.05), suggesting that cGKI may be regulated differently in the two tissues. A novel intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is thus greater cGMP responsiveness from increased cGKI-specific activity in PASM.     

cGMP signals mainly through cAMP kinase in permeabilized murine aorta

GMP affects vascular tone by multiple mechanisms, including inhibition of the Rho/Rho kinase-mediated Ca(2+) sensitization, a process identified as Ca(2+) desensitization. Ca(2+) desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca(2+) desensitization is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca(2+)] in permeabilized aortas from wild-type and cGKI-deficient mice. [Ca(2+)] increased aortic tone in the absence and presence of 50 microM GTPgammaS with EC(50) values of 160 and 30 nM, respectively. In the absence of GTPgammaS, the EC(50) for [Ca(2+)] was shifted rightward from 0.16 microM to 0.43 and 0.82 microM by 1 and 300 microM 8-bromo-cGMP (8-Br-cGMP), and to 8 microM by 10 microM Y-27632. Contractions induced by 300 nM [Ca(2+)] were relaxed by 8-Br-cGMP with an EC(50) of 2.6 microM. Surprisingly, [Ca(2+)]-induced contractions were also relaxed by 8-Br-cGMP in aortas from cGKI(-/-) mice (EC(50) of 19 microM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated "cross"-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI(-/-) mice. Indeed, the PKA inhibitor peptide (PKI 5-24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI(-/-) mice and to 65% in wild-type aortas. The thromboxane analogue U-46619 induced contraction at constant [Ca(2+)], which was only partially relaxed by 8-Br-cGMP but completely relaxed by Y-27632. The effect of 8-Br-cGMP on U-46619-induced contraction was attenuated by PKI 5-24. These results show that cGKI has only a small inhibitory effect on Ca(2+) sensitization in murine aortas.     

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

Mechanism of cGMP contribution to the vasodilator response to NO in rat middle cerebral arteries

This study examined the mechanism by which cGMP contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries (MCA). Administration of a NO donor, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate), or 8-bromo-cGMP (8-BrcGMP) increased the diameter of serotonin-preconstricted MCA by 79 +/- 3%. The response to DEA-NONOate, but not 8-BrcGMP, was attenuated by iberiotoxin (10(-7) M) or a 80 mM high-K(+) media, suggesting that activation of K(+) channels contributes to the vasodilator response to NO but not 8-BrcGMP. The effects of NO and cGMP on the vasoconstrictor response to Ca(2+) were also studied in MCA that were permeabilized with alpha-toxin and ionomycin. Elevations in bath Ca(2+) from 10(-8) to 10(-5) M decreased the diameter of permeabilized MCA by 76 +/- 5%. DEA-NONOate (10(-6) M) and 8-BrcGMP (10(-4) M) blunted this response by 60%. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (10(-5) M) blocked the inhibitory effect of the NO donor, but not 8-BrcGMP, on Ca(2+)-induced vasoconstriction. 8-BrcGMP (10(-4) M) had no effect on intracellular Ca(2+) concentration ([Ca(2+)](i)) in control, serotonin-stimulated, or alpha-toxin- and ionomycin-permeabilized vascular smooth muscle cells isolated from the MCA. These results indicate that the vasodilator response to NO in rat MCA is mediated by activation of Ca(2+)-activated K(+) channels via a cGMP-independent pathway and that cGMP also contributes to the vasodilator response to NO by decreasing the contractile response to elevations in [Ca(2+)](i).     

IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI-dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP(3)RI)-associated protein (IRAG), which decreases hormone-induced IP(3)-dependent Ca(2+) release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP(3)RI interaction and resulted in hypomorphic IRAG(Delta12/Delta12) mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol- and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG(Delta12/Delta12) mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca(2+)](i) were not decreased by cGMP in aortic smooth muscle cells from IRAG(Delta12/Delta12) mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG(Delta12/Delta12) mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP(3)RI.     

Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle

Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system. The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T. (1993) FEBS Lett. 333, 51-55). Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation. Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus. The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine.     

