Insulin increases glomerular filtration barrier permeability through PKGIα-dependent mobilization of BKCa channels in cultured rat podocytes.

Podocytes are highly specialized cells that wrap around glomerular capillaries and comprise a key component of the glomerular filtration barrier. They are uniquely sensitive to insulin; like skeletal muscle and fat cells, they exhibit insulin-stimulated glucose uptake and express glucose transporters. Podocyte insulin signaling is mediated by protein kinase G type I (PKGI), and it leads to changes in glomerular permeability to albumin. Here, we investigated whether large-conductance Ca²âº-activated K⁺ channels (BKCa) were involved in insulin-mediated, PKGIα-dependent filtration barrier permeability. Insulin-induced glomerular permeability was measured in glomeruli isolated from Wistar rats. Transepithelial albumin flux was measured in cultured rat podocyte monolayers. Expression of BKCa subunits was detected by RT-PCR. BKCa, PKGIα, and upstream protein expression were examined in podocytes with Western blotting and immunofluorescence. The BKCa-PKGIα interaction was assessed with co-immunoprecipitation. RT-PCR showed that primary cultured rat podocytes expressed mRNAs that encoded the pore-forming α subunit and four accessory ß subunits of BKCa. The BKCa inhibitor, iberiotoxin (ibTX), abolished insulin-dependent glomerular albumin permeability and PKGI-dependent transepithelial albumin flux. Insulin-evoked albumin permeability across podocyte monolayers was also blocked with BKCa siRNA. Moreover, ibTX blocked insulin-induced disruption of the actin cytoskeleton and changes in the phosphorylation of PKG target proteins, MYPT1 and RhoA. These results indicated that insulin increased filtration barrier permeability through mobilization of BKCa channels via PKGI in cultured rat podocytes. This molecular mechanism may explain podocyte injury and proteinuria in diabetes.

Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells.

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCß) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+), and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCß were PDGFRα(+) cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα(+) cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI(-/-) mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. N(ω)-nitro-l-arginine (l-NNA) abolished responses during the first 20-30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.

Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I.

AIMS: Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. METHODS AND RESULTS: To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to ß-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca(2+)]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca(2+)i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca(2+)i-handling, and contractility via cGKI. CONCLUSION: These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca(2+)i handling and contractility.

GPA-14, a Gα(i) subunit mediates dopaminergic behavioral plasticity in C. elegans.

BACKGROUND: Precise levels of specific neurotransmitters are required for appropriate neuronal functioning. The neurotransmitter dopamine is implicated in modulating behaviors, such as cognition, reward and memory. In the nematode Caenorhabditis elegans, the release of dopamine during behavioral plasticity is in part modulated through an acid-sensing ion channel expressed in its eight dopaminergic neurons. A D2-like C. elegans dopamine receptor DOP-2 co-expresses along with a Gα(i) subunit (GPA-14) in the anterior deirid (ADE) pair of dopaminergic neurons. FINDINGS: In follow-up experiments to our recently reported in vitro physical interaction between DOP-2 and GPA-14, we have behaviorally characterized worms carrying deletion mutations in gpa-14 and/or dop-2. We found both mutants to display behavioral abnormalities in habituation as well as associative learning, and exogenous supply of dopamine was able to revert the observed behavioral deficits. The behavioral phenotypes of dop-2 and gpa-14 loss-of-function mutants were found to be remarkably similar, and we did not observe any cumulative defects in their double mutants. CONCLUSION: Our results provide genetic and phenotypic support to our earlier in vitro results where we had shown that the DOP-2 dopamine receptor and the GPA-14 Gα(i) subunit physically interact with each other. Results from behavioral experiments presented here together with our previous in-vitro work suggests that the DOP-2 functions as a dopamine auto-receptor to modulate two types of learning, anterior touch habituation and chemosensory associative conditioning, through a G-protein complex that comprises GPA-14 as its Gα subunit.

cGMP and cGMP-dependent protein kinase I pathway in dorsal root ganglia contributes to bone cancer pain in rats.

STUDY DESIGN: A prospective, randomized experimental research. OBJECTIVE: To demonstrate the role of cGMP (cyclic guanosine monophosphate)-cGKI (cGMP-dependent protein kinase I) pathway in dorsal root ganglia (DRG) in bone cancer pain. SUMMARY OF BACKGROUND DATA: Treating bone cancer pain continues to possess a major clinical challenge because the specific cellular and molecular mechanisms underlying bone cancer pain remain elusive. cGMP and cGMP-dependent protein kinases pathway in DRG plays important role in nerve injury-induced hyperexcitability of DRG neurons, as well as neuropathic pain, however, whether this pathway participates in bone cancer pain is unknown. METHODS: The rat model of bone cancer pain was produced by intramedullary injection of rat breast cancer cells (Walker 256) into right tibia. Thermal hyperalgesia and mechanical allodynia were measured before and after administration of inhibitor of cGMP-cGKs pathway (Rp-8-pCPT-cGMPS). Immunofluorescence and reverse transcription-polymerase chain reaction were used to reflect expression of cGKI in DRG neurons, whereas the concentration of cGMP in DRG was tested using enzyme-linked immunosorbent assay method. Whole-cell patch clamp was used to record the hyperexcitability of small neurons in DRG with or without cGKs inhibitor after tumor cell implantation (TCI). RESULTS: TCI treatment significantly increased the concentration of cGMP in DRG and activity of cGKs in DRG and the spinal cord. TCI treatment also induced upregulation of cGKI messenger ribonucleic acid and protein in DRG, as well as enhanced hyperexcitability in DRG neurons. Spinal administration of Rp-8-pCPT-cGMPS, cGMP-cGKs inhibitor, significantly suppressed TCI-induced activation of cGMP-cGKI signaling, and hyperexcitability of DRG neurons. Meanwhile, in vivo intrathecal delivery of the Rp-8-pCPT-cGMPS significantly prevented and suppressed TCI-induced hyperalgesia and allodynia. CONCLUSION: From these results, we confirm that TCI treatment activates cGMP-cGKI signaling pathway and continuing activation of this pathway in DRG is required for hyperalgesia and/or hyperalgesia and allodynia after TCI treatment. LEVEL OF EVIDENCE: N/A.

