Modulation of Ca ²⁺ Channels by Cyclic Nucleotide Cross Activation of Opposing Protein Kinases in Rabbit Portal Vein

Abstract

Abstract-Cyclic nucleotides are known to modify voltage-gated (L-type) Ca 2ϩ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP-and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca 2ϩ channel function by using both conventional and perforated-patch-clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 mol/L), significantly increased (14% to 16%) peak Ba 2ϩ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba 2ϩ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba 2ϩ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca V oltage-dependent (L-type) Ca 2ϩ channels play a major role in excitation-contraction coupling in vascular smooth muscle cells. L-type Ca 2ϩ channels are known to be modulated by several intracellular second-messenger systems, including both the cAMP/cAMP-dependent protein kinase (PKA) and cGMP/cGMP-dependent protein kinase (PKG) pathways.1 However, for vascular smooth muscle, little information is known regarding the exact mechanism(s) by which these processes take place. Patch-clamp studies in smooth muscle cells have shown that L-type Ca 2ϩ channel activity can be enhanced by either low concentrations of 8-Br cAMP or the catalytic subunit of PKA. 2-4Stimulation of ␤-adrenergic receptors with Iso has also been shown to increase Ca 2ϩ channel currents. 2,3,[5][6][7] On the other hand, 8-Br cGMP or the NO-releasing agents sodium nitroprusside and SNAP have been reported to lead to a decrease of Ca 2ϩ channel activity. 2,8 -10The precise mechanism underlying the effects of both PKA and PKG on L-type Ca 2ϩ channels remains controversial. A previous study from this laboratory showed that a moderate increase in cAMP elicited with 1 mol/L Iso, 1 mol/L FSK, or 0.1 mmol/L 8-Br cAMP increased Ca 2ϩ channel currents. 2On the other hand, higher levels of cAMP elicited with 10 mmol/L Iso, 10 mol/L FSK, 1 mmol/L 8-Br cAMP, or 0.1 mmol/L 8-Br cGMP led to inhibition of Ca 2ϩ channel currents. Experiments that measured the time course of responses to high concentrations of Iso or FSK revealed that Ca 2ϩ channel currents were initially enhanced and subsequently inhibited. It has been suggested that moderate increases in cAMP enhance Ca 2ϩ channel currents through PKA activation, whereas higher levels of cAMP lead to activation of PKG, which then predominates over the PKA effect (ie, cross activation of PKG by cAMP). Similar findings have been recently reported in colonic smooth muscle cells. 3 In smooth muscle cells from the basilar artery, it has been shown that exposure of inside-out patches to the catalytic subunit of PKA increased L-type Ca 2ϩ channel availability. 4 In apparent conflict with these results, Sperelakis and coworkers [11][12][13] have

Intraluminal Pressure Is a Stimulus for NFATc3 Nuclear Accumulation

“…Nitric oxide and cGMP-dependent kinase (PKG) have been traditionally viewed as mediators of vasodilation through effects on large conductance, Ca 2ϩ -activated potassium (BK) channels (26), VDCC (27,28), phospholamban (29), and IRAG inositol trisphosphate receptor-associated cGMP kinase substrate, as well as by decreasing the Ca 2ϩ sensitivity of the contractile process (30 -32). Recent evidence indicates that NO/ PKG can promote or inhibit vascular remodeling (33).…”

Relaxation Patterns of Human Gastric Corporal Smooth Muscle by Cyclic Nucleotides Producing Agents

“…For the cAMP-induced relaxation: 1) decrease of Ca 2＋ influx (Bulbring and Tomita, 1980;Ozaki et al, 1992;Smith et al, 1993;Koh and Sanders, 1996;Zhu et al, 2005), and 2) regulation of sarcoplasmic reticulum (SR) Ca 2＋ uptake and Ca 2＋ extrusion (Casteels and Raeymakers, 1979;Meisheri and van Breemen, 1982;Nishimura and van Breemen, 1989). For the cGMP-mediated relaxation: 1) decrease of Ca 2＋ influx (Ozaki et al, 1992;Quignard et al, 1997;Ruiz-Velasco et al, 1998;Geeson et al, 2002;Zhu et al, 2005), 2) increase of sarcoplasmic reticulum (SR) Ca 2＋ uptake, 3) involvement of protein kinase (Kim et al, 2006;Kim and Perrino, 2007), and 4) increase of calcium activated K ＋ current (IKCa) (Yu et al, 1993;Geeson et al, 2002;Kim et al, 2006). Therefore, the inhibitory effect of cAMP and/or cGMP on smooth muscle motility seems to be associated with regulation of Ca 2＋ homeostasis.…”

“…However, there are some controversies on the inhibition pathways of smooth muscle motility through the regulation of VDCCL and cyclic nucleotides. Hot issues to be considered are: 1) whether the effect is to increase or decrease VDCCL, and 2) whether there are interactions between cAMP-and cGMP-dependent pathways (Ohya et al, 1987;Ishikawa et al, 1993;Koh and Sanders, 1996;Ruiz-Velasco et al, 1998;Zhu et al, 2005).…”

The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway

“…Although there are no reports of WT1 expression in lung epithelium, both kidney epithelial and breast epithelial cells have been reported to express this factor (24). Interestingly, WT1 is regulated by protein kinase A (PKA) (24 -26), a downstream mediator of sGC (27)(28)(29). PKA-mediated phosphorylation of WT1 induces its translocation from the nucleus to the cytosol.…”

“…Active sGC produces cGMP and the latter can mediate gene regulation through the activation of cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) (27)(28)(29). To test the role of PKG and PKA in the signal transduction of this pathway, we used the membrane soluble PKG-and PKA-specific inhibitors AD-2 and myrPKA, respectively.…”