Frequency encoding of cholinergic- and purinergic-mediated signaling to mouse urinary bladder smooth muscle: modulation by BK channels

In the urinary bladder, contractions of the detrusor muscle and urine voiding are induced by the neurotransmitters ACh and ATP, released from parasympathetic nerves. Activation of K(+) channels, in particular the large-conductance Ca(2+)-activated K(+) (BK) channels, opposes increases in excitability and contractility of urinary bladder smooth muscle (UBSM). We have shown that deleting the gene mSlo1 in mice (Slo(-/-)), encoding the BK channel, leads to enhanced nerve-mediated and neurotransmitter-dependent contractility of UBSM (38). Here, we examine the location of the BK channel in urinary bladder strips from mouse. Immunohistochemical analysis revealed that the channel is expressed in UBSM but not in nerves that innervate the smooth muscle. The relationship between electrical field stimulation and force generation of the cholinergic and purinergic pathways was examined by applying blockers of the respective receptors in UBSM strips from wild-type and from Slo(-/-) (knockout) mice. In wild-type strips, the stimulation frequency required to obtain a half-maximal force was significantly lower for the purinergic (7.2 +/- 0.3 Hz) than the cholinergic pathway (19.1 +/- 1.5 Hz), whereas the maximum force was similar. Blocking BK channels with iberiotoxin or ablation of the Slo gene increased cholinergic- and purinergic-mediated force at low frequencies, i.e., significantly decreased the frequency for a half-maximal force. Our results indicate that the BK channel has a very significant role in reducing both cholinergic- and purinergic-induced contractility and suggest that alterations in BK channel expression or function could contribute to pathologies such as overactive detrusor.     

Defects in cGMP-PKG pathway contribute to impaired NO-dependent responses in hepatic stellate cells upon activation

NO antagonizes hepatic stellate cell (HSC) contraction, although activated HSC in cirrhosis demonstrate impaired responses to NO. Decreased NO responses in activated HSC and mechanisms by which NO affects activated HSC remain incompletely understood. In normal rat HSC, the NO donor diethylamine NONOate (DEAN) significantly increased cGMP production and reduced serum-induced contraction by 25%. The guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) abolished 50% of DEAN effects, whereas the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) reiterated half the observed DEAN response, suggesting both cGMP-dependent protein kinase G (PKG)-dependent and -independent mechanisms of NO-mediated antagonism of normal HSC contraction. However, NO donors did not increase cGMP production from in vivo activated HSC from bile duct-ligated rats and showed alterations in intracellular Ca(2+) accumulation suggesting defective cGMP-dependent effector pathways. The LX-2 cell line also demonstrated lack of cGMP generation in response to NO and a lack of effect of ODQ and 8-BrcGMP in modulating the NO response. However, cGMP-independent effects in response to NO were maintained in LX-2 and were associated with S-nitrosylation of proteins, an effect reiterated in primary HSC. Adenovirus-based overexpression of PKG significantly attenuated contraction of LX-2 by 25% in response to 8-BrcGMP. In summary, these studies demonstrate that NO affects HSC through cGMP-dependent and -independent pathways. The HSC activation process is associated with maintenance of cGMP-independent actions of NO but defects in cGMP-PKG-dependent NO signaling that are improved by PKG gene delivery in LX-2 cells. Activating targets downstream from NO-cGMP in activated HSC may represent a novel therapeutic target for portal hypertension.     

Cyclic guanosine monophosphate-dependent protein kinase I promotes adhesion of primary vascular smooth muscle cells

The cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway regulates many cellular functions. The current study shows that 8-Br-cGMP stimulates the number of attached primary but not that of subcultured murine vascular smooth muscle cells (VSMCs). These effects of 8-Br-cGMP require the presence of cGKI. In agreement with previous studies, cGKI inhibited the number of cells in repeatedly passaged murine VSMCs. Activation of the cGMP/cGKI pathway in freshly isolated primary VSMCs slightly decreased apoptosis and strongly increased cell adhesion. The stimulation of cell adhesion by cGKI involves an inhibition of the RhoA/Rho kinase pathway and increased exposure of β₁ and β₃ integrins on the cell surface. Together, these results identify a novel proadhesive function of cGMP/cGKI signaling in primary VSMCs and suggest that the opposing effects of this pathway on VSMC number depend on the phenotypic context of the cells.     