Transcriptional and post-transcriptional regulation of cGMP-dependent protein kinase (PKG-I): pathophysiological significance.

The ability of the endothelium to produce nitric oxide, which induces generation of cyclic guanosine monophosphate (cGMP) that activates cGMP-dependent protein kinase (PKG-I), in vascular smooth muscle cells (VSMCs), is essential for the maintenance of vascular homeostasis. Yet, disturbance of this nitric oxide/cGMP/PKG-I pathway has been shown to play an important role in many cardiovascular diseases. In the last two decades, in vitro and in vivo models of vascular injury have shown that PKG-I is suppressed following nitric oxide, cGMP, cytokine, and growth factor stimulation. The molecular basis for these changes in PKG-I expression is still poorly understood, and they are likely to be mediated by a number of processes, including changes in gene transcription, mRNA stability, protein synthesis, or protein degradation. Emerging studies have begun to define mechanisms responsible for changes in PKG-I expression and have identified cis- and trans-acting regulatory elements, with a plausible role being attributed to post-translational control of PKG-I protein levels. This review will focus mainly on recent advances in understanding of the regulation of PKG-I expression in VSMCs, with an emphasis on the physiological and pathological significance of PKG-I down-regulation in VSMCs in certain circumstances.

Mutation of the protein kinase I alpha leucine zipper domain produces hypertension and progressive left ventricular hypertrophy: a novel mouse model of age-dependent hypertensive heart disease.

Hypertensive heart disease causes significant mortality in older patients, yet there is an incomplete understanding of molecular mechanisms that regulate age-dependent hypertensive left ventricular hypertrophy (LVH). Therefore, we tested the hypothesis that the cGMP-dependent protein kinase G I alpha (PKGIα) attenuates hypertensive LVH by evaluating the cardiac phenotype in mice with selective mutations of the PKGIα leucine zipper domain. These leucine zipper mutant (LZM) mice develop basal hypertension. Compared with wild-type controls, 8-month-old adult LZM mice developed increased left ventricular end-diastolic pressure but without frank LVH. In advanced age (15 months), the LZM mice developed overt pathological LVH. These findings reveal a role of PKGIα in normally attenuating hypertensive LVH. Therefore, mutation of the PKGIα LZ domain produces a clinically relevant model for hypertensive heart disease of aging.

The role of cGMP/cGKI signalling and Trpc channels in regulation of vascular tone.

AIMS: Signalling via cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle (SM), by which endothelial NO regulates vascular tone. Recent evidence suggests that canonical transient receptor potential (Trpc) channels are targets of cGKI in SM and mediate the relaxant effects of cGMP signalling. We tested this concept by investigating the role of cGMP/cGKI signalling on vascular tone and peripheral resistance using Trpc6(-/-), Trpc3(-/-), Trpc3(-/-)/6(-/-), Trpc1(-/-)/3(-/-)/6(-/-), and SM-specific cGKI(-/-) (sm-cGKI(-/-)) mice. METHODS AND RESULTS: α-Adrenergic stimulation induced similar contractions in L-NG-nitroarginine methyl ester (l-NAME)-treated aorta and comparably increased peripheral pressure in hind limbs from all mouse lines investigated. After α-adrenergic stimulation, 8-Br-cGMP diminished similarly aortic tone and peripheral pressure in control, Trpc6(-/-), Trpc3(-/-), Trpc3(-/-)/6(-/-), and Trpc1(-/-)/3(-/-)/6(-/-) mice but not in sm-cGKI(-/-) mice. In untreated aorta, α-adrenergic stimulation induced similar contractions in the aorta from control and Trpc3(-/-) mice but larger contractions in sm-cGKI(-/-), Trpc6(-/-), Trpc3(-/-)/6(-/-), and Trpc1(-/-)/3(-/-)/6(-/-) mice, indicating a functional link between cGKI and Trpc6 channels. Trpc3 channels were detected by immunocytochemistry in both isolated aortic smooth muscle cells (SMCs) and aortic endothelial cells (ECs), whereas Trpc6 channels were detected only in ECs. Phenylephrine-stimulated Ca(2+) levels were similar in SMCs from control (Ctr) and Trpc6(-/-) mice. Carbachol-stimulated Ca(2+) levels were reduced in ECs from Trpc6(-/-) mice. Stimulated Ca(2+) levels were lowered by 8-Br-cGMP in Ctr but not in Trpc6(-/-) ECs. CONCLUSIONS: The results suggest that cGKI and Trpc1,3,6 channels are not functionally coupled in vascular SM. Deletion of Trpc6 channels impaired endothelial cGKI signalling and vasodilator tone in the aorta.