The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca(2+) itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that β-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVβ2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca(2+)-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKIdependent 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 þ 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 D12/D12 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 D12/D12 mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca 2 þ ] i were not decreased by cGMP in aortic smooth muscle cells from IRAG D12/D12 mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG D12/D12 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.

To study the role of cGMP-dependent protein kinase I (cGKI) for cardiac contractility, force of contraction (F c ) was studied in electrically driven heart muscle from wild-type (WT) mice and from conventional and conditional cGKI knockout mice. Both 8-Br-cGMP and 8-pCPT-cGMP reduced Fc in cardiac muscle from juvenile WT but not from juvenile cGKI-null mutants. Similarly, the cGMP analogues reduced F c in forskolinstimulated ventricular muscle from WT mice but not from cGKI-null mutants. In contrast, carbachol reduced F c in both groups of animals. 8-Br-cGMP reduced F c also in heart muscle from adult WT mice but not from adult cardiomyocyte-specific cGKI-knockout mice. These results demonstrate that cGKI mediates the negative inotropic effect of cGMP in the myocardium of juvenile and adult mice.A cetylcholine and the muscarinic agonist carbachol (CCh) induce negative inotropy in human and rodent heart. The molecular basis for muscarinic inhibition of cardiac contractility is controversial. 1,2 Activation of NO synthase III leading to an increase of the cGMP level has been reported to contribute to muscarinic inhibition 3-5 as well as the irrelevance of NO, 6 NO synthase III,7,8 and cGMP/cGMPdependent protein kinase I (cGKI) 9 for this signaling pathway. The cGMP receptor potentially mediating the negative inotropic effect has not been identified. It was suggested that cGMP regulates cGMP-stimulated as well as cGMP-inhibited cAMP phosphodiesterases, thereby modulating cAMP levels and L-type calcium currents. 10,11 This type of modulation would either decrease or increase cardiac contractility. Furthermore, cGKI that is expressed in cardiomyocytes 9,12 has been implicated in the inhibitory effects of cGMP on L-type calcium current 13,14 and contraction. [15][16][17] We have investigated the role of cGMP/cGKI signaling for cardiac contractility using myocardium from conventional and conditional cGKI-knockout mice. This study shows that cGKI mediates negative inotropic effects elicited by cGMP in the absence and presence of forskolin, an activator of the ␤-adrenergic/cAMP pathway but is not involved in inhibition of cardiac contractility by CCh. Materials and MethodsThe Materials and Methods section is available online in the data supplement at http://www.circresaha.org. Results and DiscussionA conventional cGKI-null allele [(Ϫ)] was obtained by replacing the 3Ј region of exon 10 of the cGKI gene (which is essential for kinase activity) with a DNA cassette encoding CreER T recombinase. 18 The CreER T recombinase was not expressed from the cGKI (Ϫ) allele. A conditional cGKI allele (L2) was obtained by flanking exon 10 with loxP sites. Excision of exon 10 from the L2 allele by Cre-mediated recombination of the loxP sites produced an LϪ allele (Figures 1A and 1B). Heterozygous cGKI ϩ/Ϫ , cGKI, cGKI ϩ/LϪ , and cGKI LϪ/L2 mice as well as homozygous cGKI L2/L2 mice expressed cGKI protein and were phenotypically normal. Homozygous cGKI Ϫ/Ϫ and cGKI LϪLϪ mice did not express cGKI protein and were phenotypically indistingu...

Abstract-Smooth muscle expresses the I␣ and the I␤ isoforms of cGMP-dependent protein kinase I (cGKI). Inactivation of the murine cGKI gene prkg1 leads to multiple phenotypes and premature death at Ϸ6 weeks. We reconstituted mice with the cGKI␣ or -I␤ isozyme to test which isozyme was needed to support basic smooth muscle functions. Mice were generated by gene targeting. The cGKI␣ or the -I␤ coding sequences were placed under the control of the SM22␣ promoter to express either isoform selectively in smooth muscle cells (SM-I␣ or SM-I␤ transgene). To generate smooth muscle-specific cGKI␣ or cGKI␤ rescue mice, the SM-I␣ or SM-I␤ transgenes were crossed on a cGKI Ϫ/Ϫ genetic background. The levels of cGKI␣ or -I␤ expression were comparable to endogenous cGKI expression in wild-type aortic and intestinal smooth muscles. In cGKI␣ or -I␤ rescue mice, expression of the isozymes was not detectable in non-smooth muscle tissues and cells. Median survival time of the I␣ and I␤ rescue mice was 52 weeks. Both isozymes mediated the 8-bromo-cGMP-induced relaxation of precontracted jejunum and aorta muscle strips. Activation of both isozymes reduced hormone-or K ϩ -induced [Ca 2ϩ ] i levels. The cGKI␣ and cGKI␤ rescue mice did not show a significant difference in intestinal passage time of BaSO 4 in comparison with wild-type animals. Telemetric blood pressure measurements in conscious freely moving animals did not show differences between rescues and control mice in basal blood pressure and its regulation by DETA-NO, sodium nitroprusside, carbachol, or Y-27632. These results show that cGKI in smooth muscle is essential and that either cGKI isozyme alone can rescue basic vascular and intestinal smooth muscle functions. Key Words: cGMP kinase isozymes Ⅲ PKG Ⅲ nitric oxide Ⅲ smooth muscle Ⅲ blood pressure T he NO/cGMP signaling cascade plays an essential role in vascular smooth muscle (SM) relaxation, and clinical studies indicate that endothelium-derived NO is involved in normal and pathological blood pressure regulation in humans. [1][2][3] The important effector of cGMP, cGMP-dependent protein kinase I (cGKI), is highly expressed in SM. 4 Conventional deletion of the gene for cGKI in mice leads to multiple phenotypes, including severe gastrointestinal disturbances and elevated blood pressure, leading to premature death of the animals. 5 The cGKI gene generates 2 isoforms, cGKI␣ and cGKI␤, that differ only in their individual N termini (the first 90 to 100 residues), which are encoded by 2 alternatively used exons. 6,7 Both isoforms are expressed together in various SMs. 8,9 Strong evidence has been published that these isozymes interact with different proteins and affect SM relaxation through different mechanisms. 10 -13 cGKI␣ interacts specifically with MYPT1 (myosin-interacting subunit of myosin phosphatase 1) 12 and with RGS-2 (regulator of G protein signaling 2), 13 whereas cGKI␤ shows specificity for inositol 1Ј,4Ј,5Ј-triphosphate receptor-associated G kinase substrate (IRAG). 10,11 However, in vitro data from Feil et al...