Prolonged treatment of porcine pulmonary artery with nitric oxide decreases cGMP sensitivity and cGMP-dependent protein kinase specific activity

A cultured porcine pulmonary artery (PA) model was used to examine the effects of prolonged nitric oxide (NO) treatment on the response to acutely applied NO, cGMP analog, or atrial natriuretic peptide (ANP). Twenty-four-hour treatment with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) resulted in >10-fold decrease in the response to acutely applied DETA-NO. In parallel with this, the relaxant response to acutely applied cGMP analog, beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Sp isomer (Sp-8-Br-PET-cGMPS), and ANP decreased. The reduction in ANP responsiveness in PA was not associated with a reduction in cGMP levels evoked by 10(-6) M ANP. Twenty-four hours in culture and treatment with DETA-NO decreased total cGMP-dependent protein kinase (cGKI) mRNA level compared with that in freshly prepared PA (1.05 +/- 0.12, 0.42 +/- 0.08, and 0.11 +/- 0.01 amol/mug, respectively). Total cGKI protein levels were decreased to a lesser extent by 24 h in culture and further decreased by 24-h DETA-NO treatment compared with that in freshly prepared PA (361 +/- 33, 272 +/- 20, and 238 +/- 25 ng/mg total protein, respectively). Maximal cGMP-stimulated phosphotransferase activity was reduced in 24-h cultured and DETA-NO-treated PA (986 +/- 84, 815 +/- 81, and 549 +/- 78 pmol P(i).min(-1).mg soluble protein(-1)), but the cGMP concentration resulting in 50% of maximal phosphotransferase activity was not. cGKI specific activity (maximal cGMP-activated phosphotransferase activity/ng cGKI) was significantly reduced in PA treated with DETA-NO for 24 h compared with freshly prepared and 24-h cultured PA (1.95 +/- 0.22, 2.64 +/- 0.25, and 2.85 +/- 0.28 pmol P(i).min(-1).ng cGKI(-1), respectively). We conclude that prolonged NO treatment induces decreased acute NO responsiveness in PA in part by decreasing cGMP sensitivity. It does so by decreasing both cGKI expression and cGKI specific activity.     

Cyclic cytidine 3′,5′-monophosphate (cCMP) signals via cGMP kinase I

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Iα and Iβ and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositol-trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI- and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin

Human cervicalepithelial cells express mRNA for the nitric oxide (NO) synthase (NOS)isoforms ecNOS, bNOS, and iNOS and release NO into the extracellularmedium. N^G-nitro-L-arginine methylester (L-NAME), an NOS inhibitor, and Hb, an NO scavenger,decreased paracellular permeability; in contrast, the NO donors sodiumnitroprusside (SNP) andN-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increasedparacellular permeability across cultured human cervical epithelia onfilters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect wasblocked by KT-5823 (a blocker of cGMP-dependent protein kinase), butnot by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583and KT-5823 blocked the SNP-induced increase in permeability. Treatmentwith SNP increased cellular cGMP, and the effect was blocked by Hb andLY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulatedcervical cation selectivity. In contrast, both agents increasedfluorescence from fura 2-loaded cells in theCa²⁺-insensitive wavelengths, indicating that SNP and8-BrcGMP stimulate a decrease in cell size and in the resistance of thelateral intercellular space. Neither SNP nor 8-BrcGMP had an effect ontotal cellular actin, but both agents increased the fraction ofG-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823blocked the 8-BrcGMP-induced increase in G-actin. On the basis of theseresults, it is suggested that NO acts on guanylate cyclase andstimulates an increase in cGMP; cGMP, acting via cGMP-dependent proteinkinase, shifts actin steady-state toward G-actin; this fragments thecytoskeleton and renders cells more sensitive to decreases in cell sizeand resistance of the lateral intercellular space and, hence, toincreases in permeability. These results may be important forunderstanding NO regulation of transcervical paracellular permeabilityand secretion of cervical mucus in the woman.

     

A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.     

A heretical view on the role of NO and cGMP in vascular proliferative diseases

Endogenous nitric oxide (NO), and possibly NO-releasing drugs, can both inhibit and promote vascular proliferative disorders, such as atherosclerosis and restenosis. The cell types and signaling pathways that mediate these opposing effects are controversial. It is widely assumed that the NO-mediated synthesis of the second messenger cGMP and the activation of cGMP-dependent protein kinase type I (cGKI) inhibits the proliferation of vascular smooth muscle cells and, thus, vascular remodeling. However, recent data from transgenic mouse models challenge this view. Here, we propose that cGMP signaling through cGKI might promote vasculoproliferative processes and their clinical complications. This new concept has important implications for the use of cGMP-elevating drugs in humans and might help to identify novel therapeutic strategies for vascular proliferative diseases.